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.
|