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Acute pancreatitis (AP) is a common abdominal disease with no specific
treatment. The clinical manifestation of AP can vary widely from a mild selflimiting disease to a potentially life-threatening severe form. Severe acute
pancreatitis (SAP) develops in about 20-25% of patients with AP, and the
severe disease is associated with the development of such complications as
pancreatic necrosis and distant organ failure (OF). A complicated
inflammatory reaction precedes development of OF and is accompanied by
disturbances in the coagulation system.
The patients who will develop SAP need to be identified early in the
course of the disease and aggressively treated (i.e. vigorous fluid
resuscitation in intensive care unit) to prevent mortality.
The aim of this study was to investigate whether ecto-5’nucleotidase
(CD73) predicts the development of SAP and whether recombinant activated
protein C (APC) treatment alleviates the course of SAP. We evaluated also the
effects of recombinant APC treatment on occurrence of bleeding
complications, course of systemic inflammation, and coagulation cascade
disturbances in patients with SAP.
The clinical study consists of four parts. All of the patients investigated
had AP when admitted to Helsinki University Central Hospital. The first
study comprised 161 patients with AP. Their serum levels of soluble CD73
(sCD73) were analyzed by three different methods (activity, protein
concentration, and mRNA levels) and the association of the levels with
different severity grades of AP was evaluated. In addition, the ability of
sCD73 to predict SAP was examined. The second study was a pilot, doubleblind clinical trial in which 32 patients with SAP were randomized to receive
recombinant APC treatment or placebo, and the effects of APC on evolution
of organ failure and and the safety of APC treatment were analyzed. In the
third study, the effects of recombinant APC treatment on markers of systemic
inflammation were examined. The changes on humoral and cellular markers
of systemic inflammation were determined. In the fourth study, the effects of
recombinant APC treatment on coagulopathy in SAP were evaluated. The
plasma levels of selected biomarkers of coagulation and physiological
anticoagulation were determined.
The results showed that the sCD73 levels on admission to hospital
correlated inversely with the severity of AP and the sCD73 activity is able to
predict the severe form of AP among patients with no signs of OF on
admission.
We found no beneficial effects of recombinant APC treatment on
evolution of organ failures or mortality in SAP. No significant bleeding
complications occurred. Recombinant APC treatment did not alter the course
of systemic inflammation, but we could monitor the changes in parameters of
4
systemic inflammation reaction produced by SAP. We found that the SAP
patients showed coagulopathy and the recombinant APC treatment slowed
normalization of the levels of coagulation and anticoagulation markers in
SAP.
Based on these studies, sCD73 activity predicts the development of SAP.
The recombinant APC treatment does not have a significant effect on
evolution of OFs or on mortality in SAP. The effects of APC treatment on
parameters of systemic inflammatory reaction are minor. The coagulation in
SAP is hindered and APC-treated patients achieve normal homeostasis of
coagulation slower than patients receiving placebo.
5
###"
Abstract ................................................................................................................ 4
Table of Contents ............................................................................................... 6
List of original publications ............................................................................ 9
Abbreviations.................................................................................................... 10
1.
Introduction ............................................................................................. 12
2.
Review of the literature of acute pancreatitis ................................. 14
2.1
ANATOMY AND PHYSIOLOGY OF PANCREAS .............................. 14
2.2
EPIDEMIOLOGY ................................................................................ 15
2.3
ETIOLOGY .......................................................................................... 16
2.4
PATHOGENESIS ................................................................................ 17
2.4.1
Triggering factors ........................................................................... 17
2.4.2
Local inflammation ........................................................................ 19
2.4.3
Systemic inflammation .................................................................. 23
2.4.4
Coagulation and anticoagulation ................................................... 29
2.4.5
Coagulation and inflammation ...................................................... 34
2.4.6
Coagulopathy in severe acute pancreatitis .................................... 37
2.5
DIAGNOSIS ........................................................................................38
2.5.1
Main diagnostic criteria .................................................................38
2.5.2
Clinical signs and physical examination ........................................38
2.5.3
Laboratory tests..............................................................................38
2.5.4
Radiological findings ...................................................................... 39
2.6
CLASSIFICATION ............................................................................. 40
2.6.1
Atlanta classification 1992 ............................................................ 40
2.6.2
Revised Alanta classification 2012................................................ 40
6
2.7
2.7.1
Risk factors ..................................................................................... 43
2.7.2
Scoring systems .............................................................................. 43
2.7.3
Biomarkers in predicting severe acute pancreatitis ...................... 44
2.8
3.
SEVERITY ASSESSMENT .................................................................. 43
TREATMENT ...................................................................................... 45
2.8.1
Fluid therapy .................................................................................. 46
2.8.2
Causative therapy ........................................................................... 46
2.8.3
Pain management........................................................................... 47
2.8.4
Nutrition ......................................................................................... 47
2.8.5
Antibiotic prophylaxis .................................................................... 47
2.8.6
Anticoagulant therapy .................................................................... 47
2.8.7
Experimental pharmacologic therapy ...........................................48
Present investigation ............................................................................. 51
3.1
AIMS OF THE STUDY ........................................................................ 51
3.2
MATERIALS AND METHODS ........................................................... 52
3.2.1
Patients and healthy control subjects ............................................ 52
3.2.2
Designs and main objectives of each study ................................... 56
3.3
ANALYTICAL METHODS .................................................................. 58
3.3.1
Blood samples ................................................................................ 58
3.3.2
Laboratory analyses ....................................................................... 59
3.3.3
Enzyme-linked immunosorbent assay .......................................... 59
3.3.4
Whole blood flow cytometry ......................................................... 60
3.3.5
CD73 activity ................................................................................. 60
3.3.6
qPCR for CD73 mRNA ................................................................... 61
3.3.7
Thrombograms ............................................................................... 61
3.4
STATISTICAL METHODS .................................................................. 62
3.5
RESULTS ............................................................................................ 63
7
3.5.1
CD73 in predicting severe acute pancreatitis ................................ 63
3.5.2
APC treatment in severe acute pancreatitis................................... 67
3.6
DISCUSSION ...................................................................................... 70
3.6.1
Predicting severe acute pancreatitis .............................................. 70
3.6.2
Treatment of severe acute pancreatitis .......................................... 71
3.6.3
Clinical and future aspects ............................................................. 76
3.6.4
Strengths and limitations of studies .............................................. 78
3.7
CONCLUSIONS ................................................................................. 80
ACKNOWLEDGMENTS....................................................................................... 81
REFERENCES ......................................................................................................83
8
"#! $#"
This thesis is based on the following original publications, which are referred
to in the text by their Roman numerals:
I.
Maksimow M, Kyhälä L, Nieminen A, Kylänpää L, Aalto K, Elima K,
Mentula P, Lehti M, Puolakkainen P, Yegutkin G, Jalkanen S, Repo H,
Salmi M: Early Prediction of Persistent Organ Failure by Soluble CD73
in Patients with Acute Pancreatitis. Crit Care Med. 2014
Dec;42(12):2556-2564.
II.
Pettilä V, Kyhälä L, Kylänpää ML, Leppäniemi A, Tallgren M,
Markkola A, Puolakkainen P, Repo H, Kemppainen E: APCAP activated protein C in acute pancreatitis: a double-blind randomized
human pilot trial. Crit Care. 2010;14(4):R139.
III.
Kyhälä L, Mentula P, Kylänpää L, Moilanen E, Puolakkainen P, Pettilä
V, Repo H: Activated Protein C Does Not Alleviate the Course of
Systemic Inflammation in the APCAP Trial. Int J Inflam.
2012;2012:712739.
IV.
Kyhälä L, Lindström O, Kylänpää L, Mustonen H, Puolakkainen P,
Kemppainen E, Tallgren M, Pettilä V, Repo H, Petäjä J: Activated
protein C retards recovery from coagulopathy in severe acute
pancreatitis. Scand J Clin Lab Invest. 2016 Feb;76(1):10-16.
These publications have been reprinted with the permission of their
copyright holders.
9
Introduction
!%#"
AP
APA
APACHE II
APC
ARDS
AT
ATP
AUC
BISAP
CAT
CARS
CD
CD62L
CD73
CFTR
CRP
CT
DAMP
DIC
DIM
DNA
ELISA
EPCR
ERCP
ETP
FiO2
GM-CSF
Gpbar1
HAPS
HLA-DR
HMGB-1
HUSLAB
IAP
ICU
IL
IL-1ra
INF
IQR
MCP-1
MHC
acute pancreatitis
American Pancreatic Association
acute physiology and chronic health evaluation
activated protein C
acute respiratory distress syndrome
antithrombin
adenosine triphosphate
area under curve
Bedside Index for Severity in Acute Pancreatitis
Calibrated Automated Thrombography
compensatory anti-inflammatory reaction
cluster of differentiation
cluster of differentiation 62L, L-selectin
cluster of differentiation 73, ecto-5’-nucleotidase
cystic fibrosis transmembrane conductance
regulator
C-reactive protein
computed tomography
damage-associated molecular pattern
disseminated intravascular coagulation
differences in means
deoxyribonucleic acid
enzyme-linked immunosorbent assay
endothelial protein C receptor
endoscopic retrograde cholangiopancreaticography
endogenous thrombin potential
fraction of inspired oxygen
granulocyte-macrophage colony-stimulating factor
G protein-coupled bile acid receptor 1
harmless acute pancreatitis score
human leukocyte antigen – antigen D-related
high-mobility group box 1 protein
Helsinki University Central Hospital Laboratory
International Association of Pancreatology
intensive care unit
interleukin
interleukin 1 receptor antagonist
interferon
interquartile range
monocyte chemoattractant peptide 1
major histocompatibility complex
10
MLKL
MMS
MOF
MRI
mRNA
MWU
NF-kB
NT5E
OF
PaCO2
PAF
PAI
PAR
PC
PCT
PROWESS
PRSS1
PS
PT
qPCR
RCT
ROC
SAP
SD
SIRS
SOFA
SPINK
TAFI
TF
TFPI
TNF
tPA
TT
uPA
VCAM
mixed lineage kinase domain-like protein
Modified Marshall Score
multiple organ failure
magnetic resonance imaging
messenger ribonucleic acid
Mann-Whitney U-test
nuclear factor kappa-light-chain-enhancer of
activated B cells
5’-nucleotidase ecto (gene)
organ failure
arterial partial pressure of carbon dioxide
platelet-activating factor
plasminogen activator inhibitor
protease-activated receptor
protein C
procalcitonin
Protein C Worldwide Evaluation in Severe Sepsis
protease serine 1 gene
protein S
prothrombin time
quantitative polymerase chain reaction
randomized clinical trial
receiver-operating characteristic
severe acute pancreatitis
standard deviation
systemic inflammatory response syndrome
sequential organ failure assessment
serine peptidase inhibitor, Kazal type 1
thrombin-activated fibrinolysis inhibitor
tissue factor
tissue factor pathway inhibitor
tumor necrosis factor
tissue plasminogen activator
thrombin time
urokinase plasminogen activator
vascular cell adhesion molecule
11
Introduction
#!$#
Acute pancreatitis (AP) is an inflammatory disease of the pancreas and it is
one of the most common gastrointestinal-related reasons for morbidity and
hospitalization.1 The annual incidence of AP in Finland is 73 per 100 000
inhabitants.2 Usually AP is a mild, self-limited disease; however, about 2025% of patients develop a potentially life-threatening severe form of AP.3, 4
The overall mortality of AP is about 5%, but the mortality for severe acute
pancreatitis (SAP) can reach up to 20-30%.3-7
Prolonged excessive alcohol intake and gallstone disease are the most
common causes of AP.2, 8 Other causes may be complication of ERCP
(endoscopic retrograde cholangiopancreatography), metabolic disorders,
drugs, pancreas divisum, and genetic mutations.9-11 Of all AP cases, 10-25%
remain idiopathic.7
Epigastric pain, nausea, vomiting, and abdominal bloating are typical
symptoms of AP.3, 4 Patients with SAP can have hemodynamic instability and
dyspnea.4 There is no specific treatment for AP; options are supportive (i.e.
fluid replacement, enteral feeding). According to revised Atlanta criteria
(Atlanta 2012), AP is classified into three severity categories: mild AP (only
pancreatic involvement, no local complications or organ failure (OF)),
moderately severe AP (local complication and/or transient OF), and severe
AP (persistent OF).12
In pathogenesis of AP, the initial acinar injury is followed by a
complicated inflammatory reaction, which can be restricted to a local event
in mild AP or be excessive, producing a systemic inflammatory response
(SIRS) in SAP.12, 13 SIRS can lead to the evolution of vital OFs and accounts
for early mortality in SAP. A compensatory anti-inflammatory reaction
follows SIRS, resulting in secondary infections, i.e. sepsis, and accounts for
mortality in the later phase of SAP.4, 14-17 Concomitantly with activation of
inflammatory reaction, a complex coagulation cascade becomes activated.
Disseminated intravascular coagulation, consumption coagulopathy, and
increased fibrinolysis are known phenomena in SAP.18 Inflammatory
reaction and coagulation are co-players in inflammatory diseases such as
SAP.19
Cluster of differentiation 73 (CD73) is an ectoenzyme (surface-associated
ecto-5’-nucleotidase), which is expressed on the surface of vascular
endothelial cells.20, 21 CD73 affects vasculature permeability, leukocyte
extravasation, and cytokine release by adenosine metabolism.22
Activated protein C (APC) is an anti-coagulant with cytoprotective and
anti-inflammatory properties.23-27 Sepsis and SAP share similarities in
systemic inflammatory reactions and coagulopathy, 28 and recombinant APC
treatment appeared promising, decreasing mortality in patients with severe
sepsis.29
12
Severity of AP can fluctuate rapidly and predicting the course of AP is
difficult, but essential to improve outcome of patients. Patients who will
develop SAP need to be identified early in the course of disease and
aggressively treated (i.e. early intensive fluid resuscitation in ICU) to prevent
mortality. Many kinds of severity scoring systems and biomarkers have been
developed to assess severity, but a marker that could predict severe AP
precisely, easily, cost-effectively, and sufficiently early is still missing.
This clinical study investigates whether CD73 is capable of predicting the
development of SAP. We evaluated the effects of APC treatment on SAP. At
the same time, we determined effects of APC treatment on systemic
inflammation and coagulopathy associated with SAP.
13
Review of the literature of acute pancreatitis
!%&##!#$!$#
!##"
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The pancreas is a retroperitoneal organ that lies in the upper part of the
abdominal cavity. The regions of the pancreas are the head, body, tail, and
the uncinated process. The spleen is adjacent to the pancreatic tail. The distal
end of the common bile duct passes through the head of the pancreas and
joins the main pancreatic duct (also called duct of Wirsung) entering the
duodenum. The arterial blood supply of the pancreas comes from the celiac
and superior mesenteric arteries. Venous drainage of the pancreas is via the
splenic vein and the superior mesenteric vein draining into the portal vein.
The pancreas is innervated by both the parasympathetic and sympathetic
nervous systems (i.e. branches of the vagus nerve and thoracic splanchnic
nerves).30
The human pancreas has the largest capacity for protein synthesis of any
organ in the human body; mainly the synthesized products are digestive
enzymes. The pancreas is divided into an exocrine (acinar and duct tissue,
comprising 85% of the mass) and endocrine pancreas (islets of Langerhans).
The exocrine pancreas secretes digestive enzymes and NaHCO3 into the
duodenum. The function of the exocrine pancreas is tightly regulated by the
neuroendocrine system; when the meal is ingested, the secretion of digestive
enzymes is promoted by acetylcholine and cholecystokinin. Secretin
promotes the discharge of NaHCO3 into the pancreatic fluid. The endocrine
pancreas secretes hormones (insulin, amylin, glugagon, somatostatin, and
pancreatic polypeptide) into the bloodstream, and these hormones are
essential for regulating blood glucose levels.30, 31
The functional unit of the exocrine pancreas is composed of an acinus and
its draining ductule. The ductule drains into interlobular ducts, which in turn
drain into the main pancreatic ductal system. The acinar cell is designed to
synthesize, store, and secrete digestive enzymes.32 Mostly these enzymes are
proteases (i.e. amylase, lipase, and trypsin), which become active only in the
small intestine. Normally, trypsinogen (inactive zymogen) activates to
trypsin in the duodenum as the first step in the digestive enzyme cascade. In
AP, activation of zymogens occurs within the acinar cell and secretion is
inhibited.30
14
(
AP is a common potentially life-threatening disease with no specific
treatment. The incidence of AP varies in different countries. In Finland, the
incidence is one of the highest: 73 per 100 000 inhabitants annually.2 In
2009 in the United States, AP was the prevailing “gastrointestinal-related”
reason for hospitalizations (the most common principal gastrointestinal
diagnosis at discharge), the second highest cause of total hospital stays, and
the fifth leading cause of in-hospital deaths.1 In Finland, the incidence of
hospitalizations for alcohol-induced AP is from 60 to 102/100 000/year in
middle-aged men and from 5 to 21/100 000/year in middle-aged women.33
The incidence of AP has increased in many European countries and in the
United States in recent years.34-38 The reason for this increase may be partly
due to improved diagnostic capabilities of AP. The incidence of biliary AP
increases with growing obesity of the population.38, 39 Admissions for
alcoholic AP reflect rising rates of alcohol consumption.33 Alcohol-induced
AP is linked to social deprivation and has seasonal variation.40
AP has a wide spectrum of severity. Severity of AP varies from mild, selflimiting disease to severe, even fatal disease. SAP develops in about 20-25%
of patients with AP and the severe disease is associated with the development
of OF.3, 4 Respiratory failure is the most common OF in SAP.6, 41 The
incidence of SAP is also increasing.33, 42 Patients with SAP often need
prolonged stay in an intensive care unit (ICU) and impose a burden on ICU
resources; SAP patients constituted 17% of all ICU days.42
The overall mortality of AP is about 5% (3% in interstitial pancreatitis,
17% in necrotizing AP, 30% in infected necrosis), and the mortality rate for
SAP can reach up to 20-30%.3-7 The mortality for SAP is especially associated
with the development of infected necrosis and multiple organ failure
(MOF).4, 5, 37, 41, 43 Deaths manifest as two peaks; within the first 2 weeks,
deaths are generally attributed to OF, and after that they are caused either by
infected necrosis or other infection complications.4, 6
In Finland, the annual overall number of deaths from AP is between 113
and 167 (mean 142), for men between 75 and 122 (mean 102) and for women
between 29 and 47 (mean 40). Mortality is 2.1-3.7 deaths per 100 discharges
in men and 2.6-4.1 deaths per 100 discharges in women.33
Several studies have reported a slight decrease in mortality for AP,
although the incidence seems to have increased. The reason for this decrease
in mortality could be due to earlier diagnosis and improved treatment
options. On the other hand, an improved sensitivity of diagnostic modalities
leads to an increase in the diagnosis of milder forms of AP.5, 44
15
Review of the literature of acute pancreatitis
#(
Determination of the etiology of AP is important for treatment during the
initial phase of the disease and for preventing recurrence.
Prolonged excessive alcohol intake is the most common cause of pancreatitis
in Finland, accounting for around 70% of all cases.2, 8 Although AP is
associated with heavy alcohol consumption, only a minority (2-3%)45 of the
heavy drinkers develop AP. Gallstone etiology is the second most common
cause, accounting for around 20% of AP cases in Finland.2, 8
The etiology of AP differs considerably between different geographic
regions. Biliary AP predominates in many countries, e.g. in Greece (71%),
Italy, and widely in Asia.7, 46 The risk of biliary pancreatitis is about 2% in
patients with asymptomatic gallstones,47 and the risk increases if the
gallstones are small (diameter < 5 mm) or numerous.48
Alcohol administration and biliary obstruction can induce portal venous
endotoxemia, and this is correlated with complications and more severe
AP.49Idiosyncratic reaction to drugs is a rare cause for AP, overall incidence
probably ranging from 0.1% to 2% of pancreatic cases. A total of 525 different
drugs listed in the World Health Organization database are suspected to
cause AP as a side effect, but the causality for many of these drugs is based
only on case reports. For 31 drugs, a definite causality is established. The risk
of pancreatitis is highest for the use of mesalazine, azathioprine, or
simvastatin. In drug-induced AP, the disease course is usually mild or even
subclinical.10
Pancreas divisum is the most common developmental anatomic variant of
pancreatic duct (incidence 4-14% in an autopsy series).11 In case of pancreatic
divisum, the majority of pancreatic fluids drain through the duct of Santorini
into the duodenum at the minor papilla.11 The presence of pancreas divisum
is a risk factor for recurrent AP and for isolated dorsal involvement of AP.50 It
has been shown, however, that pancreas divisum alone does not cause AP,
but concomitant mutations, e.g. CFTR (cystic fibrosis transmembrane
conductance regulator) genes, suggest a cumulative effect.51
Genetic risk factors are associated with mostly recurrent AP. The
strongest risk factors, besides CFTR, include genetic mutation in protease
serine 1 gene (PRSS1) or serine peptidase inhibitor gene (SPINK).52
Rare causes associated with AP include mitochondrial cytopathy (diseases
with deletion or depletion of mitochondrial DNA, i.e. Karnes Sayre
syndrome).53 Hypercalcemia is a rare cause of AP.54
A couple of case reports have introduced rare anatomical causes of AP, e.g.
splenic aneurysm progression55 and intraluminal duodenal diverticulum.56
Of all AP cases, 10-25% remain idiopathic.7 One potential etiology of
idiopathic AP is gallbladder microlithiasis missed on transcutaneous
ultrasound examination57 and another might be sphincter of Oddi
dysfunction. 58
16
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The exact pathogenic mechanism of AP is unresolved despite decades of
intensive research. An inflammation reaction of AP is surprisingly similar,
although the etiology can differ. Multiple triggering factors sensitize the
pancreatic acinar cell to cellular injury and AP. Whatever the initiating event,
the progression of AP can be viewed as a continuum: local inflammation of
the pancreas, followed by a generalized inflammatory response, leading to
the final stage of multi organ failure.
#!!#!"
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Alcohol has a direct dose-related toxic effect on the pancreas, but additional
triggering factors are needed to initiate AP. Alcohol has direct effects on
small pancreatic ducts and on acinar cells. Alcohol increases viscosity of
pancreatic secretions and causes formation of small protein plugs within
pancreatic ducts.59 Alcohol increases digestive and lysosomal enzyme content
within acinar cells, thereby increasing the potential of intracellular activation
of digestive enzymes.60 Acute ethanol exposure decreases pancreatic blood
flow, resulting in decreased pancreatic tissue pH in animal models.61 Low
17
Review of the literature of acute pancreatitis
extracellular pH (metabolic asidosis) sensitizes acinar cells to AP by
enhancing calcium signalling.62
The toxic effect of bile acid itself on acinar cells is a possible pathogenic
factor of biliary AP. Gallstones may migrate into the distant bile ducts
causing bile and pancreatic juice obstruction. Bile acids flow upstream into
the pancreatic duct and can bind to an acinar cell surface bile acid receptor
(Gpbar-1).63 Bile acids increase intra-acinar calcium concentrations and
induce nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB)
activation and synthesis of proinflammatory cytokines.64, 65
Alcohol administration and biliary obstruction can induce portal venous
endotoxemia, and this is correlated with complications and more severe
AP.49
(/') %(#**&(/'%(* +*& )* &%*&(/
Over one hundred years ago, Hans Chiari proposed a theory that acute
pancreatitis is the result of autodigestion of the pancreas by prematurely
activated digestive enzymes.66 Under physiological conditions, the proteases
of pancreatic acinar cell become active only when they reach the small
intestine. Over the decades, man has tried to clarify mechanisms responsible
for this premature activation of pancreatic proteases.
In trypsin-related theory of AP, trypsinogen activation to trypsin within
the acinar cells appears to be an early pathogenic event in AP. Conditions
that lead to AP alter intracellular acinar cell signalling.67 Intracellular colocalization lysosomal enzymes (i.e. cathepsin B) with zymogen granules lead
to activation of pancreatic proteases in the vacuoles of acinar cells, and
normal acinar cell secretion (apical exocytosis of zymogen granules) is
inhibited.68 Autophagy function is decreased in AP and autophagic vacuoles
containing activated proteases accumulate in the acinar cells.69, 70 Inhibited
apical exocytosis of activated proteases instead leads to basolateral exocytosis
of them.71, 72 The active proteases are released into the interstitial space,
resulting in acinar cell injury.67, 73 Trypsin can also prematurely activate
precursors of other digestive enzymes.67, 73 Activation of digestive enzymes
and the subsequent progressive acinar cell injury initiate autodigestion of the
pancreas, causing hemorrhage, necrosis, edema, and destruction of the
pancreas parenchyma. Injured acinar cells release cytokines (tumor necrosis
factor alpha (TNFα), IL-1β), and this results in activation of local
inflammation cells and accelerates the proinflammatory response.14, 74
18
0%+#(*&("''# * %%%(&* ,*##)
* ,* &%
NF-κB is a “rapid-acting” transcription factor that responds to harmful
cellular stimuli. It regulates the expression of multiple inflammatory and
immune genes. Normally, NF-κB is present in the cytoplasm in an inactive
form, and many extracellular stimuli, e.g. inflammatory cytokines, viruses,
and oxidants, can activate it. Activated NF-κB can induce expression of
proinflammatory cytokines, i.e. TNFα, IL-1β, IL-6, chemokines, and
adhesion molecules.75, 76 Early intra-acinar NF-κB activation, which occurs
parallel to but independent of trypsinogen activation, might be partly
responsible for progression of local and systemic inflammation in AP.77, 78 A
mouse model lacking pathologic intra-acinar typsinogen activation showed
that inflammation can progress independently and does not require
trypsinogen activation in AP.78 The level of activation of NF-κB in acinar cells
correlates with the severity of the AP in an experimental model of AP.79 The
blocking of NF-κB activation in animal models has shown cytoprotective
effects in AP.76
#
Inflammation is a host defence mechanism for mechanical or chemical injury
or microbial infection. An inflammatory reaction is a complicated progressive
process in which numerous humoral and cellular responses participate. In
the early phase of AP, inflammation is usually localized to the pancreas
tissue, which clinically manifests as mild AP.
%(## %!+(/
Acinar cell injury and necrosis trigger inflammation. In the initial phase of
AP, premature activation of pancreatic proteases leads to acinar cell injury
and release of proinflammatory mediators (i.e. cytokines like TNFα and
IL1β).13, 80 Intracellular contents released from necrotic cells into the
extracellular space serve as damage-associated molecular patterns (DAMPs),
which can activate an inflammatory response in AP (Figure 3). High-mobility
group box 1 protein (HMBG1) is one of the DAMPs (others include DNA,
ATP, and histones). Normally inside the nucleus, HMGB1 is a nuclear DNA
binding protein, which participates in determining the structure of
chromatin and regulates the transcription.81 Intracellular HMGB1 protects
cells from Nf-κB activation and DNA damage and can mediate autophagy.81,
82 Extracellular HMGB1 is a potent proinflammatory mediator and may act
as key mediator for inflammation in AP.83 HMBG1 mediates inflammation
through Toll-like receptor 4. Toll-like receptor 4 is expressed on pancreatic
ductal and on endothelial cells and on macrophages and monocytes.84
19
Review of the literature of acute pancreatitis
Binding of the HMBG1 to its receptor on monocytes induces expression of
proinflammatory cytokines.85 Apoptosis, i.e. programmed cell death, of
acinar cells occurs also in the early phase of AP. Apoptotic cells release
nucleosomes, which are complexes formed by DNA and histone proteins.86
Normally, the nucleosomes are packed into vesicles and engulfed by
macrophages. The extraordinary apoptosis leads to overloading of
phagocytizing mechanisms and higher concentrations of nucleosomes in the
circulation.86 Higher levels of circulating nucleosomes correlate with the
inflammatory response and organ dysfunction in sepsis.87 Intracellular
HMBG1 prevents inflammatory nucleosome release in AP and blocks
inflammation.88
(+ *$%*& %#$$*&(/##)
Recruitment of inflammatory cells is a critical step in the inflammatory
reaction. The release of inflammatory signals (i.e. “first-line”
proinflammatory cytokines TNFα and IL-1β, DAMPs, and NF-κB) from
acinar cells mediates the activation of circulating inflammatory cells, such as
peripheral blood mononuclear cells, comprising lymphocytes and monocytes,
and
polymorphonuclear
neutrophils
(PMNs)
(Figure
3.).89
Monocytes/macrophages are the significant inflammatory cell population
involved in pathogenesis of AP.89 Promonocytes, precursors of macrophages,
are released from the bone marrow into the circulation. They develop into
monocytes. Monocytes leave the circulation and migrate to the tissues,
differentiating into specific types of tissue macrophages, i.e. Kupffer cells in
the liver and alveolar macrophages in the lungs.90
Monocytes/macrophages have a central role as phagocytic cells and as
antigen presenting cells in immune reaction. Macrophages are the major
resident immune cells within the pancreas and in the peritoneal cavity.89
Macrophages on pancreas tissue and in the nearby peritoneal cavity release
TNFα and IL-1β in the early stage of AP, inducing a cascade of other
cytokines and chemokines (i.e. IL-8, monocyte chemoattractant peptide
MCP-1), which leads to activation of neutrophils and amplification of the
proinflammatory response.89 Trypsin has also been shown to stimulate the
production of cytokines in peritoneal macrophages.91 The recruitment of
PMNs has also a central role in the onset and progression of inflammation in
AP. PMNs are the most numerous leukocytes in the circulation. They seek
signs of foreign organisms and antigens and are recruited to sites of infection
or inflammation.92 In the early stage of AP, activated neutrophils release high
concentrations of oxidants and cytotoxic agents, which worsens the local
damage of pancreatic tissue. Activated neutrophils also secrete
proinflammatory cytokines (i.e. IL-1β, TNFα).93
Activation of inflammatory cells is promoted by membrane-bound cell
surface receptors whose expression of them is altered during inflammation.
Activation of neutrophils and monocytes is manifested by an increased
20
expression of β2 integrin CD11B/CD18 on the cell surface. Up-regulated
expression of these adhesion molecules facilitates neutrophil extravasation at
the site of inflammation.94 Monocytes/macrophages and neutrophils express
CD14 glycoprotein on the cell surface. CD14 has been identified as a receptor
for a wide range of microbial products (LPS).95 CD14 acts as a co-receptor
with the Toll-like receptors. CD14 appears in soluble (sCD14) and
membrane-bound forms (mCD14), both of which have immune stimulatory
and inhibitory functions.96 High CD14 expression may increase susceptibility
to immune-mediated tissue injury.97 Both CD11b and CD14 expression levels
on leukocytes can be measured as markers of leukocyte activation.
+"&/*.*(,)* &%
Leukocyte extravasation from the blood into areas of inflammation is a
highly regulated phenomenon in immune response. During an early stage of
AP cytokines activate microvascular endothelium, and leukocytes can
transmigrate into the pancreatic interstitial space. A complex extravasation
cascade regulates an adhesion of leukocytes to endothelium and
extravasation to tissues. Extravasation depends on the interaction between
leukocytes and endothelium, and many kinds of adhesion molecules are
expressed on the surface of leukocytes and vascular endothelial cells (Figure
3.). Selectins mediate the initial step of adhesion cascade, promoting
tethering and rolling of leukocytes on the endothelium. For example, Eselectin is expressed on endothelial cells after an inflammatory stimulus.98
The soluble form of E-selectin can be measured in serum, and it serves as a
marker of endothelial cell activation.99 L-selectin (CD62L) is an adhesion
receptor expressed on the leukocytes that mediates the leukocyteendothelium interaction.100 Decreased monocyte and neutrophil expression
of L-selectin is a marker of phagocyte activation.101, 102 The β2 integrin
CD11B/CD18 on the surface of neutrophils and monocytes allows them to
adhere firmly to the endothelial cells.103, 104
Ectoenzymes (i.e. nucleotidases, cyclases, peptidases, proteases and
oxidases) are also involved in the extravasation cascade. Ectoenzymes are
expressed on the surface of leukocytes and endothelial cells and have their
enzymatically active domains outside of the cell membrane. In the
extravasation cascade, ectoenzymes can act as adhesion molecules or by
regulating cell recruitment through their catalytic activity.105
Cluster of differentiation 73 (CD73) is an ectoentzyme (surface associated
ecto-5’-nucleotidase) that is expressed on the surface of vascular endothelial
cells. In addition, 5-15% of blood lymphocytes express CD73.20, 21 CD73 also
appears in soluble form (sCD73) in plasma, and the soluble form is suggested
21
Review of the literature of acute pancreatitis
to be enzymatically active.106 sCD73 is produced by shedding of
lymphocytes.107
CD73 regulates the purinergic signaling cascade. CD73 regulates
extracellular ATP metabolism, dephosphorylating AMP to adenosine.108
Adenosine has anti-inflammatory effects: it decreases endothelial cell
permeability and inhibits leukocyte extravasation. Adenosine decreases
cytokine release and inhibits immune activation.22 Other non-enzymatic
functions of the CD73 molecule have also been proposed such as induction of
intracellular signaling and mediation of cell–cell and cell–matrix adhesion.107
Experimental animal models have shown that CD73 deficiency is
associated with aggravated lung injury in acute respiratory distress syndrome
(ARDS), worse hepatic, renal, and cardiac ischemia-reperfusion injury, and
increased mortality in polymicrobial sepsis.109, 110 The activity of CD73
appears to be beneficial in the fight against excessive inflammatory reactions.
Exogenously administered CD73 has protective effects in, for example, acute
lung injury and myocardial and renal ischemia. Expression of endogenous
CD73 can be upregulated by interferon beta (INF-β).109, 110 INFβ has been
shown to induce the expression and activity of CD73, thus decreasing
vascular permeability in cultured human pulmonary endothelial cells.111, 112
(&,)+#( %!+(/
Microvascular injury is partly responsible for progression of tissue injury at
the site of inflammation. Under normal circumstances, the extravasation
cascade is highly regulated and during the inflammation extravasation is
accelerated.104, 113 The transmigrated leukocytes release vasoactive factors
(i.e. endothelin-1 and phospholipase A2) and reactive oxygen species, which
contribute to the development of microvascular injury and increased
capillary permeability.114, 115 This results in edema and local ischemia, which
leads to ongoing destruction of pancreatic tissue.116 Acinar cell damage can
amplify this proinflammatory cascade.117
)*( * &%& %#$$*&(/(* &%
In mild AP, the inflammation reaction is restricted to a local event. The
termination of inflammation is an active biochemical progress that involves
the release of anti-inflammatory mediators and downregulation of NF-κB
gene activity. Additionally, cytokine and chemokine receptor antagonist (i.e.
interleukin 1 receptor antagonist, IL-1ra) limits neutrophil recruitment to the
site of inflammation.118
Simultaneously with the acinar cell injury, the cell death mechanisms
(necrosis and apoptosis) become activated.13, 80 Apoptosis is a process of
programmed cell death that is thought to limit the tissue injury in an
inflammatory reaction. Apoptosis is an active process requiring specific
22
proteins, caspases. If death of acinar cells occurs in the mode of apoptosis
instead of necrosis, the following inflammatory reaction may be milder.119
The dying cells are phagocytized by macrophages, and this is associated with
anti-inflammatory cytokine switching.120
Concomitantly with synthesis of proinflammatory cytokines, antiinflammatory cytokines and specific cytokine inhibitors are produced. IL-6 is
a cytokine known to have both pro- and anti-inflammatory properties, but
that acts predominantly as an anti-inflammatory cytokine.121 IL-6 induces
production of acute-phase proteins in the liver.122 IL-6 is an early marker of a
complicated course of AP. IL-6 concentrations peak on the first day of AP
and decrease to baseline values within 4 days.123 IL-10 is a potent antiinflammatory cytokine and a potent activator of B-lymphocytes.124
Proinflammatory cytokine synthesis is downregulated by IL-10 in activated
macrophages and neutrophils.121 IL-10 has a protective role in SAP; IL-10
might restrict the inflammation reaction. IL-1ra is an important antiinflammatory cytokine. It binds competitively to the IL-1 receptor and
prevents IL-1-mediated responses.125
During inflammation the lifespan of the circulating neutrophils is
prolonged, and induction of neutrophil apoptosis facilitates the resolution of
inflammation by normalizing the neutrophil lifespan.93
23
Review of the literature of acute pancreatitis
"("##
AP is a dynamic condition in which the severity of inflammation may change
rapidly during the course of disease. A local pancreatic inflammation in the
initial phase of AP can rapidly proceed to uncontrolled systemic
inflammation reaction in SAP.12 An ongoing overactive systemic
inflammatory reaction can progress to the development of systemic
inflammatory response syndrome (SIRS), which may further proceed to
distant organ failures and MOF and/or infection of pancreatic necrosis and
sepsis with late complications of AP (Figure 1).4, 14
20;8.
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C64@?52BI:?764D:@?C2?5C6AC:C
24
.*('%(* /*&" %'(&+* &%$'# * &%
In severe AP, loss of control results in an excessive release of
proinflammatory cytokines (i.e. TNFα, IL-1β, IL-8, and platelet-activating
factor (PAF)) from the inflamed pancreas into the systemic circulation via
portal vein and lymph fluid drainage.126, 127 Once cytokines reach the liver,
they strongly activate local macrophages, Kupffer cells. Activated Kupffer
cells release inflammatory cytokines, reactive oxygen intermediates, and
hydrogen peroxides into the circulation, causing progression of inflammatory
reaction.128 Synthesis of acute phase proteins, i.e. C-reactive protein (CRP)
and procalcitonin (PCT), is induced in the liver.129, 130 PCT levels can be
measured as a marker of systemic inflammation and the plasma levels of PCT
increase during systemic inflammation.129 Proinflammatory cytokines are
also released in the peritoneal cavity in the vicinity of the pancreas, leading to
activation of peritoneal macrophages, and this in turn amplifies the systemic
proinflammatory response. Alveolar macrophages are involved in the
lungs.89 During inflammation activated neutrophil lifespans are prolonged,
ensures their presence at the site of inflammation. Neutrophils transmigrate
from the circulation to the tissues and aggregate around the focus on
inflammation, resulting in a release of reactive oxygen species and the
production of more proinflammatory cytokines.93
)/)*$ %#$$*&(/()'&%))/%(&$
The result of this excessive expanding of local inflammation can lead to SIRS
(Figure 2).15
#*+3.
%-'(%"!-.! 2.1)),'),!)".$!")&&)1%(#)( %.%)(-
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/9:D63=@@546==4@E?D
H
=@BH
=@B:>>2DEB67@B>C
)2(2BD6B:2=A2BD:2=AB6CCEB6@742B3@?5:@H:56
)*%*&(% #+()%$+#* '#&(% #+(
Released proinflammatory cytokines activate the vascular endothelium,
leading to enhanced extravasation of leukocytes.132 Activated neutrophils and
monocytes further release proteolytic enzymes and oxygen radicals133,
contributing to the damage of endothelial cells and the loss of endothelial
integrity.134 An increase in vascular permeability in distant organs can result
in a massive infiltration of inflammatory cells and fluid to the tissues. This
25
Review of the literature of acute pancreatitis
may lead to hypotension and oxygen deficiency, resulting in dysfunction of
vital organs and finally OF.135
The most vulnerable organs are the lungs and kidneys with a high content
of vasculature tissue. Acute lung injury is a consequence of the systemic
inflammatory response with increased endothelial and epithelial barrier
permeability. The leakage of protein-rich exudate into the alveolar space and
interstitial tissues leads to compromised oxygenation and gas exchange.136
Acute lung injury can lead to a more severe disease, the ARDS. Patients with
ARDS need frequent mechanical ventilation support, and ARDS contributes
to early death in SAP.137 Secondary pulmonary infections contribute to the
late phase of deaths in SAP.138
Impairment of renal microcirculation, hypoxemia, decrease in renal
perfusion due to intra-abdominal hypertension, and hypovolemia cause
acute kidney injury in SAP.139Pancreatic phospholipase A2 released by
neutrophils and macrophages can deposit to renal proximal tubular cells, and
the measurement of pancreatic phospholipase A2 in serum may provide a test
for identifying a threatening renal complication.140 Acute kidney injury can
lead to renal replacement therapy and partly contribute to deaths in the early
stage of SAP.139
In case of more than one affected organ system, the situation is termed
multiple organ failure, MOF. Organ failures can be transient, such as in the
case of moderately severe AP, or persistent such as in SAP.12
&$'%)*&(/%* %#$$*&(/()'&%))/%(&$
In systemic inflammation, the proinflammatory phase is followed by an antiinflammatory reaction that may lead to immune suppression (Figure 2). 15-17
This paralysis of the inflammatory response is also termed a compensatory
anti-inflammatory response syndrome (CARS). The anti-inflammatory
response may control an excessive injurious inflammatory reaction (to
restrict the SIRS), resulting in the recovery of organ injuries (termed
transient organ injury in the case of moderately severe AP). CARS may be
excessive, leading to severe immune suppression.15
Monocytes express major histocompatibility complex (MHC) class II
antigens (human leucocyte antigen, HLA-DR) on the cell surface.141 HLA-DR
density is related to the capacity of monocytes to present antigen to helper Tcells.141 In immunosuppression, HLA-DR expression of monocytes is reduced
and the antigen presentation capacity is impaired. The ability of monocytes
to produce proinflammatory cytokines is decreased, and the serum levels of
anti-inflammatory cytokines (IL-10, IL-1ra, and IL-6) are increased.17, 142 Low
HLA-DR expression of circulating monocytes is a sign of
immunosuppression.142 INF-γ treatment can at least partially reverse HLADR antigen expression and help septic patients restore their immune
defenses and increase their resistance to infection.143
26
In the second phase of SAP, immune suppression enhances susceptibility
to nosocomial infections or evolution of local complications such as infected
necrosis, pseudocysts, or abscesses.12 The increase in permeability of the gut
barrier may allow translocation of bacteria and endotoxins into the
circulation. This gut barrier failure may lead to infections, including sepsis,
and ultimately to death in the late phase of SAP. 144
20;8.
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27
Review of the literature of acute pancreatitis
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28
$##$#
In case of vascular injury, an immediate response is required to limit blood
loss. Blood coagulation leads to the formation and propagation of a blood clot
at the site of vascular injury. Coagulation involves platelet-vessel interactions
related to disruption of the endothelium and the coagulation system (the
primary hemostasis, the activation of coagulation cascade, the natural
anticoagulants, and the fibrinolytic system). The formation of thrombin is an
essential phenomenon in coagulation. Thrombin can activate platelets,
convert fibrinogen to fibrin, and via feedback amplify the coagulation. In
healthy persons, coagulation is precisely controlled at the site of vascular
damage by anticoagulation and fibrinolytic mechanisms. In case of systemic
disease (i.e. systemic inflammation), the control system is disturbed,
resulting
in
coagulopathy
(i.e.
thrombosis
and
consumption
coagulopathy).145-147
( $(/$&)*) )
Primary hemostasis is the result of complicated interactions between
platelets, the vessel wall, and adhesive proteins, leading to formation of the
initial “platelet plug”. Intact endothelial lining has antithrombotic properties
that prevent plug formation. In case of vascular injury, the highly
thrombogenic subendothelial layer containing collagen and Von Willebrand
factor is exposed. Platelets adhere to collagen, and Von Willebrand factor
acts as a bridge between subendothelial collagen and platelet surface
receptors (platelet glycoprotein complex I receptors). Activated platelets
produce thromboxane A2, which stimulates further platelet aggregation,
resulting in formation of the initial plug.148
&+#* &%'*-/)&($* &%& ( %
Coagulation involves specific interactions between cellular surfaces and
plasma-derived zymogens and cofactors.
Coagulation cascade is a traditional model of coagulation in which series
of proenzymes convert into the respective serine proteases. The main step is
the conversion of prothrombin into thrombin, and the final step is the
conversion of soluble fibrinogen into insoluble fibrin, resulting in a plug. Two
pathways of the traditional coagulation cascade have been described:
extrinsic and intrinsic pathways. In the extrinsic pathway, tissue factor (TF)
(exposed by vascular injury) binds to and activates factor VII (FVII) to factor
VIIa (FVIIa) (the activation of proenzyme is depicted by the suffix “a”). The
TF-FVIIa complex in turn activates FX to FXa. In the intrinsic pathway (also
called contact activation pathway), the negative charged phospholipid
29
Review of the literature of acute pancreatitis
surfaces, usually on the activated platelet surface, result in activation of the
coagulation cascade. The activation of intrinsic pathways starts with
activation of FXII into FXIIa; the resulting activation cascade is shown in
Figure 4. These two pathways converge to form a common pathway. In the
common pathway, FXa forms a complex with its cofactor FV, leading to the
conversion of prothrombin into thrombin. Thrombin in turn cleaves
circulating fibrinogen to insoluble fibrin and activates FXIII, which
covalently crosslinks fibrin polymers. A fibrin network stabilizes the clot and
forms a definitive secondary plug.147, 149, 150
A modern model of coagulation is emphasized as sequential events
situated on the surfaces of endothelial cells (Figure 4.). The sequential
phases of coagulation are: 1) Initiation phase: TF is exposed to coagulation
factors in plasma, permitting formation of a complex between FVIIa. The
FVIIa/TF complex activates FIX and FX. FXa forms a complex with FVa, and
this complex further converts prothrombin to thrombin. 2) Amplification
phase: small-scale thrombin that is generated in the initiation phase is not
sufficient. That generated thrombin activates cofactors VIII, V, and platelets
and accelerates the activation of FII (prothrombin) by FXa and of FXa by
FIXa, 3) Propagation phase: the accumulated enzyme complexes on platelet
surface produce a large-scale thrombin burst that promotes the conversion of
fibrinogen to fibrin.147, 151
30
20;8.
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31
Review of the literature of acute pancreatitis
%* &+#* &%'*-/)
Blood clot formation is tightly regulated to prevent thrombus propagation
and vessel occlusion. Regulation of coagulation is focused on different levels
of the pathway by enzyme inhibition or by modulation of the activity of
cofactors. The endogenous anticoagulant mechanisms are: 1) antithrombin
(AT), 2) tissue factor pathway inhibitor (TFPI), and 3) protein C (PC)
pathway (Figure 4).
%* *(&$ %
Antithrombin (AT) is a liver-synthesized glycoprotein that primarily binds
and irreversibly inactivates thrombin, but also coagulation factors FXa and
FIXa. AT limits the coagulation process to the site of vascular injury and
protects the circulation from liberated enzymes. Heparin and heparin-like
molecules on the surface of endothelial cells are cofactors of AT and
stimulate its activity.150, 152, 153
))+*&(%* ))+*&('*-/ % *&(
Tissue factor (TF) (formerly known as FIII) is a transmembrane protein
present in cells surrounding vessels (adventitial but not endothelial cells
express TF).154 TF comes into contact with blood and the circulating
procoagulant factor (FVIIa) when vascular integrity is disrupted.155 TF
circulates as a free form in the bloodstream (so-called blood-borne TF), and
it was also assumed to activate the coagulation cascade.156 Free TF has been
shown to be present in the plasma of healthy individuals in low
concentrations, and the plasma levels increase in systemic inflammation, i.e.
sepsis.157 The monocytes can synthesize and express TF, and they are
supposed to be a source for TF-induced thrombosis when the endothelium is
intact.158 Several pathological conditions, e.g. sepsis, are associated with
activated monocytes, shedding TF-rich microparticles that are transferred to
activated platelets, neutrophils, and endothelial cells.159
TFPI is a serine protease inhibitor of TF that slows down the initiation of
clot formation. TFPI consists of three domains that bind and inhibit FXa and
TF-FVIIa complex.160 Endothelial cells, monocytes and macrophages
synthesize TFPI. Most of TFPI (50-80%) is bound to the endothelial surface.
In circulation, TFPI occurs as a lipoprotein-bound form (80%) and as a free
form (20%). Free TFPI has a more potent anticoagulant effect than its bound
counterpart.161
32
(&* %'*-/%* ,*'(&* %
Protein C pathway restricts the generation of thrombin. APC is an
endogenous anticoagulant (a trypsin-like serine protease) that promotes
fibrinolysis and inhibits thrombosis. Protein C, an inactive precursor, is
converted to APC by thrombin coupled to thrombomodulin.162 This process is
accelerated in the presence of endothelial cell protein C receptor (EPCR). The
binding of protein C to EPCR potentiates the activation of protein C by 20fold in vivo.163 The presence of protein S, a cofactor of PC, is required for the
anticoagulant activity of APC.164 APC inactivates the procoagulation factors
FVa and FVIIIa by shutting down the coagulation pathway; the inactivation
of FVa/FVIIIa by APC with protein S slows down thrombin generation and
inhibits thrombus growth.165 APC also inactivates plasminogen activator
inhibitor (PAI), resulting in increased fibrinolysis.162 An α1-antitrypsin and
protein C inhibitor can inhibit anticoagulant activity of APC.166
Genetic deficiencies in PC, PS, and AT predispose to thrombofilias.153, 167
( %&#/) )
Fibrinolytic system is a parallel system that is activated along with activation
of the coagulation cascade and limits the size of the clot. Fibrinolysis enables
the removal of thrombi. Fibrin promotes the generation of fibrinolytic
enzyme plasmin to degrade the clot, so fibrin acts as both a cofactor and a
substrate for the plasmin. Plasmin is activated from plasminogen by either of
two primary serine proteases, tissue plasminogen activator (tPA) and
urokinase plasminogen activator (uPA). tPA is synthesized by endothelial
cells and uPA is produced by monocytes, macrophages, and urinary
epithelium. tPA requires fibrin as cofactor. uPA acts mainly in extravascular
locations. Circulating plasmin and plasminogen activators are inhibited by
serine protease inhibitors (serpines). The most important serpines are
plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2
(PAI-2), and α2-antiplasmin. PAI-1 is mainly produced by endothelial cells
and platelets, and its expression is upregulated by proinflammatory
cytokines. In pregnancy, PAI-2 is the main plasminofen activator inhibitor.
Thrombin-activated fibrinolysis inhibitor (TAFI) is a non-serpin inhibitor
that is activated by thrombomodulin-associated thrombin, and it decreases
the affinity of plasminogen to fibrin. Fibrinolysis dissolves the fibrin clot into
fibrin degradation products, some of which have immunomodulatory and
chemotactic functions.168-170
33
Review of the literature of acute pancreatitis
$##
The inflammation and coagulation systems have bidirectional
communication with several connections at the cellular and humoral levels.
Inflammatory reaction leads to activation of coagulation and suppression of
natural anticoagulant mechanisms, and, in turn, the coagulation affects
inflammatory activation. In severe inflammatory diseases, such as sepsis and
SAP, simultaneously with SIRS, coagulation is activated.
%#$$* &% %+* ,* &%&&+#* &%
The main mediators of inflammation-induced activation coagulation are
proinflammatory cytokines.171 Inflammation upregulates TF synthesis in
endothelial cells, monocytes/macrophages, and dendritic cells.172, 173 Platelets
can be activated by proinflammatory mediators (i.e. PAF) or by formed
thrombin.174 The activation of platelets enhances the expression of TF on
monocytes by inducing NF-κB activation during systemic inflammation
reactions.175 The expression of P-selectin on platelets mediates the adherence
of platelets to leukocytes and endothelial cells and enhances the expression
of TF on monocytes.176 Binding of activated platelets to monocytes also
enhances the production of IL-1b, IL-8, MCP-1, and TNFα.177(Figure 5)
Anticoagulant pathways, like PC and AT systems, are shut down by
proinflammatory cytokines.171 The components of the protein C pathway are
also directly influenced by inflammatory mediators.19 TNF-α and IL-1β
downregulate expression of thrombomodulin on endothelial cells178 and
upregulate its surface expression on macrophages,179 leading to a deficient
protein C system.
Inflammation inhibits fibrinolysis; proinflammatory cytokines stimulate
endothelial cells to secrete PAI-1.173
*)&&+#* &%&%* ,* &%& %#$$* &%
The coagulation system can modulate inflammatory activity. Binding of
coagulation proteases or anticoagulant protein to specific receptors on
mononuclear cells or endothelial cells may affect cytokine production.172 FXa,
thrombin, and fibrin can stimulate IL-6 and IL-8 synthesis of mononuclear
and endothelial cells.180 Thrombin increases IL-8 and MCP-1 mRNA levels
and expression of E-selectins on endothelial cells.171 Thrombin can bind to
protease-activated receptors (PAR-1,3-4) on different cell types, i.e. on
platelets and endothelial cells. The resulting intracellular signaling leads to
increased production of proinflammatory cytokines. Thrombin represents
the most potent agonist of PAR-1 and PAR-4. PAR-1 activation on endothelial
cells triggers the release of chemokines and the surface expression of various
adhesion molecules. PAR-1 signaling promotes Von Willebrand factor
34
release, P-selectin exposure, and enhanced production of PAF and
prostaglandins.173, 181
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DE>@B?64B@C:C724D@B2=A92
35
Review of the literature of acute pancreatitis
//.,:96/.5-60.56;9*5:2,6*0;3*5:7*:1=*>96525/3*44*:265
Natural anticoagulant proteins suppress inflammation by several
mechanisms.
TFPI inhibits the LPS-stimulated TNFα production by monocytes and
downregulates CD11b and CD18 expression on neutrophils in vitro.173 TFPI
inhibits inflammatory TF signaling via PARs. In turn, activated neutrophils
can cleave TFPI to a less active form.182
AT has many anti-inflammatory properties, which are mediated by its
actions in the coagulation cascade, the inhibition of thrombin by AT prevents
activation of many inflammatory mediators (such as activation of platelets
and endothelial cells). AT prevents FXa-induced production of IL-6, IL-8,
and E-selectin involved in monocyte recruitment and adhesion to endothelial
cells. AT prevents FVIIa-TF complex formation and subsequent upregulation
of cytokines (i.e. IL-6, IL-8).153 AT has anti-inflammatory properties
independent of its anticoagulatory characteristics. It induces endothelial cells
to release prostacyclin, which suppresses platelet activity and inhibits
attachment of neutrophils to endothelial cells and decreases release of IL-6,
IL-8, and TNFα by endothelial cells. AT prevents leukocyte adhesion to
endothelial cells, binding receptors like syndecan-4 expressed on
neutrophils, monocytes, and lymphocytes and reduces the expression of IL-6
and TF on monocytes.171 AT reduces also IL-8 -induced chemotaxis of
neutrophils, monocytes, and lymphocytes.183 During severe inflammatory
response AT levels are decreased; synthesis is impaired, elastases from
activated neutrophils degrade AT, and ongoing thrombin generation
consumes AT.153
APC also has cytoprotective effects such as anti-inflammatory, antiapoptotic, and endothelial barrier protection effects.184 APC has an indirect
effect on inflammation; it inhibits formation of thrombin, thereby reducing
thrombin’s potent proinflammatory activities, i.e. platelet activation,
cytokine production, and upregulation of leukocyte adhesion molecules.
Administration of APC has been shown to downregulate the expression of
inflammatory cytokines and chemokines. It reduces production of
endotoxemia-induced proinflammatory cytokines (IL-6, IL-8, IL-1β, and
TNFα).185 APC inhibits TNFα production by blocking NF-kB transcription
factor in monocytes.186 APC blocks cytokine production from Th2
lymphocytes.187 APC has been shown to upregulate anti-inflammatory
mediators, like IL-10, on blood monocytes.188 The anti-inflammatory effects
of APC on endothelial cells include the inhibition of NF-κB activation, with
decreased expression of endothelial adhesion molecules (i.e. intercellular
adhesion molecule 1, E-selectin, and vascular cell adhesion molecule-1)
resulting in decreased leukocyte trafficking and reduced expression of
proinflammatory cytokines by endothelial cells.19, 189 APC increases
endothelial production of prostaglandin-2 and MCP-1.181 APC exerts antiapoptotic effects on both leukocytes and endothelial cells by reducing several
pro-apoptotic signals. The direct protective effects of APC on endothelial cells
36
are mediated in the presence of EPCR and PAR-1. The effects of APC on
leukocytes are mediated by EPCR alone or in the presence of PAR-1.24, 25
APC levels are deficient in septic patients corresponding to increased
mortality.190 APC modulates coagulation and inflammation associated with
severe sepsis.162 Sepsis decreases PC levels and it has been suggested that
treatment with human recombinant APC will increase PC levels and prevent
MOF.191
$ #("%!$# !##"
During the early phase of AP proinflammatory cytokines, chemokines, TNFα,
and PAF are released into the circulation.192 The proinflammatory cytokines
activate monocytes, neutrophils, and endothelial cells, resulting in the
production of large quantities of TF to initiate the coagulation cascade.178 AP
is often accompanied by coagulopathy, which is characterized by increased
thrombosis and fibrinolysis.18, 171. Hemostatic disorders may range from
scattered intravascular thrombosis to disseminated intravascular coagulation
(DIC).193 AP is associated with microvascular dysfunction including increased
vascular permeability, reduced blood flow, ischemia/reperfusion injury,
intravascular thrombus formation, and hypercoagulation.194 During AP
platelets accumulate intravascularly and extravascularly in the pancreas and
fibrin deposits in the connective tissue and intercellular spaces of the
pancreas.195
Coagulation abnormalities are related to severity of AP. Consumptive
coagulopathy and increased fibrinolysis are known to occur in SAP, and they
are related to its severity and OF.18 The decreased plasma levels of APC and
AT and the increased levels of D-dimer and plasminogen activator inhibitor-1
are associated with poor outcome in SAP.196 AT inhibits the secretion of
cytokines and HMGB1 and ameliorates AP in an experimental animal model
of AP.197 The high levels of plasminogen activator inhibitor-1 may contribute
to fibrin deposition in the microcirculation.196 Elevated D-dimer levels have
been shown to be a sign of SAP, and non-survivors have been found to have
higher D-dimer levels than survivors.198 Decreased platelet counts, decreased
prothrombin times (PT), and consumption of fibrinogen occur in AP.18
Modulating hemostasis may provide a therapeutic target for the treatment
of SAP. PAF is a proinflammatory mediator produced mainly by endothelial
cells. PAF can activate platelets and neutrophils and increase vascular
permeability. Increased levels of phospholipase A2 occur in AP. Pancreatic
phospholipase A2 promotes the synthesis of PAF. The PAF antagonist,
lexipafant, reduced tissue damage and OFs in AP models.199, 200 Lexipafant
was a promising treatment in SAP (phase II and phase III showed reduction
in incidences of OFs)199, 200, but later studies failed to show any such
benefit.201
37
Review of the literature of acute pancreatitis
""
"#!#!
In accordance with the Revised Atlanta classification,12 AP can be diagnosed
if at least two of the following three features are fulfilled: 1) abdominal pain
(acute onset of persistent and severe epigastric pain, often radiating to the
back), 2) serum amylase activity or plasma p-isoamylase (pancreas-specific
isoenzyme) level or serum lipase activity at least three times greater than the
upper limit of normal, and 3) characteristic findings of AP on contrastenhanced CT (MRI or transabdominal ultrasonography).12
"" ("'#
Clinical history of known cholecystolithiasis, alcohol intake, new
medications, known hyperlipidemia, previous acute pancreatitis, and
character of the pain should be evaluated. At the early stage of AP, the pain is
usually localized in the epigastric area or left upper quadrant and radiates to
the back, later it may involve the whole abdominal area. The intensity of pain
is usually severe and does not reflect the severity of AP.4 AP patients usually
experience general abdominal palpation pain and will often have rebound
tenderness as a sign of peritoneal irritation. Nausea, vomiting, and
abdominal bloating are common symptoms caused by gastric and intestinal
hypomotility.3 In severe cases, patients can have hemodynamic instability
(10%) or hematemesis and melena (5%) as signs of coagulation
disturbances.4 The Cullen sign and the Grey Turner sign manifest as
brownish-green discoloration of the umbilical region and the flank,
respectively. The skin signs are rare (1.2%) and thought to indicate an
unfavorable prognosis.202 Fever (76%) and tachycardia (65%) are common
signs, and in severe cases the patient can have dyspnea.3, 4
!#!(#"#"
Assessment of pancreatic enzymes (i.e. amylase and lipase) is the
cornerstone of clinical laboratory diagnosis. The cut-off values for both of
these enzymes are normally defined to be three times the upper limit of the
normal range. Serum or plasma amylase levels rise rapidly (within 3-6 hours)
of the onset of symptoms and can normalize rapidly because of a short halflife (12 hours) so the diagnostic accuracy time is short. Pancreatic amylase
concentration on admission is not associated with the severity of AP, and a
patient with only a slight increase can also develop SAP.203 Serum lipase
38
levels increase 3-6 h after the onset of AP, peak within 24 h, and can persist
at increased levels for 1-2 weeks. Assessment of lipase levels is today the
preferred diagnostic laboratory test over amylase levels due to its higher
sensitivity. Lipase levels also remain elevated for longer than amylase
levels.204
Other laboratory tests with diagnostic value are urine amylase, serum or
urinary trypsinogen-2, phospholipase A2, and pancreatic elastase.205-208
Valuable clinical laboratory tests in determining the severity of AP and
possible organ systems involved are white blood cell count, concentrations of
electrolytes, creatinine levels, liver function tests (i.e. aspartate
aminotransferase, alkaline phosphatase, total albumin), C-reactive protein,
blood sugar, and arterial blood gas analysis (when the patient’s oxygen
saturation is less than 95% or the patient has tachypnea).72
Clarifying the coagulation status of the patient is important to assess the
severity of coagulopathy and the dosing for anticoagulant therapy. PT (and
TT) measures the extrinsic pathway activation, and APTT (activated partial
thromboplastin time) measures the intrinsic pathway activation. Fibrinogen
levels increase within the acute-phase reactions and decrease in DIC and
when the liver function is decompensated. AT levels have been shown to
decrease in DIC. Fibrin d-dimers is a degradation product of fibrin, and the
levels are elevated in DIC and in the presence of a thrombus, i.e. deep vein
thrombosis or pulmonary embolisms.209, 210
Monitoring the CRP and leukocyte levels may provide information on the
intensity of an inflammatory reaction. CRP is a non-specific acute-phase
protein that reacts with a delay (24-48 h) to inflammation, but also to a
bacterial infection or to tissue injury.211
In unclear cases, determining the liver enzyme, calcium, and triglyceride
levels can help to uncover the etiology.212
!"
Contrast-enhanced CT is the standard and highly specific imaging technique
for evaluation of AP.213 CT scan is valuable in establishing the severity of AP;
it helps to identify local or extrapancreatic complications, e.g. necrosis,
peripancreatic fluid collections, ascites, and pleural effusions. An early CT
scan (within 4 days of symptom onset) should be obtained only when the
clinical diagnosis is unclear and other serious intra-abdominal disorders
must be excluded.213, 214 According to the 2012 IAP/APA guidelines,
indications for initial CT assessment are 1) diagnostic uncertainty, 2)
confirmation of severity based on clinical predictors of SAP, or 3) failure to
respond to conservative treatment or in the setting of clinical deterioration.
An optimal timing for initial CT is 72-96 hours after onset of symptoms.212 In
cases of patients with impaired renal function or contrast medium allergy,
the use of contrast medium is contraindicated. Magnetic resonance imaging
39
Review of the literature of acute pancreatitis
(MRI) is a viable alternative in these situations. MRI is better than CT in
distinguishing the fluid part of pseudocysts from the solid parts.215 MRI
cholangiography is used to evaluate the biliary tree and the presence of
stones in the common bile duct.216 Abdominal ultrasonography (US) has
limited utility for diagnosis and severity assessment of AP due to its poor
accuracy in obese patients and in the presence of intestinal gas.
Ultrasonography is valuable for detection of gallstones and dilatation of the
common bile duct.216 Chest radiographs can show pulmonary infiltrates and
pleural effusions in case of SAP. Panoramic abdominal radiographs can show
ileus and pancreatic calcifications if the patient has so-called acute-onchronic involvement of AP.
""#
AP is a disease with a highly variable clinical course. Classification of AP by
severity is an important tool in clinical work, aiding to identify patient with
potentially severe disease who require effective early treatment.
Classification systems are important in communication between clinicians
and researchers, enabling comparison of data from different centers.
##""#
The Atlanta classification (Atlanta 1992) has been the most widely accepted
classification system for severity of AP. It was introduced in 1992 by the
International Symposium of Acute Pancreatitis.217 Atlanta 1992 categorizes
AP patients into two groups: mild and severe AP. Mild AP is associated with a
shorter uncomplicated course of disease, without signs of significant organ
failure. Severe AP is defined by the presence of distant organ failure or local
pancreatic complications, e.g. pancreatic necrosis, acute fluid collections,
pseudocyst, and pancreatic abscess (Table 3). Diagnosis of local
complications is mostly based on CT imaging. Also poor prognostic scores
(Ranson’s score ≥3 and/or APACHE-II ≥8) result in a patient being allocated
to the severe AP group.217
To achieve better accuracy in clinical studies, patients in the severe AP
group are often categorized into two subgroups: those with only local
complications and those with OF.
40
!%"##""#
Advancements in the understanding of the pathophysiology and course of AP
and improvements in diagnostic and prognostic methods have resulted in the
need to update the classification system. In 2012 the Acute Pancreatic
Classification Working Group reported a revision of the Atlanta classification
(Atlanta 2012).12 The revised Atlanta classification is based on consensus of
11 national and international pancreatic associations.
The revised classification defines criteria for the diagnosis of acute
pancreatitis (mentioned in 2.5.1.), and it differentiates between interstitial
edematous pancreatitis and necrotizing pancreatitis. In the case of interstitial
edematous pancreatitis, an inflammatory edema leads to homogeneous
enhancement of pancreas tissue and the severity of AP is mild, usually
resolving within the first week.218 In the case of necrotizing pancreatitis (510% of AP patients), necrosis involves the pancreas and/or peripancreatic
tissues. Necrotic lesions evolve over several days and the clinical course of
necrotizing pancreatitis can be variable: necrosis may remain solid or liquefy,
remain sterile or become infected, or be radiological persistent or transient.12
According to the revised Atlanta criteria, AP is classified into three
severity categories: mild AP (only pancreatic involvement, no local
complications or OF), moderately severe AP (transient OF and/or local or
systemic complications without persistent OF), and severe AP (with
persistent OF) (Table 1). Modified Marshall Score (Table 4) is used to assess
the presence of organ failure; a score of two or more in any organ system
defines the presence of OF.
The revised criteria discriminate two overlapping time phases of AP with
two peaks of mortality: early and late phase. During the early phase, an
overactive inflammatory reaction of SIRS may lead to OF, and the
determinant of severity of AP in this phase is based on the presence and
duration of OF (transient or persistent). Transient organ failure resolves
within 48 hours. Local complications and level of necrosis are not
predominant determinants of severity during the early phase. In the late
phase of AP, patients may have signs of persistent systemic inflammation or
local complications, and in this phase SIRS may change to CARS and
increase the risk of infections. Persistent OF remains the main determinant
of severity in the late phase of AP. The late phase of AP occurs only in
patients with moderately severe and severe AP. 12 (Table 5)
The revised classification also differentiates pancreatic and peripancreatic
collections on cross-sectional imaging as acute peripancreatic fluid,
pancreatic pseudocyst, acute necrotic collections, and walled-off necrosis.12
41
Review of the literature of acute pancreatitis
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"%!#(""""#
The appropriate management of AP should be started early to prevent
mortality; nearly half of the deaths of SAP patients occur within the first
week due to the development of OF; the incidence of OF is maximal (17%) on
the first day.6, 219, 220. Therefore, it is critical to predict severity of AP in the
initial stages of disease.
!"#!"
Obesity, especially abdominal obesity, predisposes AP patients to the
development of local and systemic complications.221 Smoking and type 2
diabetes are other known risk factors for AP. 222, 223 Older patients (over 55
years) are at increased risk for severe disease.4
"!"("#"
Several multifactorial scoring systems for the prediction of the severity of AP
have been developed, and all of them have advantages and disadvantages.
One of the oldest scoring systems is a Ranson score, which was developed in
the early 1970´s.224 Ranson score has a high sensitivity and specificity (84%
and 78%, respectively), but the severity can be predicted only after 48 hours
from admission.225 Other scoring systems are Glasgow, BISAP (Bedside
Index for Severity in Acute Pancreatitis), and APACHE II. BISAP and
APACHE II were calculated using data within 24 hours of admission.
APACHE II has high predictivity, but its complexity in clinical use has stirred
criticism.226, 227 HAPS (harmless acute pancreatitis score) can rapidly identify
patients with a mild disease. According to HAPS, patients without rebound
tenderness and/or guarding, a normal hematocrit, and a normal serum
creatinine concentration have a high positive predictive value (98-99%) of
having a harmless course of AP. HAPS has a low negative predictive value, so
it is unable to predict SAP.228 Several CT scoring systems (i.e. CT severity
index CTSI, Balthazar grade) have evolved to establish severity of AP, but
their predictive accuracy is similar to clinical scoring systems.229
Several organ dysfunction scores also exist, e.g. SOFA score230 and
Modified Marshall score.231 These scores can describe the severity of OF and
are not applicable for prediction of SAP. Modified Marshall score is included
in the revised Atlanta criteria to identify patients with OF.12
43
Review of the literature of acute pancreatitis
!!" !#"%!$# !##"
Several biomarkers have been studied as potential predictors of severity of
AP. Some parameters focus on water balance and microcirculation or
presence of an inflammatory process.
C-reactive protein (CRP) is one of the acute-phase proteins most
frequently used as a single biomarker for assessment of severity of AP.
Inflammatory stimuli (i.e. IL-1 and IL-6) triggers hepatocytes to produce
CRP.232 However, production of CRP is delayed and it is a reliable predictor
only after 48 hours from symptom onset. In addition, CRP is not diseasespecific (other infectious diseases are accompanied by increased levels of
CRP such as pneumonia, infected necrosis, etc.).211 Serum creatinine has
been identified as a predictor for pancreatic necrosis.233 Also glomerular
filtration rate can predict the severity of AP.234 Blood urea nitrogen has been
shown to predict severity of AP (after 48 hours) and mortality in SAP.235
Blood urea nitrogen is included in the Ranson and BISAP scores.
Procalcitonin (PCT) is a propeptide of calcitonin and it is usually not
detectable in serum in normal controls.236 PCT is a marker of systemic
inflammation and PCT levels increase in bacterial infections and also in noninfectious SIRS.129 PCT may serve as a predictor of complicated course of
AP237, and it can be measured within hours of symptom onset.236 PCT is one
of the most promising biomarkers, but the PCT assay is expensive, hindering
its use in routine clinical practice.
Numerous cytokines have been studied regarding their relationship with
severity of AP, but most of them have proven to be mainly of
pathophysiological interest rather than of clinical utility. IL-6 is an antiinflammatory cytokine, which also has proinflammatory properties.121 IL-6
concentrations peak in the first day of onset of AP and decrease rapidly
within 2-4 days.123, 238 IL-6 is an early predictor of complicated course of AP,
with a high sensitivity and specificity, and it is in clinical use in some
hospitals.225 Drawbacks of IL-6 assay are the rapid decrease in
concentrations and the high cost and complexity of the assay.239 IL-8 is a
proinflammatory chemokine (cytokine with chemotactic properties), which
has been shown to act as an early predictive marker of disease severity in AP
and is useful in monitoring MOF.240 Recent meta-analysis showed that IL-6
has better sensitivity and specificity than IL-8.241 IL-10 is a potent antiinflammatory cytokine and its biologic effects may result in
immunosuppression.124 IL-10 is an early marker of severity of AP. Serum IL10 levels have been shown to be elevated in severe and lethal AP.242 IL-1ra is
an important anti-inflammatory cytokine. It binds competitively to the IL-1
receptor and prevents IL-1-mediated responses.125 Increased serum IL-1ra is
associated with development of MOF in AP and in sepsis.243 Serum IL-1ra
may serve as an early marker of SAP and may predict poor outcome.17, 242
Adhesion molecules on leukocytes and endothelial cells have a central role
in pathogenesis of AP, and their potential diagnostic usefulness has been
44
evaluated. The soluble form of E-selectin is a leukocyte adhesion molecule
that is expressed on vascular endothelial cells after an inflammatory
stimulus.98 Soluble E-selectin can be measured in serum and serves as a
marker of endothelial cell activation.244 Soluble E-selectin may serve as a
predictor of the severe form of AP.245 L-selectin (CD62L) is a leukocytic cell
adhesion receptor that mediates the initial step of adhesion cascade, the
leukocyte-endothelium interaction.100 Expression of L-selectin did not show
any correlation with severity of AP.246
In addition, different activation markers of leukocytes and their
association with severity of AP have been investigated. In
immunosuppression, HLA-DR expression on monocytes is reduced and the
ability of monocytes to produce proinflammatory cytokines is impaired.142
Decreased HLA-DR expression on monocytes acts as a cellular marker of
immunosuppression.247 Decreased monocyte HLA-DR expression is related
to disease severity in patients with AP and predicts the development of OF in
AP.16, 246 CD14 is a cell surface glycoprotein on mononuclear phagocytes that
has been identified as a receptor for a wide range of microbial products.95
CD14 exists in soluble and membrane-bound forms, and both have immune
stimulatory and inhibitory functions.96 Increased soluble CD14 expression is
associated with SIRS in patients with AP.248 Monocyte CD14 expression
levels were not related to AP severity.246 CD11b is an adhesion molecule
expressed on the surface of neutrophils and monocytes. Activation of these
cell types is manifested by increased integrin CD11B/CD18 expression on the
cell surface.94 Enhanced neutrophil and monocyte expression of CD11b
molecules is associated with the severity of AP.246
In addition to the above-mentioned scoring systems and markers, dozens
of other markers have been studied in severity assessment of AP. These days,
CRP and APACHE II score and PCT are the most widely used tools for
severity assessment in AP.211 An optimal test, which would predict severity of
AP precisely, easily, cost-effectively, and sufficiently early, is still missing.
#!##
A patient with mild AP can be treated in an outpatient department. Patients
with risk factors for SAP should be taken to hospital for close monitoring and
timely reassessment of disease severity. Patients with apparent OF (MMS>2)
should be taken to an advanced medical care ward, and in case of persistent
OF the patient should be treated in an intensive care setting.
No effective pharmacological treatment exists for AP despite decades of
research. Thus, supportive therapy with management of local and systemic
complications is offered. The early phase of SAP (the first 5-7 days) is critical.
OF evolves early in SAP, and unless aggressive management is provided,
mortality can reach up to 50%.249
45
Review of the literature of acute pancreatitis
$"#%#! (
In the case of alcohol- or drug-induced AP, the intake of the triggering agent
must cease.
In the case of biliary AP, the indication of ERCP depends on the degree of
obstruction of the common bile duct and the presence of cholangitis. The
2012 IAP/APA guidelines state that ERCP is not indicated in predicted mild
biliary AP without cholangitis, ERCP is probably not indicated in predicted
severe biliary AP without cholangitis, ERCP is probably indicated in biliary
AP with common bile duct obstruction, and ERCP is indicated if the patient
has biliary AP and cholangitis.212 Cholecystectomy is indicated within the
same hospital stay for mild biliary AP. If the patient has peripancreatic fluid
collections, cholecystectomy should be delayed until the collections are
resolved or after 6 weeks.212
$#! (
Early intravenous fluid therapy has been the cornerstone of treatment of AP
for years. Over the course of AP, increased vascular permeability causes
intravascular fluid loss, hypotension, and possible shock. Coagulation
cascade activates in the early phase of AP, leading to DIC and microvascular
thrombosis. Together these phenomena lead to extravasation of fluid into the
third space out of the circulation. In AP, intravenous hydration should
replace the hypovolemia that occurs secondary to vomiting, reduced oral
intake, third space extravasation, and respiratory losses.250 Correction of
hypovolemia can limit the development of necrosis and improve outcome in
SAP. Early fluid resuscitation, within the first 24 hours of admission for AP,
is associated with decreased rates of OF.212 Fluid therapy should not only
replenish blood volume, but also stabilize capillary permeability, relieve the
inflammatory reaction, and sustain intestinal barrier function in SAP.251
Patients with moderately severe or severe AP should be started on effective
fluid resuscitation. An ideal fluid for resuscitation in AP – crystalloids or
colloids – has been under debate. The recent review of Aggrawak et al.
concluded that controlled fluid resuscitation (3.0-4.0 l/24 h) should be
initiated after a bolus infusion 20 ml/kg (1000 ml over one hour), and
lactated Ringers’ is recommended as the fluid of choice. A urine output >0.5
ml/kg and a decrease in blood urea nitrogen (or creatinine level) are simple
goals of fluid resuscitation; other parameters are blood pressure, respiratory
function, and intra-abdominal pressure. Duration of fluid resuscitation
should last 24-48 hours, until signs of volume depletion disappear.252
46
#
Patient with AP usually suffer from severe pain and often require opiate
analgesics. Monitoring of oxygenation while administering high-dose opiate
medications is necessary. Epidural anesthesia may improve pancreatic
microcirculation and tissue oxygenation, so in serious cases epidural
anesthesia may be a useful pain management method (coagulation
disturbances are contraindications).253
$#!#
Previously, total oral abstinence of food was a typical treatment method in
AP. Early enteral nutrition has been shown to reduce systemic and pancreatic
infectious complications, length of hospital stay, and mortality.254 Nutritional
support should be started when the patient is unable to eat for 7 days.
Enteral nutrition stabilizes gut barrier function and reduces bacterial
translocation and infectious complications in SAP. For SAP, enteral nutrition
is better than total parenteral nutrition regarding mortality, infectious
complications, and OF.255 If the SAP patient requires nutritional support,
enteral tube feeding via either nasojejunal or nasogastric route is
recommended.212
## ! ('"
Previously, antibiotic prophylaxis was mentioned to be useful in AP, but
there is no evidence that prophylactic antibiotics reduce infectious
complications or mortality.256 Thus, antibiotic prophylaxis is currently not
recommended in AP.212
#$##! (
Thrombotic complications, i.e. portal vein thrombosis and DIC, are common,
especially in alcohol-induced and necrotizing SAP. Also systemic
microvascular disturbances, including vasoconstriction, inadequate
perfusion, increased blood viscosity, and activation of coagulation, are
involved in SAP. On the other hand, the coagulopathy in SAP is associated
with elevated risk of hemorrhage because of consumption coagulopathy and
thrombocytopenia. Sometimes major bleedings, such as bleedings from
pseudoaneurysms and direct arterial erosion, complicate the disease
course.257
47
Review of the literature of acute pancreatitis
Antithrombotic treatment in SAP is a balancing act between thrombotic
complications and elevated risk of bleedings. Usually critically ill ICU
patients, like SAP patients, have several risk factors for venous
thromboembolisms, i.e. severe illness, sedating medications, and invasive
procedures. SAP patients in the ICU require thrombus prophylaxis, i.e.
LMWH, but the treatment (including dosing and monitoring) should be
considered patient-specific.
Heparin, a highly sulphated glycosaminoglycan, is mostly known as an
anticoagulant, but it also has anti-inflammatory properties. Heparin has two
kinds of pharmaceutical forms: traditional unfractionated heparin (UFH)
and low molecular weight heparins (LMWHs), which are prepared from UFH
by different chemical or enzymatic processes. LMWHs have reduced
capability to inactivate thrombin relative to UFH; UFH can inhibit thrombin
via both antithrombin-dependent and -independent mechanisms, LMWHs
have no direct inhibitory effect on thrombin, but instead LMWHs inhibit
FXa. UFH also has direct inhibitory effects on activation of platelets and
endothelial cells.258 LMWHs have advantageous properties in clinical use
compared with UFH; LMWHs have a longer half-life, enabling a single daily
dosing, and also have more predictable dose-response and lower risk of
thrombocytopenia and bleeding complications.259 LMWHs are more widely
used today. Heparin and its derivatives have anti-inflammatory properties;
they can reduce the release of cytokines and inflammatory mediators (NF-kb,
TNF-a, IL-6).260 Heparin can diminish the formation of microthrombosis in
pancreatic tissue and attenuate inflammatory reaction. LMWH therapy has
been suggested to alleviate damage of the pancreas, lungs, kidneys, and brain
and to decrease mortality in SAP.261 LMWH therapy is also used in the
treatment of DIC.262
Abdominal venous thrombosis (mesenteric, splenic, or portal vein) is a
common complication in AP.257 Management requires therapeutic
anticoagulation with heparin infusion or subcutaneous LMWH before
transition to oral warfarin.170
Administration of AT concentrate is used in the treatment of hereditary
AT deficiency. AT has potent anti-inflammatory properties and it has shown
to ameliorate experimental AP (as mentioned in Section 2.4.6.).153
' !# !#! (
While supportive therapy is the only treatment available for AP, a better
understanding of the pathophysiology has led to emerging research of
pharmacological treatments. The target of the pharmacological therapy can
focus on different steps in the pathogenesis of AP. In the last years, the main
research focus has been on immunomodulatory therapies, the idea being that
the overactive inflammatory response should be inhibited and excessive
immunosuppression avoided in the early stages of AP.
48
Several experimental pre-clinical and clinical studies (Table 6) have
provided evidence of different medical agents with therapeutic potential.
%* )(*&(/%*)
Glucagon increases superior mesenteric blood flow and decreases pancreatic
exocrine secretion. Clinical studies have failed to show beneficial effects of
glucagon therapy in AP, instead pancreatic hemorrhage increased.263
Somatostatin inhibits exocrine secretion of the pancreas, reduces splanchnic
blood flow, and modulates the inflammatory cascade in AP.264 Although
some preclinical studies have provided promising results, clinical studies
have not shown a clinically significant mortality benefit in AP.265 Octreotidi, a
synthetic analog of somatostatin, has also been evaluated. Results of clinical
trials have been controversial, but meta-analysis (n=948) revealed no benefit
on mortality or complication rates in AP.266
(&*) % *&()
Protease inhibitors can prevent intra-acinar activation of digestive enzymes.
Clinical studies of aprotinin267, gabexate mesilate,268 and nafomostat269 have
been published, and they have shown a potential benefit in AP, but larger
studies are needed to confirm the clinical value of protease inhibitors.
$$+%&$&+#*&()
The optimal time-window for immunomodulatory therapy is narrow, and it
is difficult to know exactly a patient’s disease phase (i.e. immune status) at a
given time-point.
Blocking the inflammatory reactions by anti-inflammatory factors may be
an effective treatment for AP. Many factors (i.e. cytokines and cells) are
involved in inflammatory reactions so if the treatment is targeted only to one
factor it is possible that the therapy is insufficient to terminate the entire
overactive inflammatory reaction. Amobarbital can inhibit NF-κB activity
and in animal models attenuates AP-associated injuries in the pancreas and
lungs.270 Infliximab (monoclonal anti-TNF-α antibody) has been shown to
suppress neutrophil infiltration and ameliorate necrosis of pancreas tissue in
animal models, and to ameliorate AP in patients with active Crohn disease.271,
272 Lexipafant is a PAF antagonist. PAF amplifies the activity of key
mediators of SIRS in AP. Clinical trials of lexipafant to prevent OF in SAP
were promising273, 274, but the largest randomized trial (phase III) showed no
significant reduction in OF or local complications.275. Indomethacin inhibits
pancreatic phospholipase A2 activity and decreases neutrophil-mediated
inflammation. Clinical studies showed no decrease in inflammatory reactions
in AP276, but rectal indomethacin may prevent post-ERCP pancreatitis.277
49
Review of the literature of acute pancreatitis
Steroid therapy dampens widely the inflammation reaction; it can reduce the
inflammatory cascade and leukocyte recruitment. It has been shown to be
beneficial in autoimmune pancreatitis, but the published case reports have
mentioned steroids as inductors of AP.278
For patients with immunosuppression, proper immunostimualtion may
alleviate the disease and prevent infectious complications. In CARS, HLA-DR
expression on monocytes is reduced and the administration of IFN-γ or CMCSF can raise the HLA-DR expression on monocytes in septic patients and
shorten the course of sepsis-associated immunosuppression.279, 280The same
phenomenon has been shown in vitro on monocytes in SAP patients, but
clinical studies are needed. 281
#*+3.
( )'%3! &%(%&-./ %!-)"*$,')&)#%.,!.'!(.%(
($&#& %*
+2>A=6
(ED4@>6
C:J6
#+&%
EBB
'@67764DC@?=6?8D9@79@CA:D2=CD2I@B>@BD2=:DI
&$*&)** %
%E6?8@
64B62C65=6?8D9@79@CA:D2=CD2I72F@B23=6
492?86C:?,C42??@67764D@?>@BD2=:DI
*(&* ?5B:E==:
-9=
'@67764DC@7>@BD2=:DI@B6F@=ED:@?@74@>A=:42D:@?C
'(&* % %
2==5:?
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E9=6B
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.*$) #*
.2=56BB2>2
*65E465>@BD2=:DI:?+)
4@>A=:42D:@?C
'@67764DC@?>@BD2=:DI@B56F6=@A>6?D@7
4@>A=:42D:@?C
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):2C4:<
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*65E465>@BD2=:DI2?5?6657@BCEB8:42=:?D6BF6?D:@?
$:?8C?@BD9
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*65E4656F@=ED:@?@7(2?5=6F6=C@7"%2?5"%
*65E4656F@=ED:@?@7(
#@9?C@?
'@67764DC@?6F@=ED:@?@7(
50
!"#%"##
"#"#$(
The main goal of the study was to investigate whether ecto-5’nucleotidase
(CD73) predicts the development of SAP and whether APC treatment
alleviates course of SAP. Specific aims were as follows:
I.
II.
To examine CD73 as a predictor of SAP at the levels of mRNA, protein,
and enzyme activity.
To evaluate in a prospective randomized trial the effects of APC
treatment on (i) evolution of OF, (ii) occurrence of bleeding
complications, and (iii) course of systemic inflammation and
coagulation cascade disturbances.
51
PRESENT INVESTIGATION
#!"#"
##"#(#!"$#"
All of the patients studied fulfilled the criteria of AP and were admitted to
Helsinki University Central Hospital within 72 (I) or 96 (II, III, IV) hours
from onset of symptoms. In all studies, the diagnosis of AP was based on
typical clinical findings, including onset of epigastric pain, nausea, and
vomiting, and an elevated plasma amylase concentration of at least three-fold
the upper reference limit, and/or typical radiological appearance of AP in CT.
Patients in Study I (n=161) were retrospectively categorized into mild AP,
moderately severe AP, and severe AP according to revised Atlanta criteria
(Atlanta 2012).12 In mild AP, patients had no OF or local or systemic
complications and they were usually discharged during the early phase. In
moderately severe AP, patients had local complications and/or transient OF
(resolving within 48 hours). SAP patients had single or multiple persistent (>
48 hours) OF. The presence of OF is based on a Modified Marshall Score
(MMS) ≥ 2. Seventeen patients had MMS ≥ 2 on admission, indicating the
presence of OF. Five of them had transient OF, which recovered within 48
hours (moderately severe AP). The other 12 patients had persistent OF
(severe AP) (Figure 6).
20;8.
=@G492BD@7A2D:6?DC:?+DE5I"(B82?72:=EB6(@?25>:CC:@?G2C244@B5:?8D@
D96 &@5:7:65 &2BC92== +4@B6 &&+ 2?5 A2D:6?DCR @ED4@>6 G2C 244@B5:?8 D@ D96
B6F:C65D=2?D24B:D6B:2)24ED6A2?4B62D:D:C
52
In Studies II, III, and IV, all patients had a severe form of AP with at least
one OF.217 The additional inclusion criteria were as follows: 1) admitted to
the hospital < 96 hours from onset of pain, 2) at least one OF defined as an
organ-specific SOFA score of at least three, and 3) < 48 hours from the first
OF.
The Ethics Committee of the Helsinki University Central Hospital
approved the study protocols, and the informed consent of each patient was
obtained.
*+/
The prospective study consists of 161 patients with AP: 107 with mild AP, 29
with moderately severe AP, and 25 with severe AP (admitted between June
2003 and February 2007) (Table 7). For three patients, mRNA samples were
not available. The reference samples were obtained from 20 healthy
controls.
*+/
A total of 215 patients admitted with SAP were screened for the study
between June 2003 and August 2007. Of these 215 patients, 158 fulfilled the
inclusion criteria. After exclusions (see Figure 7), 32 patients with SAP were
randomized to receive either APC (n = 16) or 0.9% physiologic saline as a
placebo (n = 16) (Figure 6, Table 7).
*+/
Study III was a sub-study of the Study II and the study population consists of
the same patients as in Study II (Tables 8 and 9). The reference samples were
obtained from 65 healthy controls.
*+/
The study population consists of a subgroup of Study II patients who had
follow-up coagulation samples taken (APC group n=10, placebo group n=10)
(Table 10). The reference samples were obtained from 10 healthy controls.
53
PRESENT INVESTIGATION
#*+3.
$,.!,%-.%-)"*.%!(.-./ 2(
&:=5
&@56B2D6=I
+6F6B6
?
C6F6B6
?
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6>2=6
>2=6?
86I62BC>65:2?B2?86
D:@=@8I@7)?
&)$)&
%&%,2
%)*.$%
20;8.
H4=EC:@?4B:D6B:22?5B2?5@>:J2D:@?@7A2D:6?DC7@B+DE5I""+)C6F6B624ED6
A2?4B62D:D:C
54
#*+3.
$,.!,%-.%-)"*.%!(.-./ %!-( !+/!(.%&,#(%&/,!
--!--'!(.
)
)=2463@
?
?
6>2=6
>2=6?
86I62BC>65:2?B2?86
D:@=@8I@7)?
=4@9@=
:=:2BI
+(C4@B6
#*+3.
N
N
/'!,)"*.%!(.-./ 2
2C6=:?6
2I
2I
)
)=2463@
+@=E3=6>2B<6BC
6==>2B<6BC
)
)=2463@
55
PRESENT INVESTIGATION
#*+3.
/'!,)"*.%!(.-./ 2
2C6=:?6
2I
2IC
2IC
)
)=2463@
@28E=2D:@?2?5
2?D:4@28E=2D:@?>2B<6BC
,9B@>3@8B2>C
)
)=2463@
A2D:6?DCG:D9?@56D64D23=6D9B@>3:?86?6B2D:@?8B2A9CC9@G65CDB2:89D=:?6CG6B66H4=E565
"#%""#$(
*+/
Study I was a prospective cohort study. We investigated whether serum levels
of sCD73 activity, sCD73 protein concentration, and CD73 mRNA levels were
associated with the severity of AP and analyzed the ability of SCD73 to
predict the development of the severe form of AP.
*+/
Study II was a prospective randomized pilot human clinical trial where SAP
patients were randomized to receive APC or placebo. The primary efficacy
endpoint was the change in SOFA between the start of the study drug and
day 5. The sample size was determined according to the primary endpoint; a
three-point difference in change of SOFA score between the groups was the
objective. The primary safety endpoint was the number of incidents of
bleeding. Secondary endpoints were organ-failure-free days alive, days alive
outside hospital in 60 days, and changes in other laboratory values during
the first five days.
*+/
Study III was a pre-determined sub-study of Study II. The time course of the
patients’ plasma or serum levels of soluble markers (IL-8, IL-6, IL-10, IL-1ra,
56
sE-selectin, PCT) and monocyte and neutrophil cell surface (CD11b, CD14,
CD62L, HLA-DR) markers of systemic inflammatory response were
measured during the first 14 days after the randomization.
*+/
Study IV was a pre-determined sub-study of the patients in Study II who had
follow-up coagulation samples taken. Coagulation parameters, physiological
anticoagulants, thrombograms, and circulating levels of IL-6 and CRP were
determined on admission and at days 1, 3-4, and 6-7.
#*+3.
!-%#(-)"./ %!-
+DE5I
"
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+DE5I56C:8?
)B@CA64D:F6
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)B@CA64D:F6
4@9@BDCDE5I
B2?5@>:J65
CDE5I2CE3
CDE5I2CE3
A:=@D9E>2?
CDE5I@7+DE5I
CDE5I@7+DE5I
4=:?:42=CDE5I
"
"
+DE5IA6B:@5
'E>36B@7A2D:6?DC
?@7
?+)@7
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2==
5:776B6?D8B@EAC
G9@>?
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5E=DA2D:6?DC
5E=DA2D:6?DC
+2>62C+DE5I
+2>62C+DE5I
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G:D9)
G:D9+)2?5
""
""
2D=62CD@?6(
25>:DD65D@"-
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9B@?:4
+24D:F6
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3=665:?8
""
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?
?
49B@?:4
A2?4B62D:D:C
!62=D9I4@?DB@=C
?
57
PRESENT INVESTIGATION
(##"
" "
*+/
Serum samples for determining CD73 activity and CD73 protein
concentration were taken on admission to hospital. The samples were frozen
and stored at -70°C. Also venous blood samples for isolation of CD73 mRNA
were collected on admission. They were drawn directly into PAXGene tubes.
The reference samples were collected from healthy laboratory personnel
(n=20, age 28-59 years).
*+/
Blood samples were taken every day during the patient’s stay in the ICU.
Laboratory measurements for SOFA (including serum creatinine, serum
bilirubin, platelet count, and blood gas analysis for PaO2/FiO2 ratio) were
performed between the start of the study drug and day 5.
*+/
Blood samples for determination of cell markers of inflammation were
collected when a patient fulfilled the inclusion criteria. After the
randomization, follow-up samples were collected on the third, fifth, seventh,
and 14th day. Blood samples for flow cytometry and for plasma
measurements were anticoagulated with pyrogen-free acid-citrate dextrose.
Blood samples were cooled in an ice-cold water bath and kept at 0°C until
stained for flow cytometry. The plasma was separated by centrifugation at
4°C and stored at −70°C until concentrations of cytokines and sE-selectin
were determined. The reference blood samples for the analyses of cell surface
markers were obtained from 65 healthy volunteers from the hospital and
laboratory staff without medication and with no signs of infection.
*+/
Plasma samples for the determinations of the biomarkers of coagulation and
the markers of physiological anticoagulation and thrombograms were
collected before starting the APC infusion (at 0 hours) and at days 1, 3 (or 4),
and 7 (or 6) (in case the sample collection was scheduled on a bank holiday,
they were taken on the following or the previous day). The samples were
frozen and stored at −70°C. Serum CRP concentrations and blood platelet
58
counts were determined as a part of laboratory at 0 hours and 1, 3–4, and 6–
7 days later. Plasma samples at 0 hours were available from each of the 20
patients. Reference blood samples for analysis of coagulation markers and
thrombograms were obtained from healthy hospital and laboratory staff
without medication (n=10).
!#!((""
CRP (I), creatinine (I, IV), platelet count (II), blood gas analysis (II), B-hb
(II), serum conjugated bilirubin (II), serum pre-albumin (II), serum
disialotransferrine (II), and serum gamma-glutamyl-transferase (II) were
determined in accordance with the hospital’s routine laboratory practice
(Helsinki University Hospital Laboratory, HUSLAB).
The coagulation markers in Study IV were determined in accordance with
the HUSLAB laboratory practice: the TT and PT levels in plasma samples
were measured by Nycotest PT, plasma levels of PC and AT were measured
by Berichrom, and the TFPI (free and total) levels in plasma samples were
measured by Asserachrom (Diagnostica Stago). We calculated the ratio of
free and total TFPI.
)($"!#""("
In Study III, the concentrations of IL-6 (also in Study IV), IL-8, IL-10, IL1Ra, and E-selectin in plasma samples were determined by enzyme
immunoassay. The commercial reagents used were as follows: IL-6 and IL10: PeliPair ELISA, Sanquin, Amsterdam, the Netherlands; IL-8: Opt EIA,
BD Biosciences, Erembodegem, Belgium; IL-1Ra: Duo Set ELISA, R&D
Systems Europe Ltd., Abindgon, UK; E-Selectin: ELISA, HyCult
Biotechnology, Uden, the Netherlands. The detection limits and intra-assay
and interassay coefficients of variation (CV%) were as follows: IL-6: 0.3
pg/ml, 3.6% and 5.4%; IL-8: 1.6 pg/ml, 3.5%, 3.4%; IL-10: 0.3 pg/ml, 3.7%,
5.9%; IL-1Ra: 10 pg/ml, 4.2%, 5.2%; E-selectin: 20.5 pg/ml, 3.5%, 6.9%.
Procalcitonin (PCT) (III) was measured by means of a sandwich
chemiluminescent immunoassay using monoclonal antibody to fluorescein
covalently linked to paramagnetic particles and two antibodies to
procalcitonin labeled with fluorescein (ADVIA Centaur XP immunoassay
system with ADVIA Centaur BRAHMS PCT assay).
Analysis of sCD73 protein concentration (in Study I) was performed with
sandwich ELISA technique using new anti-CD73 specific monoclonal
antibodies. New mAbs against immunoaffinity-purified human CD73
molecule were generated by immunizing CD73-deficient mice. The
hybridoma generation was performed by fusing the draining lymph node
lymphocytes with SP2.0 myeloma cells. The hybridoma supernatants were
59
PRESENT INVESTIGATION
screened using immunohistochemical stainings of human tissues,
immunofluorescence stainings of transfectants expressing CD73, and
immunoblotting; the positive hybridomas were subcloned by limiting
dilution.
One of the new mabs (clone 118, mouse IgG1) was used together with an
old anti-CD73 mAb (clone 4G) for development of sandwich. In brief, 4G4
mAb was bound to the bottom of white 96-well Cliniplates, the serum
samples were added (1:10 dilution in PBS), and the bound sCD73 was
detected with biotinylated-118. Clone 118 was biotinylated with NHS-biotin
(Pierce) according to the manufacturer’s instructions. The ELISA reaction
was developed using streptavidin-horseradish peroxidase and BM
Chemiluminescence ELISA Substrate (Roche).
To validate the ELISA, serial dilutions of recombinant human CD73 (RD
Systems) and CD73-depleted sera were used. CD73 was removed from serum
by three sequential incubations of normal pooled sera with CnBr-activated
sepharose beads armed with an anti-CD73 mAb 4G4.
CD73 activity, protein concentration, and mRNA levels were determined
in a research laboratory at the University of Turku.
&&(##!(
In Study III, monocyte expression of CD14, CD11b, CD62L, and HLA-DR and
neutrophil expression of CD62L and CD11b were determined using whole
blood flow cytometry.17, 246 Monoclonal antibodies (mAbs) were as follows:
phycoerythrin (PE) and fluorescein isothiocyanate (FITC) conjugates of antiCD14 mAb (IgG2b, clone MFP9), PE conjugates of anti-HLA-DR mAb
(IgG2a, clone L243), anti-CD11b mAb (IgG2a, clone D12) and control mouse
IgG2a, mAb, and FITC conjugate of anti-CD62L mAb (IgG2a, clone SK11).
Staining of aliquots of the whole blood sample at 0°C for flow cytometry was
carried out as described previously.17, 246 Data acquisition and analyses were
done with a FACSCalibur flow cytometer and Cell Quest software (BD
Sciences, San Jose, CA, USA). Neutrophils were identified by the light
scattering properties and monocytes by the clonal marker CD14. Monocyte
HLA-DR expression was determined as the proportion of HLA-DR-positive
monocytes.289 Fluorescence intensity is presented as relative fluorescence
units (RFUs).
#%#(
CD73 activity in the sera was determined using radioactive enzyme assays290
in a research laboratory at the University of Turku. The measurement of
CD73 activity was based on chromatographic analyses with 3H-labeled
nucleotides and bioluminescent measurement of ATP metabolism. Serum
60
(typically 10 μl) was incubated with 300 μM AMP along with tracer [2-3H]
AMP (Quotient Bioresearch, GE Healthcare, UK) at 37ºC in a final volume of
60 μl RPMI1640 medium supplemented with 5 mM β-glycerophosphate.
Large excess of β-glycerophosphate as an alternative phosphorylated
substrate in the enzyme assays allowed us to exclude the potential
contribution of non-specific phosphatases (e.g. alkaline phosphatase) in the
measured activities. The incubation times (30-60 min) were chosen so that
the amount of hydrolyzed AMP was <15% of the initially added substrate to
ensure linearity. Sample aliquots were then applied to Alugram SIL G/UV254
sheets (Macherey-Nagel, Germany), and separated by thin-layer
chromatography
using
isobutanol/isoamyl
alcohol/23
ethoxyethanol/ammonia/H20
(9:6:18:9:15).
[2- H]AMP
and
its
dephosphorylated 3H-nucleoside derivatives were visualized in UV-light and
quantified by scintillation β-counting. The CD73 activities are reported as
nanomoles AMP hydrolyzed by one milliliter of serum in one hour.
!!!
CD73 mRNA levels were analyzed in blood leukocytes by using a qPCR
(quantitative polymerase chain reaction) technique in a research laboratory
at the University of Turku. Leukocyte mRNA was isolated from the blood
samples collected into PAXgene tubes using PAXgene Blood RNA kit
(PreAnalytix) according to the manufacturer’s instructions. mRNA was
reverse transcribed into cDNA using an iScriptTM cDNA Synthesis kit
(BioRad). The CD73 mRNA levels were determined using a TaqMan Gene
Expression Assay (Applied Biosystems) for CD73 (NT5E, assay
Hs00159686_m1) as the primer/probe set. The PCR reactions were carried
out as suggested by the supplier using the Applied Biosystems 7900HT Fast
Real-Time PCR System in the Finnish DNA Microarray Center, Turku Center
of Biotechnology, Turku, Finland. All samples were run in triplicate, and the
expression values were normalized using a house-keeping gene (GAPDH) as
the endogenous control. The results were analyzed with SDS 2.3 software.
Changes in cycle threshold levels (ΔCT) were calculated by subtracting the
average of GAPDH CT values from the average of target gene CT values.
Relative expression of the gene analyzed was estimated using the formula:
relative expression = 2-ΔCT. The quantity of CD73 mRNA was expressed as a
percentage of GAPDH mRNA after multiplying the relative target gene
expression by a factor of 100.
#!!"
The thrombin generation of patients was analyzed by thrombograms in Study
IV. The thrombograms were obtained through Calibrated Automated
61
PRESENT INVESTIGATION
Thrombography (CAT, ThrombogramTM, Synapse B.V., the Netherlands) in
the Hematology Laboratory of Helsinki University Central Hospital. The
parameters of the thrombogram are the lag time (reflecting the same
physiological phenomena that largely determine various clotting time-based
assays), the area under the curve (AUC=endogenous thrombin potential,
ETP), the peak height (thrombin burst), and the time to reach the peak.291
Platelet-poor plasma (PPP) was used. Coagulation was triggered by
recalcification in the presence of 5 pM tissue factor.
"##"##"
Study I was a preliminary study, which included prospectively enrolled AP
patients to the emergency unit at Helsinki University Central Hospital
between June 2003 and February 2007. The sample size for Study II was
calculated according to the primary endpoint: the change in SOFA.230 We
assumed that critically ill SAP patients would have an average admission
SOFA score of 7 and that the mean (SD) increase would be 1 (± 3) point in
the placebo group.41 Calculations according to established methods revealed
that a sample of 16 patients per group would allow us to detect a clinically
meaningful three-point difference in change of SOFA score (7 to 8 in placebo
group vs. 7 to 5 in APC group with an estimated SD of 3.0 and with P < 0.05
and a power of 80%). The sample size was also assumed to be adequate for
testing the differences in patient-related change in laboratory parameters in
this pilot trial (no preliminary data were available for calculations). Studies
III and IV were pre-determined sub-studies of Study II.
The results (summary statistics) were expressed as means (II, III, IV),
differences in means (II), medians (I, III, IV), ranges (I, IV), or inter-quartile
ranges (IQR) (I,III,IV).
Normal distribution of the data was tested with skewness (I) and
Kolmogorov-Smirnov test (continuous variables) (II, III, IV). In Study I,
statistical tests with non-parametric assumptions were used because most of
the data showed non-normal distribution (skewness of the data). In the other
studies, the data were normally distributed.
Comparison of continuous data was tested with the Mann-Whitney U-test
(between two groups) (I, III), the Kruskall-Wallis test (between three groups)
(I), or the Jonkheere-Terpstra test for trend (I). In Study II, comparisons
between the study groups were performed using patient-related changes
(before and after the study drug, days 0 and 5) in laboratory values and the
calculation of differences in means (differences in means (DIM) (95% CIs))
according to evidence-based medicine guidelines for randomized studies292,
and regarding categorical variables with Fisher’s exact test.
The Wilcoxon signed-rank test was used in comparisons of repeated
measurements (comparisons of the follow-up samples) (III).
62
The correlations between two continuous variables were determined
using Spearman’s rank correlation (I). In Study IV, the general estimating
equations with exchangeable correlation matrix were used to detect
differences between the groups in variables with repeated measurements.
The Friedman test was used to find statistically significant differences, and
the Page test to find statistically significant trends in variables with ordered
categories with repeated measurements. In Study I, logistic regression
analysis was used to assess the prognostic value of independent predictors
(CD73, CRP, and creatinine) in predicting the development of SAP.
In Study I, the area under the receiver-operating characteristic (ROC)
curve (AUC), together with the corresponding sensitivity (y-axis) and
specificity (x-axis), was used to measure the predictive accuracy with
different threshold values. Clinically optimal cut-off values were chosen to
analyze the sensitivity and specificity of sCD73 activity and other markers in
predicting the development of SAP. Clinically optimal cut-off point of a ROC
curve is a point at which the slope R satisfies the equation R = C/Bx(1-P)/P,
where C/B is the ratio of the net cost of treating non-diseased individuals and
the net benefit of treating diseased individuals and P is the prevalence of the
disease. The prevalence of OF among patients with AP is approximately 10%,
but the C/B ratio in this scenario is not clear. Since ICU beds are limited, we
chose a cut-off point where the number of false positives is as low as possible
(specificity >90%) by selecting a threshold value at a point where the longest
increase in the sensitivity of the slope declines, as described elsewhere. 293
P-values < 0.05 were considered significant in all pair-wise comparisons.
In Study I with multiple comparisons, the Bonferroni correction was used in
order to avoid the effects of mass significance.
The statistical analyses were performed using the SPSS (SPSS Inc.,
Chicago, IL, USA) and the Stat Exact v4.0 software (I) (Cytel Software
Corporation, Cambridge, MA, USA).
!"$#"
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Medians of serum CD73 activity (320 nmol/ml/h, IQR 195-635) and protein
concentration (49.7 ng/ml, IQR 33.4-90.9) were significantly higher in
patients with AP than in healthy reference subjects (115 nmol/ml/h, IQR 139181, p<0.0001 and 33.2 ng/ml, IQR 23.1-38.9, p=0.0002). CD73 activity,
protein concentration, and mRNA levels were associated inversely with
severity of AP (Table 12).
63
PRESENT INVESTIGATION
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The medians of CD73 activity, protein concentration, and mRNA did not
differ significantly between the moderately severe and severe group (p for
CD73activity = 0.053, Mann-Whitney U-test, p for protein concentrations
0.538 and p for mRNA levels 0.086).
We examined whether the patients with transient OF differed from those
with persistent OF. Seventeen patients had MMS ≥ 2 on admission,
indicating OF (Figure 6). Five of the patients had transient OF, ultimately
belonging to the moderately severe AP group, and 12 persistent OF,
ultimately belonging to the severe AP group. sCD73 activity, protein
concentrations, and mRNA levels did not differ significantly between
transient and persistent OF groups (Table 13).
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The sCD73 activity on admission predicted the development of SAP
among different subgroups of the patients. AUC value for sCD73 activity was
0.79 (95% CI 0.69-0.88) among all patients (Fig. 8). AUC value for CD73
activity was 0.65 (95% CI 0.51-0.80) among a subgroup of patients
comprising moderately severe and severe disease (Fig. 9) and 0.75 (95% CI
0.60-0.89) among patients with no signs of OF (MMS< 2) on admission (Fig.
10). The AUC values for CRP and creatinine were lower than that for sCD73
activity in all subgroups; thus, sCD73 activity was better than CRP or
creatinine in predicting the severe form of AP.
64
20;8.
*646:F6B@A6B2D:?8492B24D6B:CD:4*(4EBF6C7@BAB65:4D:?8C6F6B624ED6
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20;8.
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A2?4B62D:D:C)2>@?8A2D:6?DCG:D9>@56B2D6=IC6F6B6@BC6F6B6)
65
PRESENT INVESTIGATION
20;8.
*646:F6B@A6B2D:?8492B24D6B:CD:4*(4EBF6C7@BAB65:4D:?8C6F6B624ED6
A2?4B62D:D:C)2>@?8A2D:6?DCG:D9>@56B2D6=IC6F6B6@BC6F6B6)
The clinically optimal cut-off values on the ROC curves were chosen to
analyze the sensitivity and specificity of sCD73 activity (and CRP and
creatinine) in predicting the development of SAP. Among patients with
moderately severe and severe AP, the sensitivity and specificity of sCD73
activity (cut-off 139 nmol/ml/h, sensitivity 0.40, specificity 0.828) were
comparable with those of CRP (cut-off 224 mg/ml, sensitivity 0.40,
specificity 0.724) and creatinine (cut-off 164 μmol/l, sensitivity 0.40,
specificity 0.862). The combination of sCD73 and CRP (sensitivity 0.60,
specificity 0.89) or sCD73 and creatinine (sensitivity 0.60, specificity 0.90)
improved the sensitivity. The clinically optimal cut-off values obtained from
the whole population or from the patients with moderately severe and severe
AP were almost identical. Logistic regression analyses showed that sCD73
activity was an independent predictor of SAP among all patients (cut-off 138,
OR 6.578, 95% CI 1.834-23.592). sCD73 activity showed no correlation with
creatinine (R=0.015, p=0.913), but a negative correlation with CRP on
admission (R=-0.291, p=0.033). Among the patients with MMS<2 on
admission, the sCD73 activity (cut-off 139, sensitivity 0.308, specificity
0.939) had a better combination of sensitivity and specificity in predicting
SAP than did CRP (cut-off 224, sensitivity 0.077, specificity 0.954) or
creatinine (cut-off 164, sensitivity 0.077, specificity 1.00). Logistic regression
analyses showed that sCD73 (cut-off 139, OR 6.639, 95% CI 1.245-35.409)
66
(in contrast to CRP, cut-off 224, OR 0.763, 95% CI 0.066-8.802) was an
independent predictor of SAP among patients with MMS<2.
Thus, among all groups (all AP patients, patients with moderately severe
and severe AP on admission and patients with no signs of OF on admission)
sCD73 activity is better in predicting SAP than CRP or creatinine.
#%# !# #!##"%!$#
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The efficacy of APC treatment was evaluated by analyzing its effects on OF.
We analyzed changes in SOFA scores (primary endpoint), ventilator-free
days, renal replacement therapy-free days, and vasopressor-free days. No
significant difference in SOFA score changes between the APC (from 6.5, SD
± 4.0, range 2 -14, to 8.2,SD ± 5.0, range 0-16) and placebo groups (from 6.3,
SD ± 3.1, range 3-11) to 5.7 (SD ± 4.6, range 0-16) was found (DIM 2.3, 95%
CI -0.7 to +5.3). The median hospital stay was 23 days (range 2 - 40 days),
and the median duration of ICU stay 11 days (range 0 - 43 days). Altogether
21/32 patients (66%) needed mechanical ventilation, 12/32 (38%) needed
dialysis, and 20/32 (63%) needed vasopressor treatment during their stay at
the ICU. No differences in ventilator-free days, in renal replacement therapyfree days, or in vasopressor-free days were detected between the groups.
Safety of APC treatment in clinical use was also evaluated. No serious
bleedings were detected clinically or by CT scans in either the APC group or
the placebo group (incidence 0%, 95% CI 0 to 18%, DIM 95% CI -17 to +17%).
Minor bleeding (from mouth, nose, or urinary tract) occurred in four patients
in the APC group and in two patients in the placebo group (p=not significant,
NS). The administered red blood cell units during the APC infusion did not
differ between the groups (seven in APC vs. eight in placebo group, p=NS).
No significant differences in plasma hemoglobin levels or platelet counts
were found. We evaluated the effects of APC treatment on liver function tests
(serum bilirubin, serum pre-albumin, serum disialotransferrine, serum
gamma-glutamyl-transferase) and serum creatinine levels. Administration of
APC was associated with an increase in serum bilirubin (DIM 28.4 mmol/L,
95% CI 3.6 to 53.1), and serum conjugated bilirubin (DIM 25 mmol/L, 5.6 to
44.4). No significant differences in other liver function tests or creatinine
levels were found. The 30-day mortality in the APC group was 3/16
compared with 0/16 in the placebo group (absolute risk reduction, ARR 19%, 95% CIs -38% to 0). The non-survivors had MOF (SOFA scores 10, 13,
and 14), which was also the autopsy-confirmed cause of all deaths (on days 4,
11, and 14). No significant differences in days alive outside the hospital were
detected (DIM -17.4, 95% CI-0.1 to -34.9).
67
PRESENT INVESTIGATION
Thus, no differences in the evolution of MOF between APC and placebo
groups were found.
*)&*(*$%*&%*&+()&)/)*$ %#$$* &% %
),(+*'%(* * )*+/
Characteristics of the patients are given in the Materials and Methods section
and in Table 8 and the numbers of the patients at different time-points in
Table 9. Study III was a sub-study of Study II. The effects of APC treatment
on immunologic parameters in patients with SAP were investigated.
The inflammatory response was detected by analyzing the changes in
plasma concentrations of proinflammatory IL-8. IL-8 levels of all patients
decreased during the first five days after admission to hospital (at day 0: 264
pg/ml vs. at day 5: 110 pg/ml, p=0.001). The APC had no significant effect on
the changes in IL-8 concentrations during the 14-day follow-up. Plasma
concentrations of pro-/anti-inflammatory cytokine IL-6 and antiinflammatory cytokines IL-10 and IL-1Ra decreased during the first five days
(IL-6 at day 0: 670 pg/ml vs. at day 5: 215 pg/ml, p=0.001; IL-10 at day 0:
12.7 pg/ml vs. at day 5: 11.3 pg/ml, p=0.001; IL-1Ra at day 0: 2890 pg/ml vs.
at day 5: 1250 pg/ml, p=0.007) in both groups. The APC treatment did not
have any significant effect on the anti-inflammatory response (changes in IL6, IL-10, and IL-1Ra concentrations) during the follow-up. Endothelial
response in terms of plasma concentrations of soluble E-selectin decreased
over the course of the disease (at day 0: 45.5 ng/ml vs. at day 5: 38.2 ng/ml,
p=0.031) in both groups, and the APC treatment did not have any effect on
changes. Serum concentrations of PCT, a marker of systemic inflammation,
showed no altered concentrations during the first five days (at day 0: 0.97
ng/ml vs. at day 5: 0.66 ng/ml, p=0.487), and administration of APC had no
effects on the changes in PCT concentrations.
As a marker of immune suppression, monocyte HLA-DR expression was
not altered significantly during the first five days of the follow-up period (at
day 0: 54% vs. at day 5: 58%, p=0.316) in either group, and the APC had no
effect on the HLA-DR expression. The cell surface expression levels of CD11b,
CD14, and CD62L were measured as markers of monocyte activation. The
monocyte surface expression of CD11b, CD14, and CD62L was downregulated
during the first five days (CD11b at day 0: 291 RFU vs. at day 5: 200 RFU,
p=0.001; CD14 at day 0: 168 RFU vs at day 5: 150 RFU, p=0.028; CD62L at
day 0: 217 RFU vs. at day 5: 135 RFU, p= 0.001) in both groups, and the APC
treatment had no effect on the expression levels. Neutrophil activation was
evaluated by analyzing the expressions of CD11b and CD62L: both were
downregulated during the first five days of follow-up (CD11b at day 0: 325
RFU vs. at day 5: 259 RFU, p=0.001; CD62L at day 0: 146 RFU vs. at day 5:
81 RFU, p=0.001), and the APC treatment had no effects on expression
levels.
68
&+#* &%*)& %),(+*'%(* * )*+/
Coagulation markers In the placebo group, the rate of change in PT was
negative (p<0.001), indicating decreasing temporal levels, whereas in the
APC group such a decrease was not detected (p=0.997). The rate of change
was different between the groups (p<0.001). The temporal levels of TT
increased in the placebo group (p<0.001 for slope), but not in the APC group
(p=0.521). The rate of change between the groups differed (p<0.001). As to
D-dimer levels, the rates of change in the two groups showed increasing
temporal sequence (p<0.001 and p<0.039, respectively) and differed
between the groups (p=0.019). The temporal levels of platelet count in
placebo and ACP groups were increasing during the follow-up (both
p<0.001); the rates of increase were comparable (p=0.609). The rates of
change in fibrinogen levels were positive in the placebo and APC groups
(both p<0.001), indicating increasing temporal values. The rates of change
were comparable between the groups (p=0.148) (Study IV: Figure 1, Panel
A).
In the placebo group, there were significant changes in DIC scores
between the measurement days (p=0.008), but the trend was not significant
(p=0.064, Page test). In the APC group, the changes in DIC scores between
the different measurement days were not significant (p=0.505, Friedman
test) (Study IV: Table III).
Physiological anticoagulation markers As to PC levels, the rates of
change were positive in the placebo and APC groups (p<0.001 and p=0.027,
respectively), indicating increasing temporal values. They were comparable
between the groups (p=0.067). As to AT levels, the rates of change were
positive (p<0.001 for both groups) and differed between the groups
(p=0.0111). The alterations of TFPI free and TFPI total levels or TFPI free
/TFPI total ratios were not significant (Study IV: Figure 1, Panel B and Table
II, Panel B).
Inflammation markers The rates of change in IL-6 levels and in CRP
levels were negative in the placebo and APC groups (Study IV: Figure 1, Panel
C and Table II, Panel C).
Thrombograms The shapes of the thrombograms were deranged in the
patients with SAP relative to the healthy reference subjects. Of the patients,
10/20 had no detectable thrombin response, and their thrombogram graphs
showed straight lines (“flat curve”) at time-point 0. At days 1 and 3-4, nine of
the patients (5 in the APC group and 4 in the placebo group) had detectable
thrombin generation. The patients with a “flat curve” were excluded from
further analyses of the thrombograms. The rates of change in lag time and
time to peak values of the placebo group were positive (p=0.013 and
p=0.007, respectively). The rate of change in ETP values was positive in the
APC group (all p-values 0.05) (Study IV: Figure 1, Panel D, Table II, Panel
D).
69
PRESENT INVESTIGATION
"$""
!#"%!$# !##"
The spectrum of severity of AP ranges from a mild, self-limiting disease to a
highly fatal SAP with MOF. Of AP cases, 75-80% remain mild3, 4 and require
only supportive care (i.e. intravenous fluid replacement and pain
management). Overall mortality of AP is still high (5%), reaching 30-40% in
cases with infected necrosis or those with MOF.3-7 Mortality in AP manifests
in two peaks; nearly half of the deaths occur within the first week due to
overwhelming SIRS, and septic complications account for the deaths in the
late phase.4
Severity of AP can fluctuate rapidly, and predicting the course of AP is
difficult. The patients who will develop SAP need to be identified early in the
course of disease and aggressively treated to prevent mortality. On the other
hand, proper identification of mild cases of AP is necessary to avoid
overtreatment and to reduce financial implications, i.e. high ICU costs. As
mentioned earlier (Section 2.7.2), many kinds of multifactorial scoring
systems have evolved to assess the severity of AP. The scoring systems have
several limitations, e.g. Ranson score takes 48 hours to complete and
APACHE II is cumbersome in clinical use.294 It is difficult to find a single
biochemical parameter that could predict the severity of AP early enough in
the course of the disease; if the inflammatory reaction has already started, it
is difficult to terminate. For example, the widely used CRP has a delayed
peak (48-72 hours),294 and it is a non-specific inflammatory marker in AP
(i.e. cholangitis and pneumonia increase CRP levels). An ideal predictor
should be easy to obtain, widely available, cost-effective, associated with high
sensitivity/specificity, have high positive and/or negative predictive value,
and be able to predict the severity of AP as early as possible (within the first
24-48 hours of symptom onset).
Our main finding in Study I was that the circulating levels of sCD73 are
significantly higher in patients with AP than in healthy control subjects, and
the sCD73 levels on admission to hospital correlate inversely with the
severity of AP. Even more importantly, the sCD73 activity predicted the
development of SAP among all patients and also among patients without
signs of OF on admission (MMS<2). Thus, AP patients with increased sCD73
on admission more often had the mild disease than patients with less
strongly elevated sCD73 levels on admission. sCD73 activity has better
predictive accuracy than CRP and creatinine levels, and the combination of
CD73 activity and CRP showed improved predictive performance. sCD73
levels can be measured by using a single blood test, performed within 7
hours, with reagent costs of about 3 euros per sample.
At present, the mediators for sCD73 induction and the cell type from
which sCD73 levels derive in AP remain uncertain. Tissue hypoxia, a
70
common feature in patients with SAP, is known to increase the expression of
CD73.112, 295 Also cytokines, such as IFN-α and -β, promote CD73
expression.111 Soluble CD73 is mainly derived by shedding of lymphocytes
rather than endothelial CD73, and our study showed decreased CD73 mRNA
synthesis in leukocytes of SAP patients.107. An intriguing question is whether
the patients with SAP are low responders in terms of CD73
induction/shedding, with their sCD73 levels therefore being lower than those
of the patients with mild AP. Genetic deficiency of CD73 has been shown to
cause arterial and periarticular calcification phenotypes296, but the possibility
of involvement of polymorphisms of the CD73 gene in AP is not known and
requires further studies.
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Several pre-clinical studies have shown that APC has potentially beneficial
effects on inflammation.19, 162, 184, 185, 187, 297 The endogenous protein C levels
have been shown to correlate with positive outcome in severe sepsis,298 and
the recombinant APC administration has a protective effect in animal models
in Escherichia coli-induced sepsis.299 The precise mechanism of the
cytoprotection is unknown, but possible effects of APC administration may
include attenuation of proinflammatory cytokine storm, re-balancing
dysregulated coagulation, or degradation of cytotoxic extracellular
histones.300, 301
The PROWESS trial (Protein C Worldwide Evaluation in Severe Sepsis)
showed beneficial effects of recombinant APC (drotrecogin alpha, Xigris®)
on mortality in sepsis patients (included 1690 patients with severe sepsis, 28day mortality rate of 25% in patients treated with recombinant APC
compared with 31% in those treated with placebo, 6.1% reduction in
mortality).29 Drotrecogin alpha was approved for use as a pharmaceutical
therapy for severe sepsis by decision of the Food and Drug Administration in
2001 in the United States.
Sepsis and SAP share similarities in systemic inflammatory reactions and
coagulopathy, although both of these diseases also have their own unique
characteristics.28 Therapeutic use of APC in SAP was a potential idea, and the
thought was supported by an earlier trial of our study group showing that
patients with SAP have low PC levels and high endogenous APC to PC
ratios.302 Also in experimental animal pancreatitis models, APC has been
shown to have beneficial effects; i.e. APC improved the severity of pancreatic
tissue histology, superinfection rates, and serum markers of inflammation in
acute necrotizing AP.303-305
71
PRESENT INVESTIGATION
Study II was a prospective randomized double-blind trial of APC in 32
patients with SAP. In this study, we found no difference in the evolution of
MOF (the change in SOFA between day 0 and day 5) or in OF-free days
(ventilator-free-days, renal replacement-free-days, vasopressor-free days) or
days alive outside hospital in 60 days between APC-treated patients and
those receiving placebo. We found no serious bleeding in either group.
An interesting question is had a higher dose of APC been used or the
duration of infusion extended, would the APC have had a more obvious effect
on SAP. The therapeutic use of an anticoagulant like APC in SAP is a
balancing act between the beneficial effects on inflammation and the
increased risk of bleeding. The anticoagulant nature of APC may enhance the
SAP patients’ already high risk of bleeding, and therefore, increasing the dose
is challenging. Clinical trials, done after the PROWESS study, used the same
standard protocol in administration of recombinant APC as suggested by
Bernard et al. (24 μg/kg/h for 96 hours).29 Effectiveness of APC has varied,
but the risk of bleeding complications is documented.306, 307
A randomized double-blind placebo-controlled multi-center trial
published in 2012 (the PROWESS SHOCK trial, required by the European
Medicines Agency) found no evidence suggesting that APC treatment reduces
the risk of death in severe sepsis.307 The pharmaceutical form of APC
(drotrecogin alfa, Xigris®) was withdrawn from the pharmaceutical market
in 2011.
Sepsis studies of APC treatment are interesting examples of numerous
possibilities of biases and errors in randomized clinical trials (RCTs) and
their interpretations. In addition, these studies raise ethical questions related
to RCTs. The PROWESS study was stopped early for benefit. The Data and
Safety Monitoring Board, an independent agency that mainly consists of
biostatisticians, clinicians, basic scientists, and bioethicists, is required to
minimize conflicts of interest in many large Phase 3 trials.308 The Data and
Safety Monitoring Board of the PROWESS study made the decision to
terminate the trial at the second interim analysis because of the significant
mortality benefit detected.309 It has been shown that trials stopped early for
benefit (so-called truncated RCTs for benefit) may overestimate treatment
effects.309 and may miss important adverse drug reactions (if they became
underpowered).310 It is possible that the PROWESS study had
methodological faults and interpretation of the trial was overly optimistic.
On the other hand, the withdrawal of drotrecogin alfa has attracted
criticism. Kalil et al. questioned the big differences in resulting mortality of
the PROWESS and PROWESS SHOCK trials, which were carried out by the
same manufacturer of medicine, the same drotrecogin alfa dose, and the
same placebo-controlled design.311 In the PROWESS study, the decrease in
mortality was 6.1%, and in the PROWESS SHOCK study no survival benefits
were found.191, 307 Kalil et al. performed an analysis of the clinical and
statistical heterogeneity between the trials. They noted that clinical variables
(i.e. baseline characteristics and co-interventions) presented significant
72
heterogeneity and the PROWESS SHOCK study was underpowered, and they
concluded that these trials are not comparable.311 Kalil et al. also published in
2012 a meta-analysis of drotrecogin alfa treatment in sepsis (over 40 000
patients) and concluded that APC treatment was associated with a significant
reduction in hospital mortality (18%) and increased rates of bleeding (5.6%)
in patients with severe sepsis.312
After several trials and interpretations, open questions remain concerning
the benefit of APC treatment in severe sepsis.
*)&* ,*'(&* %*(*$%*&%*&+()&)/)*$ %#$$* &% %),(+*'%(* * )
Originally, the APC treatment was proposed mentioned to be a promising
target for the treatment of sepsis based largely on its anticoagulant
properties. Later, mounting evidence with other anticoagulants (AT,
recombinant TFPI) failed to show a benefit in severe sepsis,313, 314 resulting in
the suggestion that the role of APC as an anticoagulant does not explain its
benefits in systemic inflammation.
APC has been demonstrated to have direct cytoprotective effects such as
anti-inflammatory, anti-apoptotic, and endothelial barrier protection
functions. Anti-inflammatory activity of APC is mediated by its interaction
with endothelial cells and leukocytes.184
Several in vitro and animal studies have revealed that APC has antiinflammatory effects. APC has been shown to inhibit leukocyte infiltration.
Administration of APC inhibits NF-κB activation on cultured endothelial
cells, resulting in suppressed expression of adhesion molecules (i.e. ICAM-1,
E-selectin, and VCAM-1) and consequent decreased leukocyte trafficking.19,
189 APC also reduces expression of proinflammatory mediators from
endothelial cells (i.e. IL-6, IL-8, and MCP-1).315 Administration of APC has
been shown to downregulate the expression of inflammatory cytokines and
chemokines by leukocytes.181 APC has direct effects on leukocytes, and the
effect is mainly mediated by EPCR. EPCR receptors are located on the
surface of monocytes, neutrophils, and eosinophils.316, 317 APC directly
inhibits chemotaxis of neutrophils by its association with EPCR.189, 318 APC
has been demonstrated to reduce production of endotoxemia-induced
proinflammatory cytokines (IL-6, IL-8, IL-1β, and TNFα) by monocytes.185
APC inhibits TNFα production by blocking NF-κB transcription factor in
monocytes.186 APC also inhibits cytokine production from Th2
lymphocytes.187 APC upregulates anti-inflammatory mediators, e.g. IL-10 on
blood monocytes.188
No previous clinical trials have scrutinized the effects of APC in
inflammation markers in SAP. In the PROWESS study, they found decreases
in IL-6 levels in sepsis patients’ plasma and that has been taken as evidence
of an anti-inflammatory action of APC.191 HLA-DR expression has been
shown to correlate with PC and APC levels in SAP.302
73
PRESENT INVESTIGATION
In Study III, we examined effects of APC on systemic inflammation
response. We analyzed the effect of APC on soluble and cellular markers
during a two-week follow-up and found no significant differences between
the groups. The result showed that recombinant APC treatment of patients
with SAP did not alter the course of systemic inflammation, in accordance
with the clinical findings in Study II.
Recent studies on inflammation effects of APC have revealed that the
anti-inflammatory effects are mainly mediated by cell signaling. APC triggers
an array of signaling pathways via distinct receptor interactions on different
cell types. For example, binding the APC to EPCR facilitates activation of
PAR1 and PAR3.27, 319 APC-mediated PAR1 signaling is distinct from the
traditional G-protein-coupled signaling (thrombin uses PAR1 receptor to
mediate proinflammatory effects) 320, PAR1 mediates APC’s endothelialbarrier protection and anti-apoptotic and anti-inflammatory activities.321 The
enhanced knowledge of APC’s cytoprotective mechanisms is a step forward in
fully utilizing APC or its variants in clinical use.
&+#* &%*)&* ,*'(&* %*(*$%* %),(+*
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The hypothesis in Study IV was that APC treatment should alleviate
consumptive coagulopathy and increased fibrinolysis in SAP. Our results
showed that recombinant APC standard protocol treatment did not have an
alleviating effect on coagulopathy; instead APC treatment interfered with
normalization of coagulopathy in SAP.
TT (thromboplastin time) monitors the interaction of FII, FVII, and FX,
and TT decreases in the DIC.322 In our study, TT levels in the placebo group
showed a trend towards normal in the placebo group, but not in the APC
group. PT (prothrombin time) depicts the extrinsic pathway of coagulation. It
monitors the function of fibrinogen, thrombin, FV, FVI, and FX.322 PT levels
showed the same phenomenon as TT levels in our study.
Our results indicated that SAP patients’ platelet counts were reduced, and
an increasing trend was observed in both groups (APC treatment showed no
effect on the trend). Consumptive coagulopathy and increased fibrinolysis
are known to occur in SAP, and they are related to its severity and OF.18
Fibrin D-dimers are degradation products of fibrin, and they are released as a
result of fibrinolysis to the circulation. D-dimer levels increase when
coagulation and fibrinolysis are accelerated, i.e. in severe inflammatory
reactions and DIC.323 D-dimer levels depend upon both thrombin formation
and fibrinolytic activation. In this study, the D-dimer levels showed an
increasing trend in both groups, and the rate of change was greater in the
placebo group (reflecting that coagulation and fibrinolysis remained
accelerated in the placebo group relative to the APC group).
Fibrinogen levels increase within the acute phase reactions and decrease
in DIC and when liver function is decompensated.210, 324 In this study,
74
fibrinogen levels were elevated in both groups throughout the follow-up, and
the fibrinogen levels increased as pancreatitis progressed. This may indicate
that acute phase reactions predominate over consumption of fibrinogen in
SAP.
Protein C (PC) is the inactive precursor of APC. PC is a K-vitamindependent inhibitor of coagulation, and PC levels decrease in DIC.325
Decreased PC levels are associated with the severity of AP.326 In our study,
PC levels of SAP patients were low and showed an ascending trend; the
administration of APC had no effect on the trend.
Antithrombin (AT) is an anticoagulant that forms an inactive complex
with thrombin. AT levels have been shown to decrease in DIC. Secondary AT
deficiency is associated with OF in SAP patients.327 In this study, SAP
patients showed secondary AT deficiency, and in the APC group
normalization of the levels was delayed.
TFPI (tissue factor pathway inhibitor) is a specific inhibitor of TF, and it is
released in endothelial cells as a result of thrombin generation.328 TFPI
occurs in plasma as a lipoprotein-bound form (ca. 80%) and as a free form
(5-20%).329, 330 Free TFPI has a more intense anticoagulant effect than the
bound form.329 Elevated TFPI levels have been detected in plasma of AP
patients.327 Our earlier study showed a rising trend in free TFPI and total
TFPI levels and in ratios of free/total TFPI associated with severity of AP. 327
This study did not find a significant trend in TFPI levels in either group.
Thrombograms probe exogenous TF-triggered thrombin generation in
plasma samples and can be used to diagnose hyper- and hypocoaguable
states of patients.291 Our earlier study showed, for the first time, that the
thrombin generation of AP patients was highly variable and the shapes of
thrombograms were disturbed. Elevated free TFPI levels of SAP patients
appear to inhibit exogenous TF in CAT measurements. The conclusion of the
study was that failure of TF-initiated thrombin generation in the
thrombogram assay can be explained by high levels of circulating free TFPI
that may be associated with OF and mortality in AP.327 In the recent study, all
of the patients had a severe form of AP and their TF-triggered thrombin
generation was generally disturbed and shapes of the thrombograms were
abnormal. We found no association between patients with “flat curves” (no
detectable thrombin response) and mortality, nor did we observe any
significant differences in levels of TFPI between patients with “flat curves”
and the others. SAP itself had more powerful effect on thrombograms than
APC, and in vivo standard dose of APC had only minor effects on
thrombograms. A possible explanation for the differences between these
findings and those of the previous study is that the samples of this study were
collected later, when patients already had severe pancreatitis, not on
admission to hospital.
To our knowledge, there are no previous clinical studies on the effects of
APC on coagulation parameters in SAP. Dhainaut et al. investigated host
coagulopathy responses of APC in sepsis patients (n=1690) in the PROWESS
75
PRESENT INVESTIGATION
study. They showed that APTT and PT were prolonged in the APC-treated
sepsis patients compared with placebo-treated patients, and the sepsis
patients D-dimer levels were decreased during the APC infusion time.331 Our
study revealed a similar phenomenon; TT normalization was delayed and Ddimer stayed lowered in the APC group. Dhainaut’s study showed that the AT
levels were significantly higher in the APC group than in the placebo
group.331 In our patients, AT levels remained depressed.
It is difficult to know exactly which state of coagulation (hypo- or
hypercoagulation) is favorable for SAP patients at different time-points. SAP
patients may suffer from DIC and microthrombosis, and during this phase
strengthening of anticoagulant mechanisms may be beneficial. On the other
hand, the consumptive coagulopathy and increased fibrinolysis may lead to
hemorrhagic complications, and these patients may then benefit from
amplification of coagulation mechanisms. Increasing knowledge of
coagulopathy in SAP helps clinicians to target anticoagulant treatment to the
patients who need it.
$#$!" #"
The most important aspect in predicting the course of a potentially severe
disease is in clinical work, i.e.the ability of the predictor to predict severity of
the disease so early that clinicians have the possibility to influence the course
of the disease by the treatment methods available. According to the revised
Atlanta criteria, mild AP pertains to patients with MMS<2 on admission. In
addition, this is the group (population of AP patients) to which prediction
should be directed. Prediction is not important in the group that already has
clinical complications of disease, i.e. OF. The patients with OF should be
admitted to the intensive care unit, and intensive care is terminated if OF
proves to be transient (does not matter if the correction of the clinical state is
due to a milder course of AP or due to the treatment in ICU). We were the
first to take this approach in predicting SAP, and our results showed that
sCD73 is the kind of marker that can predict the severity of AP among
patients with no signs of OF on admission (MMS<2). Our group has also
investigated with the same principle the predictability of cytokines and
nucleosomes in SAP.332 Future studies on predictors should have this
approach so that clinicians can gain useful tools in clinical practice.
CD73 may have therapeutic potential in SAP. CD73 yields antiinflammatory adenosine333, which mediates anti-inflammatory effects such
as the prevention of vascular permeability and the inhibition of leukocyte
recruitment to the site of inflammation.105, 334 These protective mechanisms
are enhanced in the presence of hypoxia.335 INF-β has been shown to induce
sCD73 and alleviate acute lung injury following ischemia-reperfusion in a
mouse model.111 CD73/adenosine has been established to decrease vascular
permeability in cultured human pulmonary endothelial cells. 111 A recently
76
published preliminary clinical study showed that administration of INF-β
upregulates human lung CD73 expression and is associated with reduction in
mortality in patients with ARDS .336 In addition to vascular permeability,
CD73 may modify epithelial permeability in the gut and improve the bowel
barrier function.112, 337 In patients with SAP, increased vascular permeability
and tissue edema and hypoxia are universal phenomena.338, 339 Impaired
bowel barrier function leads to infection complications in SAP.144 At this
stage, depressing systemic inflammation and improving barrier functions
without increasing the risk of secondary infections are attractive goals. Our
study showed that low levels of CD73 are associated with the development of
vital organ dysfunction in SAP. The possibility that patients with SAP have a
shortage of sCD73 and would therefore benefit from administration of
exogenous sCD73 or induction of sCD73 by INF-β warrants further research.
Despite the failure of recombinant APC (drotrecogin alfa, Xigris®), the
cytoprotective functions of APC give a reason to continue searching for new
therapeutic applications. Recombinant APC has been shown to be beneficial
in numerous animal models of diseases in addition to sepsis and AP, i.e.
inflammatory bowel disease340, motor-neuron degeneration341, and ischemic
stroke.342 Genetically altered animal models with the separated anticoagulant
and cytoprotective activities of endogenous APC have been developed and
their use has helped to identify APC’s dual activity. These models had a major
role in clarifying APC’s cytoprotective effects.343, 344 Anticoagulant function of
APC is not required for the protective effects on endotoxemia or stroke.342, 344
Several kinds of APC variants have been generated by targeted mutagenesis.
These novel approaches to engineer APC eliminate either its anticoagulant or
cell signaling activity.345 For example, a manipulation of the APC serine
protease domain via the introduction of a new disulfide bridge inhibits
anticoagulation activity of APC.346 APC variants that have limited
anticoagulant function but normal cytoprotective activity represent a
potentially safer alternative. These variants may enable increasing dosage of
APC with reduced risk of serious bleeding complications. APC has a short
half-life in the circulation (15-20 min) and may require a longer infusion
time or repeated boluses for full efficacy, thus, another approach with new
APC variants is extending plasma half-life of recombinant APC.345
Despite early difficulties with therapeutic use of recombinant APC, it is
not time to bury it. Protein engineering can provide second-generation APC
mutants, which may have a safer and more effective impact, with a reduced
bleeding risk.
Recent research has extended knowledge of pathogenic mechanisms
involved in inflammation. An interesting aspect is related to elucidated
pathways of cell death, especially the role of necroptosis in the inflammatory
reaction. Traditionally, necrosis is considered to be the primary form of cell
death caused by inflammation, but recent studies have demonstrated the
existence of multiple pathways of regulated necrosis, i.e. necroptosis.
Necroptosis is a pathway of regulated necrosis, a programmed form of
77
PRESENT INVESTIGATION
necrosis, that is mediated through a pathway dependent on receptorinteracting protein kinase 3 and mixed lineage kinase domain-like protein.347
Necroptosis has characteristics of both necrosis and apoptosis, i.e.
necroptosis is actively regulated by multiple genes and accomplished through
activations of a specific death-signaling pathway. Recent studies have
confirmed the existence of acinar cell necroptosis in AP.348 The discovery of
necroptosis may provide a potential target for regulating the inflammatory
reactions in AP. In the future, it may be possible to switch the acinar cell
necrosis to necroptosis, which may block the overactive systemic response
leading to SIRS. An interesting future objective may be determining whether
the early vigorous ICU treatment favors the evolution of necroptosis instead
of necrosis, resulting in transient OF instead of persistent OF.
"#!#"##"#"#$(
Some strengths and limitations of the Studies I-IV are noteworthy.
The approach of the sCD73 study has clear clinical relevance; the
prediction of SAP is meaningful among patients who have no signs of OF on
admission. The clinical APC study (Study II) was a pilot double-blinded trial
and the concomitant treatment did not change during the study period.
Patients not included in the study were registered according to the
CONSORT guidelines.349 The patients in the placebo group received the best
available standard treatment, thus avoiding potential bias caused by undertreatment of this group. The mortality in the placebo group was < 20%,
which is within the range of the best available practice. The same standard
protocol in administration of recombinant APC was used as in the PROWESS
study, as assessed by Bernard et al. (24 μg/kg/h for 96 hours).29 Sub-studies
of the clinical APC study (Studies III and IV) provide new information on the
complicated pathophysiological inflammatory reaction and coagulopathy in
patients with SAP.
The studies also have some limitations. The severity of AP was classified
according to the original Atlanta classification (Studies II-IV), which was
later revised. In addition, categorizing patients to the different severity
groups involves some difficulties owing to the dynamic nature of AP. In the
clinical APC study (Study II), the disease of some of the patients turned out
to be milder than originally thought (thrombocytopenia resulted in
classification into the SAP group, but no signs of OF manifested). The study
groups were not totally comparable after randomization; patients in the APC
group had higher plasma amylase levels. The study patients were not divided
into subgroups according to the etiology; however, the etiology of AP was
alcohol in all patients in the APC group, and in the placebo group the etiology
was alcohol in all but one patient. Some of the patients had high SOFA scores
on admission, and thus, it is possible that patients with SAP will present with
different phases and severity of the disease at ICU admission (at the time of
78
randomization). In general, ICU patients are a very heterogeneous group
(chronic illnesses, medications, etc.), and this should be taken into account
when interpreting the results. It is also possible that the dose of APC was
insufficient to have an effect on the excessive inflammatory reaction involved
in SAP. In a pilot study such as Study II, it is safer to use the same dose as in
previous sepsis trials. Sample size was determined according to SOFA
change. This enabled us to find a difference in incidences of serious bleeding
exceeding 17%; we were unable to exclude possible smaller differences in
incidences of bleeding.
Because of the preliminary nature of Study I, the expected levels of sCD73
in different severity groups were unknown, and thus, proper power analyses
and sample size calculations could not be performed.
The sample sizes for Studies III and IV were determined according to a
primary endpoint (Study I) and not according to systemic inflammatory
response or changes in coagulation markers between the groups. In addition,
some follow-up samples were missing in Studies III and IV. Therefore, it is
not possible to exclude type II error in these studies (Studies III and IV).
79
PRESENT INVESTIGATION
$""
Based on these studies, the following conclusions may be drawn:
I.
CD73 levels are increased in patients with AP and correlate negatively
with the severity of AP. sCD73 activity predicts the development of
SAP among patients with no signs of OF on admission (MMS<2).
II.
APC treatment had no effect on evolution of OF among patients with
SAP. We found no differences in ventilator-free days, renal
replacement-free days, vasopressor-free days, or days alive outside the
hospital over a 60-day period between the study groups. No serious
incidents of bleedings occurred in SAP patients receiving APC.
III.
APC treatment did not alter the course of systemic inflammation, as
determined using soluble and cellular markers of inflammation.
IV.
Patients with SAP receiving APC reached normal homeostasis of
coagulation slower than patients receiving placebo.
80
&#"
This study was carried out at the Department of Gastrointestinal Surgery of
the Abdominal Center at Helsinki University Hospital and University of
Helsinki during 2007-2016.
My sincere gratitude and respect are owed to the former and current
academic heads of our department, Professor Eero Kivilaakso and Professor
Pauli Puolakkainen, for creating an inspiring academic environment and for
the opportunity to carry out this study. They have created a valuable tradition
for medical research, especially in the field of pancreatitis.
I am very grateful to my supervisor Docent Leena Kylänpää for guiding
me in the world of research. I greatly admire her clinical and scientific
expertise. Without her support and practical advice, I would not have
reached the desired goal.
I am deeply indebted to my other supervisor Docent Heikki Repo for his
generous support and warm and encouraging attitude. His enthusiasm for
scientific research is contagious, and I hold his analytical thinking and
writing skills in high esteem. I am grateful for the inspiring discussions that
we had during the revision phase. His positive thinking carried me through
the darker days of this work.
I sincerely thank the official reviewers of this thesis, Docent Juhani Sand
and Docent Elina Armstrong, for valuable advice and constructive comments
for improving this manuscript. I also warmly thank Professor Juha Grönroos
for agreeing to be my opponent.
I am indebted to my superiors Esko Kemppainen, Jukka Sirén, and Leena
Halme for kindly supporting my work on this thesis. I am grateful for the
opportunity to step away from clinical work in order to complete this
dissertation.
All of my coauthors are thanked for their collaboration and kind help
during the course of these studies. I am especially grateful to Outi Lindström
for helping me in numerous ways with practical issues and for reviewing my
manuscripts. I warmly thank Anne Penttilä for collaborating in collecting
patient data. I am deeply indebted to Panu Mentula and Harri Mustonen for
their patient help with the statistical analyses. I also owe a special thanks to
Marko Salmi for collaborating in the interesting process with CD73, Ville
Pettilä for the opportunity to take part in the clinical study of APC and for
valuable revision of APC studies, and Jari Petäjä for professional and
generous help with hematological issues. I am grateful to the staff of the
Hematology Laboratory of Helsinki University Hospital and the Department
of Bacterilogy and Immunology, Haartman Institute, University of Helsinki,
for their collaboration, especially Eine Vironen and Kaisa Kuukka.
Carol Ann Pelli is acknowledged for precise and enthusiastic editing of the
language of this thesis.
81
acknowledgments
I thank all of my friends and colleagues at the Department of
Gastrointestinal Surgery at Helsinki University Hospital for providing a
pleasant working atmosphere. I am especially grateful to colleagues and
nurses at the PÄIKI ward of the Surgical Hospital for companionship during
the last few years.
My friends, I thank for help and support over the years and for reminding
me that there is life outside work and science. Special thanks go to my longtime friends from Nivala, Merja and Sari, for being there for me for decades.
I also thank “my first real colleague” Katja for joyful moments and shared
experiences over many years. My heartfelt thanks go to my friends from
medical school, the ”Lehmät” group: Hanna, Katriina, Mari, and Tuomo, for
their friendship and the shared ups and downs of our clinical and private
lives.
I owe my deepest thanks to my parents Marja and Erkki for their
unconditional love and support and for always believing in me. I am grateful
to my sister Hanna for unstinting support and friendship throughout my life.
I also thank my dear goddaughter Saga for bringing joy to our lives and for
being such a wonderful cousin to Hilla. I thank my brother Kari and his
family for their support. I am grateful to my mother-in-law Pirjo for helping
us in numerous ways in our everyday lives.
Most of all, I thank my family: Markus and our daughter Hilla. Markus,
your endless love and understanding made this possible. Thank you for
sharing your life with me. Hilla, you are the most important person to me,
filling my life with purpose and joy. I love you both!
Financial support from the Professor Martti I. Turunen Fund, the Mary
and Georg Ehrnrooth Fund, the Finnish Association of Gastroenterology, and
the Helsinki University Central Hospital Research Funds (EVO) is gratefully
acknowledged.
Helsinki, June 2016
Lea
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