BIOMARKER INVESTIGATIONS IN ACUTE BRAIN INJURY. Tihamér Molnár M.D.

BIOMARKER INVESTIGATIONS IN ACUTE BRAIN INJURY Tihamér Molnár M.D. PhD Thesis Department of Anesthesiology and Intensive Therapy University of Pécs, Hungary Leader of the project: Assist. Prof. Zsolt Illés M.D., Ph.D. Leader of program: Prof. Sámuel Komoly M.D., DSc. Leader of Doctoral School: Prof. Sámuel Komoly M.D., DSc. Pécs 2009

CONTENT ABBREVIATIONS... 5 I. INTRODUCTION: Background of Theses... 6 The ideal biochemical serum marker... 6 Biomarkers of brain tissue damage... 6 Neurone specific enolase... 6 S100beta protein... 7 Biomarkers of inflammation... 8 C-reactive protein... 8 Procalcitonin... 8 Role of leukocytes in acute ischemic stroke... 9 Leukocyte antisedimentation rate... 9 N-terminal prohormone of brain derived natriuretic peptid... 10 Transient Ischemic Attack... 11 Acute ischemic stroke... 11 Postoperative cognitive deficit... 12 Post-cardiac arrest syndrome... 12 REFERENCES... 13 II. AIM OF THE STUDIES... 18 I. The role of innate immune response in patients with acute ischemic stroke complicated by post-stroke infection... 18 II. Measurement of brain derived biomarkers in the sera of cardiac patients with accidentally provoked transient neurological signs by intravenous dipyridamole (DP) stress test... 19 III. Measurement of biomarkers of endothel and platelet activation in the sera of patients with non-cardiac surgery... 19 IV. Measurement of biomarkers in the sera of successfully resuscitated cardiac arrest victims... 20 2

III. SUMMARY OF PAPERS SUPPORTING THESES... 21 I. THE ROLE OF INNATE IMMUNE RESPONSE IN PATIENTS WITH ACUTE ISCHEMIC STROKE COMPLICATED BY POST-STROKE INFECTION... 21 Paper 1... 21 Deficient leukocyte antisedimentation is related to post-stroke infections and outcome Paper 2... 22 Relationship between C-reactive protein and early activation of leukocytes indicated by leukocyte antisedimentation rate (LAR) in patients with acute cerebrovascular events Paper 3... 23 Impaired function of innate T lymphocytes and NK cells in the acute phase of ischemic stroke Paper 4... 24 Immune responses and neuroimmune modulation in the pathogenesis of acute ischemic stroke and post-stroke infections II. MEASUREMENT OF BRAIN DERIVED BIOMARKERS IN THE SERA OF CARDIAC PATIENTS WITH ACCIDENTALLY PROVOKED TRANSIENT NEUROLOGICAL SIGNS BY INTRAVENOUS DIPYRIDAMOLE (DP) STRESS TEST... 24 Paper 5... 24 Dipyridamole stress test in the early evaluation of cerebral circulatory disorders? Paper 6... 25 Cerebrovascular stressing : drug-induced S100B elevation and transient neurological signs predict ischemic cerebrovascular events III. MEASUREMENT OF BIOMARKERS OF ENDOTHEL ACTIVATION IN THE SERA OF PATIENTS WITH NON-CARDIAC SURGERY... 26 Paper 7... 26 Increased levels of baseline biomarkers reflecting platelet and endothelial activation predict early cognitive dysfunction after lung surgery IV. MEASUREMENT OF BIOMARKERS IN THE SERA OF SUCCESSFULLY RESUSCITATED CARDIAC ARREST VICTIMS... 28 3

Paper 8... 28 Prognostic role of serum S100β and procalcitonin in patients after cardio-pulmonary resuscitation Paper 9... 29 NT-proBNP as early marker of septic complications in patients after cardiopulmonary resuscitation IV. SUMMARY OF THESES... 31 V. BIBLIOGRAPHY... 34 Articles... 34 Congress abstracts... 36 Awarded lectures... 42 Chapter... 43 VI. ACKNOWLEDGEMENTS... 44 VII. PAPERS... 45 4

ABBREVIATIONS AIS CI CNS CPC CPR FasL GCS GOS hscrp IFN-γ Ig IL IQR LAR MCP-1 MMSE NIHSS NK cells NKT-like cells NSE OR PCT POCD scd40l sp-selectin svcam-1 TBI TIA TNS tpa WBC WHO acute ischemic stroke confidence interval central nervous system cerebral performance category cardio-pulmonary resuscitation Fas ligand Glasgow Coma Scale Glasgow Outcome Scale high-sensitivity C-reactive protein interferon-γ immunoglobulin interleukin interquartile range leukocyte antisedimentation rate monocyte chemoattractant protein-1 Mini Mental State Examination National Institute of Health Stroke Scale natural killer cells natural killer T-like cells neurone specific enolase odds ratio procalcitonin postoperative cognitive dysfunction soluble CD40 ligand soluble P-selectin soluble vasculare cell adhesion molecule-1 traumatic brain injury transient ischemic attack transient neurological sign tissue plasminogen activator White Blood Cell World Health Organization 5

I. INTRODUCTION Background of Theses Biomarkers The ideal biochemical serum marker. In 1983, Bakay and Ward (1) suggested that an ideal serum marker should have high specificity for the brain, high sensitivity for brain injury, be released only after irreversible destruction of brain tissue, have a rapid appearance in serum, and be released in a time-locked sequence with the injury. The age- and sex-related variability should be low to ensure a predictable relationship between the serum concentration and the amount of brain injury. Furthermore, reliable assays for immediate analyses should be available. Finally, and most importantly, it should show clinical relevance. These prerequisites remain the most important properties of a biochemical serum marker of acute cerebral damage. In clinical practice a biomarker can serve several objectives. There are a large number of studies assessing the value of biomarkers in the prognosis of i. infection, ii.. acute focal brain injury (stroke, traumatic brain injury-tbi), iii. global hypoxic brain damage due to cardiac arrest. However, biomarkers can provide additional information to the clinical evaluation namely in the diagnosis of infection, minor head injury, TIA/minor stroke (when neuroimaging is not sensitive enough), furthermore they are suitable for risk stratification and assessment of the response to a certain therapy (antibiotics, immunmodulators, fibrinolytics, therapeutic hypothermia etc.). Biomarkers are not static but dynamic, presenting marked changes in response to different inflammatory stimulus, specifically, bacterial infection and unspecifically various type of tissue necrosis. Consequently, serial measurements could be more informative than a single one. Additionaly, there is a continous search for novel markers to better predict the outcome of patients with acute cerebral insults. Biomarkers of brain tissue damage Neuron specific enolase (NSE). NSE was originally described by Moore and McGregor in 1965 (2). Enolases are glycolytic enzymes occurring as a series of dimeric isoenzymes made of three immunologically distinct subunits, the α, β, and γ chains (3) The isoforms γγ and αγ are restricted to neurons, peripheral neuroendocrine tissue, and tumors of the amine precursor uptake and degradation system, and referred to as NSE (3). The major distinctive feature of NSE compared with other enolases is its high degree of stability. NSE is 6

located in the cytoplasm of neurons and is probably involved in increasing neuronal chloride levels during the onset of neural activity (4) The molecular mass of NSE is 78 kda and the biologic half-life is probably more than 20 hours (5) Serum NSE levels of more than 10 µg/l are considered pathologic (6,7). In clinical practice, NSE is predominantly used as a marker for tumors of the amine precursor uptake and degradation system, such as small-cell lung cancer, neuroblastoma, and melanoma (3) NSE is also released in the blood by hemolysis, which may be a serious source of error (8). S100beta protein. The S100 protein, whose name is derived form its solubility in 100% saturated ammonium sulfate at neutral ph, was first described by Moore in 1965 (9). These proteins are a group of small Ca 2+ -binding modulator proteins with a molecular mass of about 21 kda. Members of this protein family have been implicated in the Ca 2+ -dependent regulation of a variety of intracellular activities such as protein phosphorylation, enzyme activities, cell proliferation and differentiation, the dynamics of cytoskeleton constituents, the structural organization of membranes, intracellular Ca 2+ homeostasis, inflammation, and in protection from oxidative cell damage (10). S100 is a mixture of similar proteins composed of two immunologically distinct subunits, the α and β chains, which combine to yield αα(s100a0), αβ(s100a), and ββ (S100B) forms. The isoforms αβ and ββ are predominantly present in astroglial cells of the CNS and commonly referred to as the brain-specific S100B protein. S100B is eliminated by urinary excretion and renal metabolism (11). The biologic halflife has not been exactly established. Recent studies suggest that it is well below 60 minutes (12). Concentrations of S100 have been determined in CSF and in blood. Van Engelen and Nygaard reported that S100 concentrations in CSF increase with age (13,14). This was recently confirmed by Wiesmann et al.,(15) who determined the concentration of S100 in blood in 200 healthy blood donors between 18 and 65 years of age. The median plasma concentration of S100b was 0.05 µg/l. There was no difference between men and women. Serum S100 level >0.2 µg/l is considered pathologic (16). In line with the possibility that astrocytes respond to brain insults with increased S100B release is the observation that serum levels of S100B increase significantly after head injury (Rothoerl et al., 1998), cardiac arrest (Rosen et al., 1998), cardiac surgery (Kumar et al., 1997) and stroke (Buttner et al., 1997). Other evidence suggests that serum S100B may be a marker of blood brain barrier opening that is not necessarily related to neuronal damage (17). It is reasonable to hypothesise that S100B accumulates in the extracellular space after astrocyte death or due to increased release by activated astrocytes, or after cellular disintegration of the damaged parenchyma. 7

Biomarkers of inflammation C-reactive protein (CRP). This acute phase protein is produced by the liver (18) and by adipocytes (19). It is a member of the pentraxin family of proteins. C-reactive protein was originally discovered by Tillett and Francis in 1930 as a substance in the serum of patients with acute inflammation that reacted with the C polysaccharide of pneumococcus (20). Plasma CRP rises whenever an inflammatory process is present and characteristically, its serum concentration depends only on the intensity of the stimulus and on the rate of synthesis (21-27). Recently, it was shown that elevated CRP levels independently predict the risk of future stroke and transient ischemic attack in the elderly (28). After acute stroke, a single CRP concentration measured within 72 hours was found to be an independent predictor of survival in a subgroup analysis from a prospective observational study (29). In this study survival in patients with CRP levels >1.01 mg/dl was significantly worse (29). Di Napoli et al (30) studied the 1-year prognostic influence of CRP measured within 24 hours after ischemic troke and described an association between increased levels of CRP and unfavorable outcome. A recent investigation analyzed the association between CRP measured within 24 hours after ischemic stroke, after 48 to 72 hours, and at hospital discharge and long-term outcome (31). The authors found that CRP concentration at discharge was better related to outcome than earlier measurements. One can conclude that a reactive acute phase response due to an accompanying infection could not be ruled out if blood samples were taken within 24 hours after symptom onset. Similarily, the CRP level measured within 12 hours after symptom onset of an acute ischemic stroke is not independently related to long-term prognosis. In contrast, a CRP increase between 12 and 24 hours after symptom onset predicts an unfavorable outcome and is associated with an increased incidence of cerebrovascular and cardiovascular events (32). Procalcitonin (PCT). PCT is classifeid as hormokine since it has simultaneously hormone and cytokine properties (33). Procalcitonin is a prohormone physiologically transformed into calcitonin by the medullary cells of the thyroid. However, the origin of this substance can be extrathyroidal. High PCT levels were found in thyroidectomized septic patients, while calcitonin levels were undetectable. Furthermore, the rise in PCT in the absence of an increase of mature calcitonin has been described during bacterial infection (34). Inflammatory response is a principal early component in the pathophysiology of stroke (35). PCT a marker of septicemia and infection severity (36) has also been proposed as an indicator of systemic inflammatory response in noninfectious situations (37,38). There are 8

only few studies related to PCT in stroke. A pilot study on the prognostic value of PCT in acute stroke found no correlation between serial serum PCT levels and stroke mortality or neurologic outcome at discharge (39). Studies have correlated laboratory parameters with prognosis in acute stroke, sometimes with conflicting results (40-42). Prognostic utility of laboratory parameters in acute stroke is limited by the need to perform serial measurements, together with the interrelation of at least some of the inflammatory markers. Hence, determination of stroke severity by clinical criteria and appropriate imaging techniques remains the main prognostic tool in acute stroke settings (43), and the optimal biochemical marker (if any) is still unknown. Role of leukocytes in acute ischemic stroke Leukocytes accumulate in the region of cerebral ischemia in the early stage of stroke, within hours (44). The neurological outcome was shown to be worse and the infarct larger in patients with severe polymorphonuclear leukocyte (PMNL) accumulation (45). A significant correlation between stroke volume or stroke severity and an acute increase of white blood cell (WBC) in the peripheral blood have been reported (46-48). Increased aggregation of peripheral leukocytes in the absence of WBC elevation was also reported in major stroke (47). Increased in vitro adhesive properties, activation of leukocytes indicated by increased plasma oxidation of adrenaline to adrenochrome and higher plasma levels of cytokines and proteases have been shown in ischemic stroke and TIA (49-52). While these studies addressed the detrimental effect of leukocyte activation, it may also play a major role in defense against pathogens, and participate in combating infections. A deficient activation of leukocytes may thus contribute to an increased susceptibility to post-stroke infections. Leukocyte antisedimentation rate (LAR). We have recently established a simple test to examine activation of leukocytes by measuring upward floating in a tube during one hour of gravity sedimentation. The LAR indicates the percentage of leukocytes crossing the middle line of the blood column upwards during 1 hour of sedimentation (53). In short, Westergreen blood sedimentation rate technique was modified for measuring leukocyte motion during gravity sedimentation of the whole blood. After one hour sedimentation, the leukocyte counts of upper and lower half sections were measured with an automatic cell counter (Coulter Counter CBC5, Coulter Electronics Ltd, Luton, UK) and results were expressed as a rate (53,54). 9

Figure 1. Calculation of leukocyte antisedimentation rate 10.000/µl 8.000/µl 6.000/µl 0 60 LAR: 25% By definition, LAR can be calculated after 1 hour gravity sedimentation by the following equation: [upper-lower/upper+lower]x100 = LAR (%) (upper=content of wbc in the upper half, lower=content of wbc in the lower half of the blood column.) It has been shown that a raised LAR is positively correlated with enhanced leukocyte adherence proportional to activation, with an increased cell volume and higher vacuole content of polymorphonuclear leukocytes (54). LAR correlated significantly with serum procalcitonin (PCT) and C-reactive protein (CRP) concentration in critically ill patients with severe sepsis (55). The simple LAR test was a good positive predictor of bacteraemia in critically ill patients presenting their first febrile episode without preceding antibiotic treatment (56,57). N-terminal prohormone of brain derived natriuretic peptid (NT-proBNP) Natriuretic peptides play an importent role in the regulation of cardiovascular homeostasis and fluid volume. Increased plasma levels of natriuretic peptide hormones have been identified as predictors of cardiac dysfunction and death in many critical care settings, including congestive heart failure, myocardial infarction and septic shock (58-60). The prohormone brain natriuretic peptide (pro-bnp) is produced by ventricular myocytes in response to wall stress (61). In the circulation the biologically active hormone is separated from the N-terminal part of the prohormone termed NT-proBNP. It promotes natriuresis and diuresis, acts as vasodilator and aldosterin antagonist and also inhibits the endothelin release and smooth cell proliferation (62). Because BNP- or NT-proBNP-guided therapy of heart failure could reduce the occurance of cardiac events (63) and thereby decrease mortality (64), the marker itself seems to be an early predictor of prognosis and myocardial dysfunction in patients with septic shock (60), investigation of their levels and prognostic significans in patients after CPR with or without sepsis is of great interest. 10

Transient Ischemic Attack (TIA) Neurological event with the signs and symptoms of a stroke, but which go away within a short period of time (less than 24 hours). Also called a mini-stroke, a TIA is due to a temporary ischemia to the brain. This is often caused by the narrowing (or, less often, ulceration) of the carotid arteries. TIAs typically last 2 to 30 minutes and can produce problems with vision, dizziness, weakness or trouble speaking. If not treated, there is a high risk of having a major stroke in the near future. People who have a TIA have a 25% greater risk of having a stroke or other serious complication within 90 days (65). In one study of people followed for 3 months after a TIA, about 10% had strokes, half of them in the 2 days after their TIA (66). This was more than 50 times the stroke rate than expected in people of their age. One fifth of the strokes were fatal and nearly two-thirds were disabling. Acute ischemic stroke The World Health Orginazation s (WHO) standard definition of stroke is a focal (or at times global) neurological impairment of sudden onset, and lasting more than 24 hours (or leading to death) and of presumed vascular origin. Approximately 80% of cases are ischemic, 20% are hemorrhagic. In addition to stroke being the third leading cause of death, many survivors of stroke have to adjust to a life with varying degrees of disability (67). Stroke has a high incidence worldwide in the United States 700,000 people are affected each year leading to over 150,000 death (68). In Hungary, approximately 40,000 patients per year are admitted to hospital with the diagnosis of stroke and stroke mortality is extremely high in Central-Eastern European countries (69). While the direct neurological deficit cause early death, infectios complications prevail in the postacute phase of stroke contributing to the poor outcome. 18-28% of patients developed a poststroke infection in different studies. In addition, infectious complications (predominantly chest and urinary tract infections) have been frequently reported within the first few days after stroke resulting in an increased mortality rate and worse clinical outcome (70). Acute infections may also increase the risk of stroke, particularly during the first 3 days of infection (74,75). Immunodepression has been reported in other potentially life-threatening conditions, such as myocardial infarction, polytrauma or major surgery, causing an increased risk of infectious complications (76,77). Immunodepression after stroke could be partially overcome by suppression of the sympathetic nervous system resulting in enhanced cellular immune responses and prevention of bacterial infections (78,79). Thus, early identification of those at increased risk of post-stroke infection should represent a significant contribution to 11

health improvement so that interventions (early antibiotic therapy) can be targeted to those most likely to benefit. Perioperative cognitive dysfunction (POCD) Advances in anaesthetic and surgical techniques have led to the assumption that postoperative cognitive decline is currently less common than previously. The benefits of such technological advances, however, may have been offset by inclusion of older patients with more comorbidity. The neurobehavioural outcomes range from the well-documented incidence of stroke to the less well-delineated postoperative delirium, cognitive difficulties (such as memory loss and visuospatial deficits), and depression. Prolonged postoperative cognitive dysfunction (POCD) is reported to occur frequently after cardiac surgery (80). However, it is rarely assessed in routine clinical practice and receives little attention after noncardiac major surgery. The incidence of cognitive dysfunction has been reported to be between 1% and 60%, depending on the type of operation (81). Multiple etiologies, including age, preexisting cerebrovascular disease, prior functional and American Society of Anesthesiology (ASA) status, urgency of operation, general versus regional anesthesia, have been proposed to explain cognitive dysfunction but the causative factors are unknown and the severity of dysfunction is unpredictable (82-85). Few previous studies have focused on thoracic and vascular surgical patients as a primary study group with a long-term follow-up, despite aortic aneurysm and noncardiac thoracic operations were found to be high-risk procedures for delirium with 7% of incidence (82). Even less data can be found concerning POCD related to lung surgery, where one-lung anesthesia should cause transient hypoxia, which could be associated with cognitive dysfunction, nevertheless there was no association of intraoperative oxygenation with cognitive dysfunction (86,87). Post-cardiac arrest syndrome (PCAS) Among cardiac arrest survivors, brain injury is the major determinant of functional outcome and quality of life. A major goal is to identify patients destined for poor neurological outcomes (88). The failure of injured structures to recover leads to a spectrum of neurological dysfunction including the inability to perform tasks independently, persistent vegetative state, coma, and brain death (Cerebral Performance Category: 3,4 and 5 respectively)(89). The need for protracted high-intensity care of neurologically devastated survivors presents an immense burden to healthcare systems and society in general (90). To limit this burden, clinical factors and diagnostic tests are needed to prognosticate functional outcome. Many studies have 12

identified (pre-, intra- and post-cardiac arrest) factors associated with poor functional outcome after resuscitation, but no studies have shown a reliable predictor of outcome. Beside others, biochemical markers have been used to prognosticate functional outcome after cardiac arrest. Numerous studies show varying thresholds of NSE < from 30 to 65 µg/l for poor outcome and mortality (91-94). In cardiac arrest survivors, S100B similarily showed high specificity with low sensitivity (93,95,96) The prognosis of survival in patients with cardiac arrest still remains poor due to the whole-body ischemia/reperfusion syndrome, with alteration in immuno-inflammatory profile and consequently serious infectious complications (97). The importance of early recognition of systemic inflammatory response, leading to an exacerbation of the inflammatory balance is not questionable. During the last years procalcitonin (PCT) has become a well established marker to determine severe infections or sepsis in the early stage (98). The non brainoriginated PCT was found in successfuly resuscitated out-of-hospital cardiac arrest victims as sensitive and specific as the brain originated S100B regarding the neurological outcome becoming a surrogate marker for hypoxic brain damage, reflecting the severity of tissue oxigen depth (99). Thereafter, PCT levels per se should be interpreted with caution regarding the diagnosis of infection after cardiopulmonary resuscitation (CPR). Similarily, the diagnostic role of PCT in contrast to CRP as a marker for ventilator associated pneumonia (VAP) after successful CPR was also proven (100). There is a great need for further biomarkers or their combination in order to distinguish the acute phase response due to tissue injury and the infectious complications which might lead us to new therapeutic approaches in postresuscitation care. REFERENCES 1. Bakay RA, Ward AAJ. Enzymatic changes in serum and cerebrospinal fluid in neurological injury. J Neurosurg. 1983;58:27 37. 2. Moore BW, McGregor D. Chromatographic and electrophoretic fractionation of soluble proteins of brain and liver. J Biol Chem.1965;240:1647 1653. 3. Cooper EH. Neuron-specific enolase. Int J Biol Markers. 1994; 4:205 210. 4. Marangos PJ, Schmechel DE. Neuron specific enolase, a clinically useful marker for neurons and neuroendocrine cells. Ann RevNeurosci. 1987;10:269 295. 5. Johnsson P. Markers of cerebral ischemia after cardiac surgery. J Cardiothorac Vasc Anesth. 1996;10:120 126. 6. Nygaard Ø, Langbakk B, Romner B. Neuro-specific enolase concentrations in serum and cerebrospinal fluid in patients with no previous history of neurological disorder. Scand J Clin Lab Invest. 1998;58:183 186. 7. Raabe A, Grolms C, Keller M, Döhnert J, Sorge O, Seifert V. Correlation of computed tomography findings and serum brain damage markers following severe head injury. Acta Neurochir (Wien). 1998;140:787 792.). 8. Johnsson P. Merkers of cerebral ischemia after cardiac surgery. J Cardiothorac Vasc Anesth. 1996;10:120 126. 13

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II. AIM OF THE STUDIES I. The role of innate immune response in patients with acute ischemic stroke complicated by post-stroke infection Cerebrovascular diseases of acute onset, including ischemic stroke, are associated with diverse immune repsonses. The high incidence of infections in stroke patients is likely to be a result of an impaired immune function. Therefore, we investigated different biomarkers in patients within hours after onset of acute ischemic stroke and TIA to characterize the innate immune response and its relation to post-stroke infection and functional outcome. Furthermore, the aim of our study was to evaluate the most reliable markers in identifying a subset of stroke patients, which later develop infection. Therefore, we addressed, whether: 1. What is the relation of leukocyte activation to post-stroke infections? 2. Does post-stroke leukocyte activation depends on the duration of ischemia and the extent of infarct? 3. Can we use simple bed-side tests to predict outcome and susceptibility for poststroke infections? 4. Does serial measurement of leukocyte activation show differences in acute ischemic stroke and TIA? 5. Does serial measurement of leukocyte activation show differences in patients with or without post-stroke infection? 6. Is there a relationship between leukocyte activation and functional outcome? 7. Does plasma hscrp of patients with acute ischemic stroke differ from either TIA or healthy controls? 8. Are there differences in plasma hscrp as marker of stroke in first-ever or recurrent stroke subpopulation? 9. Is there an association between plasma hscrp level and NIHSS indicating the severity of symptoms? 10. Is there an association between the inflammatory aspect of atherogenesis represented by hscrp and inflammation mediated by leukocytes? 11. Does serial measurement of hscrp show any differences in patients with or without post-stroke infection? 18

12. Is there a relationship between hscrp and functional outcome of acute ischemic stroke? 13. What is the best cut-off value of hscrp in prediction of infectious complications after acute ischemic stroke? 14. To analyze functional alterations in cytokine production and cytotoxicity of innate lymphocyte subsets capable of shaping adaptive T cell responses in the acute phase of ischemic stroke. II. Measurement of brain derived biomarkers in the sera of cardiac patients with accidentally provoked transient neurological signs by intravenous dipyridamole (DP) stress test Intravenously administered DP can induce short lasting and reversible minor neurologic deficit in high-risk vascular patients, that can be detected by using brain SPECT imaging providing a very early assessment of patients at risk of threatening cerebrovascular event. The aim of this prospective study was to determine: 15. Does DP-stress impact biomarkers (S100B, NSE, hscrp)? 16. What is the frequency of transient neurological signs (TNS) during DP-stress? 17. Do induced biomarkers and TNS predict manifest cerebrovascular events? What is the incidence of a new cerebrovascular event in the high risk population selected by DP stress test and/or elevated S100B after DP stress within 7 years? 18. What is the best predictor of future cerebrovascular events in patients with coronary artery disease? III. Measurement of biomarkers of endothel and platelet activation in the sera of patients with non-cardiac surgery Cardiac operations are frequently complicated by postoperative cognitive decline. Less common and less studied is postoperative cognitive decline after noncardiac surgery. Therefore, we examined: 19. What is the incidence of POCD after lung surgery at the Department of Surgery, University of Pécs, Hungary? 20. Is MMSE suitable for determination of POCD? 21. Could biomarkers reflecting immuno-endothelial dysfunctions be related to ischemia and neurocognitive alterations after lung surgery? 19

22. Which molecules are the best predictors of susceptibility for POCD before major surgeries? IV. Measurement of biomarkers in the sera of successfully resuscitated cardiac arrest victims Increased plasma levels of natriuretic peptide hormones have been identified as predictors of cardiac dysfunction and death in many critical care settings. To date no studies were addressed to investigate the relationship between NT-proBNP and PCT in the serum of patients sucessfuly resuscitated after in-hospital cardiac arrest with or without concomitant infection or sepsis. Therefore, we performed a serial analysis of S100B, PCT and NT-proBNP in the first 72 postresuscitation hours in cardiac arrest victims to examine the role of these markers in prediction of septic complication: 23. Are there any difference in plasma level of PCT, S100B and NT-proBNP of patients resuscitated due to either ventricular fibrillation or asystole/pulsless electrical activity? 24. Are there any differences in plasma level of PCT, S100B and NT-proBNP of cardiac arrest victims with or without septic complication? 25. Is there any relationship between the measured biomarkers and clinical outcome? 20

III. SUMMARY OF PAPERS SUPPORTING THESES I. THE ROLE OF INNATE IMMUNE RESPONSE IN PATIENTS WITH ACUTE ISCHEMIC STROKE COMPLICATED BY POST-STROKE INFECTION Paper 1 Deficient leukocyte antisedimentation is related to post-stroke infections and outcome (J Clin Path, 2008, 61:1209-1213) Patients with stroke are more susceptible to infections suggesting possible deficiencies of early immune responses, particularly of leukocytes. Here, we examined whether postischemic activation of leukocytes is related to duration of ischemia and extent of infarct. We also addressed, if dsyregulated leukocyte activation might be related to post-stroke infection and worsen outcome. We used leukocyte antisedimentation rate (LAR) to detect activation of leukocytes and correlated LAR with clinical and laboratory parameters. An additional aim was to test simple bed-side investigations in predicting outcome and susceptibility for poststroke infections early. LAR, a simple test to detect activation of leukocytes was serially examined and correlated with blood level of S100β related to extent of infarct, procalcitonin indicating infection and outcome in patients with acute ischemic events. Venous blood samples were taken from 61 healthy volunteers and 49 patients with acute ischemic events: 38 patients with acute ischemic stroke, AIS, and 11 patients with transient ischemic attack, TIA where symptoms disappear in 24 hours and cranial CT scan does not indicate infarct. Sampling was done within 6 hours, at 24 and 72 hours after onset of symptoms. LAR was significantly higher in acute ischemic events within 6 hours after onset of stroke regardless of post-stroke infections. In addition, elevation of LAR was delayed and attenuated in TIA in contrast to AIS and we also observed a positive correlation between LAR and S100β at 72 hours after the onset of ischemic stroke both indicating that the extent of tissue injury correlates with the magnitude of innate immune responses. Importantly, a deficiency in early elevation of LAR was associated with post-stroke infections and a poor outcome measured by Glasgow Outcome Scale in AIS. We conclude that an early activation of leukocytes indicated by elevation of LAR is characteristic of acute ischemic cerebrovascular events. A delayed and ameliorated leukocyte 21

activation represented by LAR is characteristic to TIA in contrast to definitive stroke. Our data suggest that acute activation of leukocytes, which has been regarded detrimental so far, serves also to prevent post-stroke infections. Our data imply that concept about the postischemic role of leukocytes should be changed and dissected: recrution of leukocytes in the CNS may be damaging but should be separated from the systemic activation, which may prevent post-stroke infections. A disregulated early immune response or deficient leukocyte activation may result in an increased susceptibilty to infections in some patients with stroke. *** Paper 2 Relationship between C-reactive protein and early activation of leukocytes indicated by leukocyte antisedimentation rate (LAR) in patients with acute cerebrovascular events (Clin Hemorheol, in press.) A non-specific inflammatory response occurs after stroke and the degree of inflammation is predictive of outcome. The purpose of this study was to determine the relationship between the acute phase reactant C-reactive protein (CRP) and leukocyte antisedimentation rate (LAR) as a specific test to detect early activation of leukocytes providing the first line of defence against infections. Here, we examined relationship among LAR, astroglia specific S100B indicating the extent of brain tissue damage and hscrp in 49 patients within hours after onset of acute ischemic stroke (AIS) and transient ischemic attack (TIA) to characterize the innate immune response and its relation to the outcome. Serum levels of hscrp on admission was significantly higher in patients with acute ischemic stroke (AIS) compared to healthy subjects and were higher in patients with recurrent to first ever ischemic stroke. Increased basal levels of hscrp also correlated with extent of infarct reflected by S100B levels in sera, but did not correlate with post-stroke infections. However, a higher rate of infection was observed among patients, in whom hscrp was elevated at 72 hours but LAR did not increase. Therefore, such late elevation of hscrp may indicate pre-clinical infections due to deficient leukocyte activation. Indeed, our data indicate that early (within 6 hours) and late (after 72 hours) changes in serum levels of hscrp may reflect different pathological processes in stroke. We can conclude that simple tests like LAR and hscrp may help in predicting outcome and high risk of infectious complications. 22