Your browser does not support JavaScript!

Home    Search  

Results - Details

Search command : Author="Μπριασούλης"  And Author="Γεώργιος"

Current Record: 21 of 96

Back to Results Previous page
Next page
Add to Basket
[Add to Basket]
Identifier 000447632
Title Ανίχνευση της ενδοκυττάριας πρωτεϊνικής ενεργοποίησης των αποπτωτικών κασπασών και της αντιαποπτωτικής survivin με κυτταρομετρία ροής σε ενήλικες και παιδιατρικούς ασθενείς με σήψη
Alternative Title Detecting intracellular protein activation of apoptotic caspase and antiapoptotic survivin by flow cytometry in adult and pediatric patients with sepsis
Author Μπαστάκη, Καλλιόπη
Thesis advisor Μπριασούλης, Γεώργιος
Reviewer Ηλία, Σταυρούλα
Κονδύλη, Ευμορφία
Abstract Background Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. According to recent data, the global burden of sepsis is estimated at around 48.9 million cases and 11 million sepsis-related deaths worldwide, which accounted for almost 20% of all global deaths. The pathophysiology of sepsis is characterized by an early hyperinflammatory reaction of the host to an infection, that is followed by an immunodeficient state. Sepsis related death occurs either primarily at the hyperinflammatory state, or late at the stage where the hyperinflammation is persistent or at the immunodeficient state. The predominance of sepsis leads to a cascade of cellular paths of protein molecules, that define the survival of the cell. The interest of the current study is turned to those cellular phenomena. Specifically, apoptosis is activated during sepsis, which is a programmed cell death. Apoptosis is induced by caspases, which are a family of proteolytic enzymes. Caspases are separated in two categories, the initiators and executioners of apoptosis. The initiator caspases are activated by the two distinct paths of apoptosis, the extrinsic and intrinsic pathway. The activation of the initiators of apoptosis leads to the activation of the executioner caspases and the apoptosis of the cell. On the contrary there are the inhibitors of apoptosis, which are a family of proteins with an antiapoptotic effect. Survivin is a member of that family, that plays an important role during the apoptosis of the cell at sepsis, inhibiting key points of the apoptotic pathways and leading to the survival of the cell. The apoptotic and antiapoptotic phenomena seem to happen alongside during sepsis and their prevalence defines the progress of the cell. The study of apoptotic (caspases) and antiapoptotic (survivin) molecules could highlight key elements that mediate in the stimulatory and inhibitory pathways of the cascades in sepsis. Objective The purpose of the study is to investigate the hypothesis of the stimulation of caspase 1, 3, 7, 8 and 9 at the mononuclear cells of the peripheral blood (PBMCs) and survivin at the cells of the blood of patients with sepsis comparatively to patients with non-infectious inflammation (trauma, postoperative inflammation) and to healthy controls. The evaluation of the intracellular expression of the apoptotic caspases and antiapoptotic survivin was performed by the method of flow cytometry. Secondary goals of the study are to compare the results of the measurements with clinical and laboratory indicators of the severity of the illness and the outcome and to evaluate their ability to identify sepsis, as well their prognostic value. Also, a secondary goal of the study is to compare the results of the analysis between the two aging groups of the study (children and adults). Methods This is a prospective single-centre study measuring apoptotic and anti-apoptotic proteins associated with sepsis in adult and pediatric patients of intensive care units and their correlation with clinical-laboratory indicators. The study sample consists of adult and pediatric patients with sepsis and non-infectious inflammation / trauma of the Intensive Care Unit (ICU) and Pediatric Intensive Care Unit of the University Hospital of Heraklion, compared with healthy controls. The patients of the study were selected based on the criteria of sepsis and systemic inflammatory syndrome (SIRS) and the patients were matched for age and sex. Patients with chronic diseases, immunodeficiencies, malignancies, and those receiving immunosuppressive and immunomodulatory therapy were excluded. Patient laboratory, clinical, and demographic data were collected from the medical files of the patients. For the laboratory part of the study, whole blood samples were collected during the first 24 hours of sepsis or SIRS documentation and additional samples were taken on the third and seventh day of ICU stay. The expression of caspases and survivin in patients’ blood cells was determined by flow cytometry. Specifically, the expression of caspases was detected in peripheral blood mononuclear cells (PBMCs) using the technique of binding specific fluorescent labeled inhibitors of caspases (FLICA- fluorochrome labeled inhibitors of caspases), which bind to activated caspases. While the intracellular expression of survivin was calculated in the whole blood cells of patients with the technique of staining cells with a specific conjugated monoclonal antibody (Mouse Anti-Human Survivin PE-conjugated Monoclonal Antibody) with affinity for this protein. The results of the study were compared between the groups of the patients, separately considering the age groups, and with clinical data on disease severity and outcome. The appropriate statistical tests of the statistical package SPSS 26 were used for the statistical analysis of the data. Results The results of the study show an increased expression of survivin in lymphocytes (S=0,65% (0,17-2,05), I=0,4% (0,25-1,05), H=0,2% (0,1-0,95), p=0,370), monocytes (S=3,05% (1,42-7,3), I=1,1% (0,9-6), H=3% (1,12-6,6), p=0,567), and neutrophils (S=8,2% (1,02-17,5), Ι=5,8%(2,1- 26,9), Η=3,4% (1,5-6,3), p=0,672) in septic adult patients, as well as a long-term increase in its expression during sepsis in both adult and pediatric patients noting monocytes to be superior in survivin expression compared to lymphocytes. At the same time, there was an increased expression of caspases 1, 3, 7, and 8, namely the main initiators and executors of apoptotic cell death throughout the course of sepsis in monocytes of adult patients. While in the pediatric population with sepsis the expression of caspases 3 and 7, which are the executors of apoptosis occurs mainly in lymphocytes (p=0,215). The course of sepsis is characterized by necrosis of apoptotic cells with the main representative apoptotic lymphocytes that express caspases 1 (S=1,75%(0,97-5,15) vs I=0,7% (0,5-1,1), H=0,85% (0,62-1,4), p=0,018), 3, 7 (S=1,95% (1,37-3,32) vs I=1% (0,85-1,8), H=1,05% (0,62-1,32), p=0,015), and 8 (S=1,65% (0,97- 7,05) vs I=0,8% (0,55-1,05), H=0,6% (0,3-0,72), p=0,002) which seem to die to a greater extent in septic adult patients, and consequently, their necrosis is particularly increased in patients who did not survive compared to those who survived (necrosis of apoptotic lymphocytes expressing caspase 1 (1,9% (1,75-5,8) vs 0,9% (0,62-1,22), p=0,003), caspase 3/7 (2% (1,35- 4,75) vs 1,25%(0,9-1,5), p=0,011) and caspase 8 (1,9% (1,15-9,6) vs 0,65% (0,52-1,07), p=0,006). Thus, necrosis of apoptotic lymphocytes significantly predicts mortality in adult patients. In addition, necrosis of apoptotic lymphocytes expressing caspase 8 is associated with clinical and laboratory markers of sepsis such as elevated body temperature, white blood cell count (WBC), and C-reactive protein (CRP) in septic adult patients. Conclusions The expression of survivin is increased in patients with sepsis, initially and over time, expressing an important defense mechanism of the cell to survive. In addition, the parallel increased expression of intracellular caspases of septic patients, as well as the overtime increase in necrosis of the apoptotic cells, shows the aggressive and self-destructive attitude of the cell towards sepsis, so that the organism to survive. Both caspases and survivin are important protein molecules that play a key role in sepsis, the diagnostic and prognostic value of which requires further research.
Language Greek
Subject Αποπτωτικός
Κασπάσες
Issue date 2022-03-30
Collection   School/Department--School of Medicine--Department of Medicine--Post-graduate theses
  Type of Work--Post-graduate theses
Permanent Link https://elocus.lib.uoc.gr//dlib/3/d/0/metadata-dlib-1650015376-678087-27462.tkl Bookmark and Share
Views 358

Digital Documents
No preview available

Download document
View document
Views : 19