Martin Ploder

Andreas Spittler

Katharina Schroecksnadel

Gabriele Neurauter

Linda E Pelinka

Erich Roth

Dietmar Fuchs
email: dietmar.fuchs@i-med.ac.at

Fuchs Dietmar

Tryptophan degradation in multiple trauma patients: survivors versus non-survivors

Keywords: tryptophan, kynurenine, indoleamine (2,3)-dioxygenase, trauma, survival Clinical Science Immediate

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Introduction

The inflammatory response in trauma patients is crucial for the fate of the patients. In such patients significant activation of various immune system compartments is demonstrated, which however is accompanied by diminished functional immune response and immune paralysis [START_REF] Zedler | The impact of endogenous triggers on trauma-associated inflammation[END_REF]. Enzyme indoleamine 2,3-dioxygenase (IDO) converts tryptophan to kynurenine and is strongly inducible by pro-inflammatory stimuli and preferentially by cytokine interferon-γ (IFN-γ) [START_REF] Taylor | Relationship between interferon-gamma, indoleamine 2,3-dioxygenase, and tryptophan catabolism[END_REF][START_REF] Brown | Implications of interferon-induced tryptophan catabolism in cancer, autoimmune diseases and AIDS[END_REF]. Activated IDO diminishes the availability of the essential amino acid tryptophan and is considered as an immune defence mechanism, which suppresses the growth of intracellular bacteria, viruses and parasites like toxoplasma and of malignant tumor cells [START_REF] Brown | Implications of interferon-induced tryptophan catabolism in cancer, autoimmune diseases and AIDS[END_REF][START_REF] Pfefferkorn | Interferon gamma blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan[END_REF][START_REF] Carlin | Interferons and indoleamine 2.3-dioxygenase:role in antimicrobial and antitumor effects[END_REF]. But also T-cell proliferation can be inhibited by IDO activity [START_REF] Mellor | Tryptophan catabolism and T-cell tolerance: Immunosuppression by starvation?[END_REF], and thus, immune response may be suppressed when tryptophan is diminished. In patients after multiple trauma, tryptophan deficiency was found to be associated with the decline of lymphocytes in numbers [START_REF] Pellegrin | Enhanced enzymatic degradation of tryptophan by indoleamine 2,3dioxygenase contributes to the tryptophan-deficient state seen after major trauma[END_REF].

Pro-inflammatory stimuli like Th1-type cytokine IFN-γ induce IDO and neopterin production in human monocyte-derived macrophages and dendritic cells (DC) [START_REF] Weiss | Modulation of neopterin formation and tryptophan degradation by Th1-and Th2-derived cytokines in human monocytic cells[END_REF][START_REF] Wirleitner | Monocyte-derived dendritic cells release neopterin[END_REF]. In several groups of patients, e.g. suffering from virus infections, auto-immune syndromes and also arteriosclerosis and cancer, enhanced tryptophan degradation concurs with increased neopterin formation [START_REF] Fuchs | Increased endogenous interferon-gamma and neopterin correlate with increased degradation of tryptophan in human immunodeficiency virus type 1 infection[END_REF][START_REF] Wirleitner | Interferon-γ-induced conversion of tryptophan: immunologic and neuropsychiatric aspects[END_REF][START_REF] Pertovaara | Indoleamine 2,3-dioxygenase enzyme activity correlates with risk factors for atherosclerosis: the Cardiovascular Risk in Young Finns Study[END_REF][START_REF] Pertovaara | Mechanisms dependent on tryptophan catabolism regulate immune responses in primary Sjogren's syndrome[END_REF]. In trauma patients, plasma concentrations of neopterin are able to predict outcome [START_REF] Zedler | The impact of endogenous triggers on trauma-associated inflammation[END_REF][START_REF] Strohmaier | D-erythro-neopterin plasma levels in intensive care patients with and without septic complications[END_REF][START_REF] Hensler | The clinical value of procalcitonin and neopterin in predicting sepsis and organ failure after major trauma[END_REF]. From the data one can expect that in multiple trauma patients increased neopterin production will be paralleled by activation of IDO, which could represent a key to understand the negative contribution which immune activation and IFN-γ production might have in patients with trauma.

In this study, concentrations of tryptophan and kynurenine and kyn/trp were analyzed in patients with trauma and compared to the outcome. In addition, results on tryptophan metabolism were compared to concentrations of neopterin.

Materials and Methods

Patients

Fifteen patients post-trauma (12 males, 3 females; aged mean + S.D.: 40.4 + 17.2 years, range: 20 -77 years; Apache score: 17.5 + 6.5 range: 8-34; Injury Severity Score (ISS): 39.1 + 13.1, range: 18-57) were included in this study who either were admitted to the Intensive Care Unit (ICU) of the Medical University of Vienna or to the ICU of Lorenz Boehler Trauma Center, Vienna (Table I). Inclusion criteria were age between 18 and 80 years and evidence of a multiple trauma (ISS >16). Exclusion criteria were known immunoseppressive therapy, known HIV infection or any other chronic disease. All patients were otherwise healthy before the trauma. Average length of stay in the ICU was 25.3 + 20.1 days. During follow up 6 patients died on days 7, 10, 14, 17, 26 and 37. Serum specimens were collected every third day during 12 -14 days of follow up. For statistical analyses, specimens were divided into five groups: one specimen collected between days 1-2 of each patient was referred to group I, day 3-5 = group II, day 6-8 = group III, days 9-12 = group IV, and days 13-14 = group V. In total every patient contributed 3-5 specimens to the total number of 69 sera analyzed, which corresponds to 6 missing specimens over the whole period. All patients received standard parenteral nutrition (1700 kcal/d, 100g amino acids/d) after the end of hypodynamic shock. Samples of 49 healthy blood donors (21 women, 28 men, aged 35.2 + 13.5 years) served as a reference group [START_REF] Widner | Simultaneous measurement of serum tryptophan and kynurenine by HPLC[END_REF].

The study was performed according to the Helsinki declaration. The protocol was approved by the local ethics committee, and written consent was granted by the next of kin. The observed values of all study parameters had no influence whatsoever on the course of the therapy.

Measurements

Concentrations of tryptophan and kynurenine were determined by HPLC [START_REF] Widner | Simultaneous measurement of serum tryptophan and kynurenine by HPLC[END_REF][START_REF] Laich | More rapid method for simultaneous measurement of tryptophan and kynurenine by HPLC[END_REF], kyn/trp was calculated and expressed as µmol kynurenine per millimol tryptophan (µM/mM). Concentrations of Neopterin (BRAHMS, Hennigsdorf/Berlin, Germany; detection limit of 2 nM) were measured by commercially available ELISA according to the manufacturer's instructions.

Statistical Analysis

Demographic parameters were compared using the Student's t-test or Χ²-test. Results of measurements were expressed as mean + SD. Because not all the data sets showed normal distribution, non-parametric statistics were applied for data analyses, Kruskal Wallis H-test was used to look for differences in the median values between the 5 time groups, Mann Whitney U-test was subsequently applied for direct comparisons between grouped data. Spearman's rank correlation coefficients (r s ) were calculated for regression analyses. Changes during follow-up were calculated by paired rank test. Repeated measure analysis of variance with 5 steps was calculated concerning the 5 days of measurement. The Statistical Package for the Social Sciences (version 14 SPSS, Chicago, IL, USA) was used. P-values below 0.05 were considered to indicate significant differences.

Results

Kyn/trp and concentrations of kynurenine were increased and concentrations of tryptophan were decreased in patients compared to the reference group (gray boxes) (Fig. 1). None of the parameters correlated with Apache scores of patients. During follow-up, significant changes of kyn/trp (H = 21.3, p <0.001) and kynurenine concentrations (H = 21.2, p <0.001) were observed (Fig. 1). Kyn/trp and kynurenine concentrations increased from group I to II and remained higher than baseline throughout the further follow-up period (kyn/trp -I vs. II: p <0.01, vs. III: p <0.01, vs. IV: p <0.01, vs. V: p <0.06; kynurenine -I vs. II: p <0.01, vs. III: p <0.01, vs. IV: p <0.01, vs. V: p = 0.06). Concentrations of tryptophan did not significantly differ from day I throughout the study period.

Patients who died during follow-up presented with significantly higher kyn/trp (p <0.05, in groups II, IV and V; Fig. 1) and higher kynurenine concentrations (p <0.05, group IV). Tryptophan concentrations were lower in non-survivors compared to survivors but did not differ significantly. When solely the concentrations measured in non-survivors were compared to baseline, the increases of kyn/trp and kynurenine became significant in group III (p <0.05), the decline of tryptophan became nearly significant in group V (U = 1.82, p = 0.068). Repeated measure analysis of variance showed a significant difference between survivors and non-survivors concerning kynurenine concentrations (p <0.05) and kyn/trp (p <0.01).

Average concentrations of the additionally measured marker neopterin (mean + SD: 19.7 + 21.7 nM) were increased in patients compared to the reference group. Patients who died during follow-up had higher neopterin (p <0.05, groups II and III; p <0.01, groups IV and V) concentrations compared to survivors. In the whole data set, there existed a positive correlation between kyn/trp and neopterin (r s = 0.590, p <0.001; data not shown).

Discussion

This study shows that tryptophan metabolism is altered in patients suffering from trauma compared to healthy controls. Compared to healthy controls, kynurenine concentrations and kyn/trp were elevated in patients, whereas tryptophan concentrations were decreased. During follow-up, a higher rate of kynurenine accumulation and of tryptophan degradation as indicated by higher kyn/trp was found in non-surviving trauma patients compared to survivors. This data confirm and extend earlier observations on enhanced degradation of tryptophan by IDO in an independent set of patients after major trauma [START_REF] Pellegrin | Enhanced enzymatic degradation of tryptophan by indoleamine 2,3dioxygenase contributes to the tryptophan-deficient state seen after major trauma[END_REF].

In parallel to the increase of kynurenine concentrations and of kyn/trp, an increase of neopterin formation was observed. Because increase of neopterin is associated with Th1-type cytokines as IFN-γ [START_REF] Murr | Neopterin as a marker for immune system activation[END_REF], which induces both, tryptophan degradation via IDO and neopterin production in parallel [START_REF] Weiss | Modulation of neopterin formation and tryptophan degradation by Th1-and Th2-derived cytokines in human monocytic cells[END_REF][START_REF] Wirleitner | Interferon-γ-induced conversion of tryptophan: immunologic and neuropsychiatric aspects[END_REF], we assume that accelerated tryptophan degradation is due to enhanced IDO activity in the patients. Low tryptophan concentrations are unlikely to be related to a reduced dietary intake of this essential amino acid, because in this case also a decrease of kynurenine and no change of kyn/trp would be expected. Increased IDO activity may indeed account for lowered tryptophan concentrations, and because of the significant association between neopterin concentrations and kyn/trp, also tryptophan degradation in our patients seems likely to be induced by pro-inflammatory stimuli, of which IFN-γ is the strongest inducer of IDO in macrophages and DC.

Measurement of circulating IFN-γ concentrations in serum/plasma of patients is usually very insensitive. That is why we refrained from analyzing IFN-γ concentrations in our patients. Like other cytokines, IFN-γ rapidly sticks to its specific receptors on target cells or their shed soluble forms and the therefore low concentrations in the blood are limiting the diagnostic application.

Post trauma, immunocompetent cells appear to respond against non-self structures, and cells produce cytokines including IFN-γ aimed at halting growth of pathogens. Activation of IDO as one out of several important antimicrobial mechanisms triggered by IFN-γ however not only affects microbes, it potentially may also counteract growth and development of T-cells [START_REF] Mellor | Tryptophan catabolism and T-cell tolerance: Immunosuppression by starvation?[END_REF]. A kind of equilibrium might develop between the activation degree of immunocompetent cells, their suppressive effect on microbes and also on themselves. In some patients the bactericidal effect of IDO will be able to stop the infectious process, in others immunocompetent cells are suffering from IDO activity more than the microbes, and then depletion of T-cells and immunosuppression is a consequence of the host's immune response against invading pathogens [START_REF] Wood | Interferon gamma: a crucial role in the function of induced regulatory T cells in vivo[END_REF][START_REF] Schroecksnadel | Interferon-γ for counteracting T-cell activation[END_REF]. Certainly this study is too preliminary to give a final answer to this question.

A similar relationship between enhanced tryptophan degradation and immune activation was found earlier in several other chronic diseases, like HIV-1 infection or malignancy [START_REF] Wirleitner | Interferon-γ-induced conversion of tryptophan: immunologic and neuropsychiatric aspects[END_REF][START_REF] Giusti | Differential patterns of serum biomarkers of immune activation in human T-cell lymphotropic virus type I-associated myelopathy/tropical spastic pararesis and adult T-cell leukemia/ lymphoma[END_REF][START_REF] Brandacher | Prognostic value of indoleamine 2,3-dioxygenase expression in colorectal cancer: effect on tumorinfiltrating T cells[END_REF][START_REF] Weinlich | Decreased serum tryptophan concentration predicts poor prognosis in malignant melanoma patients[END_REF][START_REF] Boasso | How does indoleamine 2,3-dioxygenase contribute to HIV-mediated immune dysregulation[END_REF]. Moreover, in patients with HIV-1 infection and with cancer, enhanced tryptophan degradation and increased neopterin production were found to strongly predict shortened survival [START_REF] Giusti | Differential patterns of serum biomarkers of immune activation in human T-cell lymphotropic virus type I-associated myelopathy/tropical spastic pararesis and adult T-cell leukemia/ lymphoma[END_REF][START_REF] Brandacher | Prognostic value of indoleamine 2,3-dioxygenase expression in colorectal cancer: effect on tumorinfiltrating T cells[END_REF][START_REF] Weinlich | Decreased serum tryptophan concentration predicts poor prognosis in malignant melanoma patients[END_REF]. Also in patients with HIV-1 infection, antimicrobial/antiviral activity of IDO is suggested to contribute to immunodeficiency [START_REF] Boasso | How does indoleamine 2,3-dioxygenase contribute to HIV-mediated immune dysregulation[END_REF][START_REF] Schroecksnadel | Indoleamine-2,3-dioxygenase and other interferon-γ-mediated pathways in patients with human immunodeficiency virus infection[END_REF] and may explain why immune activation markers are strong predictors of outcome. From in vitro studies it was concluded that not only the decline of tryptophan concentrations but also the increase formation of toxic tryptophan catabolites could be involved in the development of T-cell unresponsiveness [START_REF] Terness | Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites[END_REF]. Interestingly in our study, only the increase of kynurenine concentrations but not the decline of tryptophan was significantly associated with the outcome of the patients. Thus, data may favour a role of tryptophan catabolites rather than tryptophan lowering in the immune deviations which might be of relevance for the fatal outcome of some of our patients. A c c e p t e d M a n u s c r i p t patients after trauma [START_REF] Pellegrin | Enhanced enzymatic degradation of tryptophan by indoleamine 2,3dioxygenase contributes to the tryptophan-deficient state seen after major trauma[END_REF]. In our study, the decrease of tryptophan concentrations did not reach the level of significance. Standard parenteral nutrition regimen which contains tryptophan as well as other essential amino acids was initiated early in our patients. It may have counteracted to some degree the loss of tryptophan despite further accelerating degradation of the amino acid and further increase of kynurenine concentrations.

Tryptophan deficiency resulting from its accelerated catabolism could also be involved in the increased risk for developing anemia, cachexia and neuropsychiatric abnormalities [START_REF] Wirleitner | Interferon-γ-induced conversion of tryptophan: immunologic and neuropsychiatric aspects[END_REF][START_REF] Widner | Neopterin production tryptophan degradation and mental depression: what is the link?[END_REF][START_REF] Myint | Kynurenine pathway in major depression: evidence of impaired neuroprotection[END_REF]. Still, the relevance of this assumption needs to be clarified in further extended studies.

In conclusion, accelerated tryptophan degradation is found in non-surviving patients post trauma. The same was true for higher concentrations of neopterin, which confirms and extends earlier data [START_REF] Strohmaier | D-erythro-neopterin plasma levels in intensive care patients with and without septic complications[END_REF][START_REF] Hensler | The clinical value of procalcitonin and neopterin in predicting sepsis and organ failure after major trauma[END_REF]. Tryptophan degradation may represent one important aspect in the development of the post traumatic failure of the immune system to respond appropriately. More extended and follow-up studies examining the impact of tryptophan degradation and its relationship with cellular immune activation in patients suffering from trauma may provide interesting new data, which should be helpful to define new therapeutic intervention strategies. Larger clinical studies are necessary to find out more about the potential prognostic expressiveness of tryptophan metabolism in patients suffering from trauma. A c c e p t e d M a n u s c r i p t

LEGENDS TO THE FIGURES

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Clinical

  Science Immediate Publication. Published on 05 Nov 2008 as manuscript CS20080319 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080319A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Recently Pellegrin et al. showed that a decline of tryptophan was associated with the drop of T-lymphocytes in Clinical Science Immediate Publication. Published on 05 Nov 2008 as manuscript CS20080319 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080319

Fig. 1

 1 Fig. 1 Serum kynurenine to tryptophan ratio (upper), kynurenine (middle) and tryptophan (lower) concentrations in plasma samples of 15 patients post trauma during follow-up for 2 weeks (9 survivors, indicated by straight lines, black box with white cross indicating mean values; 6 non-survivors, dotted lines, black triangle indicating mean values; black circles indicating mean of all patients; gray fields in the background showing control values of a reference group, mean + SD; numbers of subjects (survivors/non-survivors) in bottom line; *p <0.05 survivors compared to non-survivors)

Fig. 1

 1 Fig. 1 Serum kynurenine to tryptophan ratio (upper), kynurenine (middle) and tryptophan (lower) concentrations in plasma samples of 15 patients post trauma during follow-up for 2 weeks (9 survivors, indicated by straight lines, black box with white cross indicating mean values; 6 non-survivors, dotted lines, black triangle indicating mean values; black circles indicating mean of all patients; gray fields in the background showing control values of a reference group, mean + SD; numbers of subjects (survivors/non-survivors) in bottom line; *p <0.05 survivors compared to non-survivors)

Table I .

 I Characteristics of all patients, survivors versus non-survivors. Data are presented as mean values ± SD if not stated otherwise (*Student's t-test, # Pearson's chi-square test, all not significant).

		Clinical Science Immediate Publication. Published on 05 Nov 2008 as manuscript CS20080319
		Survivors	Non-Survivors	p-value
	Number of patients	9	6
	Age	34.3 ± 10.4	50.3. ± 21.8	0.141*
	Gender (female/male) Length of stay in ICU (days) Received inotropes (yes/no) Apache score Injury Severity Score (ISS) A c c e p t e d M a n u s c r i p t 2/7 1/5 0.605 # 32.9 ± 21.3 14.0 ± 12.1 0.137* 8/1 6/0 0.412 # 15.0 ± 4.7 20.8 ± 7.3 0.154* 35.2 ±10.9 44.8 ± 14.9 0.170*	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080319

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

Support by the "Stiftung Propter Homines, Vaduz -Fürstentum Liechtenstein", is gratefully acknowledged. We thank Miss Astrid Haara for excellent technical assistance. Clinical Science Immediate Publication. Published on 05 Nov 2008 as manuscript CS20080319 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080319

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.


Antonio Pinto

Antonino Tuttolomondo

Alessandra Casuccio

Domenico Di Raimondo

Riccardo Di Sciacca

Valentina Arnao

Giuseppe Licata

; Di

Di Sciacca

Immuno-inflammatory predictors of stroke at follow-up in patients with chronic non-valvular atrial fibrillation (NVAF)

Introduction

Atrial fibrillation (AF) is the most common of the serious cardiac rhythm disturbances and it is responsible for substantial morbidity and mortality in the general population [1]. Men and women with AF are at a substantially increased risk of stroke. [START_REF] Fibrillation | Echocardiographic predictors of stroke in patients with atrial fibrillation: a prospective study of 1,066 patients from 3 clinical trials[END_REF]. Nevertheless, the atrial fibrillation population is heterogeneous in terms of ischemic stroke risk and there has been little agreement on how to predict the risk of stroke owing to the fact that clinical schemes to predict risk of stroke do not seem fully exhaustive [START_REF] Baruch | Can patients at elevated risk of stroke treated with anticoagulants be further risk stratified? A comparison of seven stroke prediction schemes[END_REF] On this basis an intriguing opportunity could be to evaluate the possible predictive value of some biomarkers towards ischemic stroke so as to integrate clinical schemes to improve their prognostic power.

Previous studies [START_REF] Inoue | Prothrombotic Activity Is Increased in Patients With Nonvalvular Atrial Fibrillation and Risk Factors for Embolism[END_REF][START_REF] Lip | Fibrin d-dimer and beta-thromboglobulin as markers of thrombogenesis and platelet activation in atrial fibrillation: effects of introducing ultra-low-dose warfarin and aspirin[END_REF][START_REF] Lip | Fibrinogen and fibrin d-dimer levels in paroxysmal atrial fibrillation: evidence for intermediate elevated levels of intravascular thrombogenesis[END_REF] have shown that hypercoagulability was present in patients with non valvular atrial fibrillation (NVAF) by determining the levels of haemostatic markers. Moreover, evidence is growing that inflammation may be associated with AF [START_REF] Chung | C-reactive protein elevation in patients with atrial arrhythmias: inflammatory mechanisms and persistence of atrial fibrillation[END_REF] and it may underlie pathogenesis of the arrhythmia [START_REF] Frustaci | Histological substrate of atrial biopsies in patients with lone atrial fibrillation[END_REF][START_REF] Conway | Prognostic significance of raised plasma levels of interleukin-6 and C-reactive protein in atrial fibrillation[END_REF] and of cerebrovascular events. On this basis, we hypothesized that increased plasma levels of some immuno-inflammatory biomarkers would be associated with an increased risk of stroke in patients with NVAF. To test our hypothesis, we undertook a prospective study to determine rates of stroke in patients with NVAF, evaluating the relationship between plasma levels of some immuno-inflammatory variables at admission and the occurrence of stroke during a three year follow-up. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Methods

Patient Selection

Chronic atrial fibrillation was definied [START_REF] Lévy | International consensus on nomenclature and classification of atrial fibrillation; a collaborative project of the Working Group on Arrhythmias and the Working Group on Cardiac Pacing of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology[END_REF] according to its temporal pattern based on the history of recurrent episodes and the duration of the current episode of AF with regard to two recognised patterns of chronic disease: "persistent atrial fibrillation" describes an episode of sustained atrial fibrillation (usually >7 days) that does not convert to sinus rhythm without medical intervention, with the achievement of sinus rhythm either by pharmacological or electrical cardioversion; "permanent atrial fibrillation" where episodes of persistent (usually >1 year) atrial fibrillation, in which cardio-version is not attempted or is unsuccessful, with atrial fibrillation accepted as the long term rhythm for that person.

Patients were excluded if they had: rheumatic AF or mechanical heart valves, acquired or congenital valvular disease (except mitral annulus calcification or mitral valve prolapse without mitral regurgitation), atrial fibrillation <7 days, echocardiographic evidence of intracardiac thrombosis or tumour, left ventricular aneurysm, severe congestive heart failure (New York Heart Association functional class >III), a recent (<3 months) myocardial infarction, unstable angina or stroke, carotid endarterectomy or coronary or peripheral revascularization procedures performed during the previous 6 months.

Other exclusion criteria were: neoplasms, connective tissue disorder, other chronic inflammatory diseases or a history of recent infection, patients who consumed regular nonsteroidal antiinflammatory drugs, corticosteroids, or hormone replacement therapy.

The study was approved by the Ethics Committee of the "Policlinico P.Giaccone" of Palermo.

Written informed consent was obtained for all patients.

Baseline

At entry, each patient's medical history was taken and he/she underwent a physical examination.

The following diagnostic procedures were also performed: routine blood laboratory tests, baseline was defined as a value of LV mass indexed for height ≥51 gr in both genders [START_REF] Devereux | Evaluation of cardiac and vascular structure and function by echocardiography and other non-invasive techniques[END_REF].

Patients were classified as known diabetic if they had been diagnosed as diabetics according diagnostic criteria of the Expert Committee on The Diagnosis and Classification of Diabetes Mellitus (i.e. a fasting glucose level of ≥ 7 mmol/L or a plasma glucose level of ≥11.1 mmol/L after glucose ingestion) [START_REF]Expert commettee on the diagnosis and classification of diabetes mellitus. Report of the Expert Commettee on the diagnosis and classification of diabetes mellitus[END_REF] or if they are treated by diet, oral hypoglycemic drugs or insulin before hospital admission, whereas new-diagnosed diabetes were classified as 1997 ADA criteria [START_REF]The Expert Comittee on the Diagnosis and Classification of Diabetes Mellitus: Report of the Expert Comittee on the diagnosis and classification of diabetes mellitus[END_REF].

Impaired fasting glucose tolerance (IFGT) was not considered .

Hypertension was defined according to 1993 World Health Organization criteria [systolic blood pressure (SBP) >140 and/or diastolic blood pressure (DBP) >90 mmHg in subject who are not taking antihypertensive medication or antihypertensive treatment on admission] [START_REF] Zanchetti | The 1993 guidelines for the management of mild hypertension: memorandum from a WHO/ISH meeting[END_REF] Hypercholesterolemia was defined as total serum cholesterol ≥5.1 mmol/L and hypertrygliceridemia as total serum trigliceride ≥1.7 mmol/L on the basis of NCEP-ATP III reports [START_REF] Drown | New guidelines for blood cholesterol by the National Cholesterol Education Program (NCEP). National Cholesterol Education Program (NCEP)[END_REF][START_REF]National Cholesterol Education Program Panel. Second Report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel)II[END_REF] that define this cut off for optimal total serum cholesterol and triglyceride levels.

We defined heart failure according to European Society of Cardiology criteria, namely as appropriate symptoms or signs of congestive heart failure (New York Heart Association functional class II or worse) plus objective evidence of cardiac dysfunction [START_REF]Task Force on Heart Failure of the European Society of Cardiology. Guidelines for the diagnosis of heart failure[END_REF].

Atrial fibrillation duration was assessed by careful review of previous hospital records and, where necessary, liaison with general practitioners.

Follow-Up

Patients were periodically monitored (every 6 months) to assess compliance and clinical status and a record was made of the occurrence of outcome event (ischemic stroke). A c c e p t e d M a n u s c r i p t Stroke was defined as rapidly developing signs of focal or global disturbance of cerebral function, lasting ≥24 hours, or leading to death, with no apparent cause other than that of vascular origin [START_REF] Aho | Cerebrovascular disease in the community: results of a WHO collaborative study[END_REF].

The occurrence of stroke was determined on the basis of clinical manifestations and confirmed by

CT or MRI

Outcome Events

Patient events ( ischemic stroke at follow-up) were determined by careful review of hospital records and, where necessary, liaison with general practitioners to determine dates of stroke occurring during the intervening period (since initial blood sampling) during a three year follow-up.

Blood Collection and Laboratory Analysis

Blood samples from each subject enrolled were drawn in the morning within 72 hours of admission after an overnight fast from an antecubital vein by venipuncture into plastic tubes with 3.2% sodium citrate. They were centrifuged (10,000 g) and the resulting supernatant was immediately frozen at -80°C until analysis was completed. We evaluated plasma levels of IL-1β, TNF-α, IL-6, IL-10, Eselectin, P-selectin, ICAM-1, VCAM-1 and vWF. The minimum detectable concentrations for the diagnostic tests are: TNF alpha: 1.6 pg/mL; IL-1β: <1 pg/mL; IL-6: <0.70 pg/mL; IL-10: <3.9 pg/mL; ICAM-1: <0.35 ng/mL; VCAM-1: 0.6 ng/mL; E-Selectin: <0.1 ng/mL; P-Selectin: <0.5 ng/mL; vWF: 1.0%.

Intraassay and interassay coefficients of variation were: TNF alpha:4.2% and 4.6%; IL-1β: 3.3% and 4.2%; IL-6:1.6 % and 3.3%; IL-10: 4.3% and 7.5%; ICAM-1: 4.8% and 6.1%; VCAM-1: 3.5% and 7.7%; E-Selectin: 4.8% and 5.7%; P-Selectin: 4.9% and 8.8%; vWF: 5% and 10%.

Statistical Analysis

For normally distributed variables, results are expressed as mean and standard deviations and for non-normally distributed data as median and interquartile range (IQR) values. Mann-Whitney U test and univariate analysis of variance test (ANOVA) were performed in order to compare nonnormally and normally distributed continuous variables, respectively. Chi-square and chi-square test for trend were used for categorical variables.

A Cox proportional-hazards regression model was used to evaluate adjusted and unadjusted predictive values of each clinical and laboratory variable at baseline to occurrence of the end point stroke during follow-up. The following variables, assessed at the baseline evaluation, were considered as potential predictors of ischemic events: age, male sex, hypertension, diabetes, dyslipidemia, CHF, previous stroke, plasma levels of TNFα, IL6, IL-1β, IL-10, E-Selectin, P-Selectin, ICAM-1, V-CAM-1 and vWF.

Only variables that resulted significant on univariate analysis were included in the final multivariate model.

The best predictive model was composed using backward variable selection strategy, in which a probability value of P < 0.05 was applied for entering or removing a variable. In order to assess the predictive rate of different values of some studied continuous immuno-inflammatory variables for the determination of stroke outcome, a receiver operating characteristic (ROC) curve with their area under the curve (AUC) was constructed.

Survival curves free from stroke onset in relationship with quartiles of immuno-inflammatory variable plasma levels were generated by the Kaplan-Meier method.

All the analyses were evaluated in relation of anticoagulation or antiplatelet treatment and with regard of sex and previous stroke history.

Sample size calculation indicated a sample size of 350 to detect a minimal relative prognostic risk of 2.6 for patients with different levels of cytokines with a alfa error of 0.05 and a power of 0.80 and so we enrolled 373 patients.

Results

We enrolled 451 patients with atrial fibrillation consecutively admitted to our Department of Internal and Specialist Medicine of the University of Palermo. We excluded, on the basis of exclusion criteria, 78 patients (18 for recent cardiovascular event, 30 for paroxysmal or atrial fibrillation with a duration <7 days atrial fibrillation, 10 for mechanical heart valves, 7 for inflammatory disease, 3 who used nonsteroidal anti-inflammatory drugs and 10 for cancer), so we limited biochemical evaluation of immunoinflammatory variables at baseline to 373 patients. The mean duration of atrial fibrillation at baseline was 3.2±1.8 years. 187 (50,1% ) were treated at the time of blood sampling with antivitamin-k antagonists and 88 patients (23.5%) were treated with antiplatelet drugs (table 1).

Median values of TNFα, IL6, IL-1β, IL-10, E-Selectin, P-Selectin, ICAM-1, V-CAM1 and vWF in patients with chronic atrial fibrillation are shown in table 2. No significant difference were observed between subjects with and without anticoagulant treatment. No significant difference was observed between enrolled subjects in relation to clinical risk factors (diabetes, hypertension, smoking, cholesterol, CAD, carotid plaque, left ventricular hypertrophy previous stroke) with regard to plasma levels of biomarkers.

Patients were followed up for 36 months ; the mean duration of follow-up at stroke onset was 22 ±9.7 months. During this follow-up period, there were 51 (13.6%) ischemic strokes ( see table 1). 38 (74,05% ) were detected by a CT scan, whereas 13 (25,49%) by MRI .

Univariate Analysis

Among clinical and laboratory variables age, prior stroke, hypertension, diabetes congestive heart failure (CHF) and CRP were significantly associated with stroke onset at follow-up (see table 3) With regard to cytokine plasma levels, IL6, TNF-α, vWF and IL1β were significantly associated with new onset stroke at follow-up (see table 3). Hazard ratios for quartiles with an analysis of p for trend are given in table 3.

Multivariate analysis

Multivariate data from Cox proportional hazards analysis for stroke are given in table 3 By incorporating as putative predictors all significant variables at univariate analysis, among clinical variables, only age and hypertension were associated with stroke at follow-up. Moreover high levels of IL6 (HR: 2.43; 95% CI = 1.78-3.05; p=0.0001); TNF-α (HR: 2.81; 95% CI=1.80-3.70; p=0.0001) and vWF (HR: 3.69; 95% CI=1.96-4.53; p=0.0001) remained significantly associated with ischemic stroke at follow-up.

The relationships between chosen biomarkers and stroke risk at follow-up were the same for the groups with and without anticoagulant treatment,with and without antiplatelet treatment, for males and females and for the groups with and without previous stroke

Kaplan Meyer Curves

Kaplan-Meier curves were constructed to compare the survival curves of patients in the highest quartile of those immuno-inflammatory variables significantly associated with stroke at follow-up on multivariate analysis versus patients of the other quartiles. As shown in figures 1,2,3 higher plasma levels of IL-6, vWF and TNF-α are significantly associated with ischemic cerebrovascular events at follow-up.

Receiver-Operating Characteristic (ROC) curves

To provide a further demonstration of the predictive value of immuno-inflammatory markers that showed a significant association with new stroke incidence at follow-up we developed a multivariable logistic regression model. The plasma values of TNF-α, IL6 and vWF showed a significant area under the ROC curve (respectively 0.77, 0.74, 0.81). The best cut-off values for TNF-α (35 pg/mL) had a 72.5% sensitivity and 75.7% specificity to predict new-onset stroke at follow-up. Cut-off values of 17 pg/mL and 6 pg/mL maximized sensitivity (66.6% and 78.43%) and specificity (77.6% and 74.2%) for IL-6 and vWF, respectively.

Discussion

In our study we report that baseline plasma levels of TNF-α, IL-6 and vWF are associated with new onset ischemic stroke at follow-up in patients with chronic non-valvular atrial fibrillation (NVAF).

Previous studies suggested that markers of inflammation are elevated in patients with atrial fibrillation (AF) [START_REF] Chung | C-reactive protein elevation in patients with atrial arrhythmias: inflammatory mechanisms and persistence of atrial fibrillation[END_REF][START_REF] Frustaci | Histological substrate of atrial biopsies in patients with lone atrial fibrillation[END_REF][START_REF] Marcus | Interleukin-6 and atrial fibrillation in patients with coronary artery disease: data from the Heart and Soul Study[END_REF]. Nevertheless, no study, to our knowledge, evaluated the role of TNF-α in prediction of stroke in patients with NVAF, whereas some studies previously reported the association of IL-6 [START_REF] Conway | Prognostic significance of raised plasma levels of interleukin-6 and C-reactive protein in atrial fibrillation[END_REF] and vWF baseline plasma values [START_REF] Lip | e d M a n u s c r i p t Table 2: laboratory and immuno-inflammatory variables at baseline in NVAF patients Continous variables are expressed as mean±ds; normally distributed variables were expressed as median and percentiles (first and third quartiles). CRP: C-reactive protein; Tumor Necrosis Factor α; IL-1 β: Interleukin-1β; IL-6: Interleukin-6; IL-10: Interleukin 10; ICAM-1: intercellular adhesion molecule-1; V-CAM-1: vascular cell adhesion molecule-1; vWF: von Willebrand Factor. Total Cholesterol (mmol/L) (n: 373) LDL-cholesterol ( mmol/L ) (n: 373) HDL-cholesterol (mmol(L)[END_REF]21] with stroke at follow-up in these subjects.

However, in addition to these findings [START_REF] Conway | Prognostic significance of raised plasma levels of interleukin-6 and C-reactive protein in atrial fibrillation[END_REF][START_REF] Lip | e d M a n u s c r i p t Table 2: laboratory and immuno-inflammatory variables at baseline in NVAF patients Continous variables are expressed as mean±ds; normally distributed variables were expressed as median and percentiles (first and third quartiles). CRP: C-reactive protein; Tumor Necrosis Factor α; IL-1 β: Interleukin-1β; IL-6: Interleukin-6; IL-10: Interleukin 10; ICAM-1: intercellular adhesion molecule-1; V-CAM-1: vascular cell adhesion molecule-1; vWF: von Willebrand Factor. Total Cholesterol (mmol/L) (n: 373) LDL-cholesterol ( mmol/L ) (n: 373) HDL-cholesterol (mmol(L)[END_REF]21], our study, underlined the predictive role of three inflammatory variables such as TNF-α, IL-6 and vWF as markers of inflammation and endothelial dysfunction involved in thrombogenesis. So our results encompass three main events of vascular damage: inflammation, endothelial dysfunction and thrombosis as predictive markers of future cardioembolic ischemic events and this could represent a novel finding. In particular, in our study, ROC curve analysis emphasizes the role of vWF plasma.

Cytokines such as IL-6 can induce a prothrombotic state by increasing expression of fibrinogen, tissue factor, factor VIII and von Willebrand factor, as well as by activating endothelial cells and increasing platelet production [22]. These findings may provide a link between inflammation and thrombosis and may explain the association of IL-6, TNF-α and VWF with ischemic stroke at follow-up in our patients with atrial fibrillation.

Nevertheless, it is possible to hypothesize that inflammatory markers could express not only a cardiac inflammatory background, but also inflammation and endothelial dysfunction of the recipient cerebral vessels and microvessel, in a context of atherosclerotic systemic vascular inflammation, so as to predispose brain arterial occlusion due to an embolus arising in the heart.

A c c e p t e d M a n u s c r i p t

What is the possible clinical and therapeutic involvement of this reported predictive power of some immuno-inflammatory markers in NVAF setting?

The rate of stroke in nonvalvular atrial fibrillation (AF) ranges widely and depends on the presence of prior cerebral ischemia, comorbid conditions, and use of antithrombotic therapy. Without antithrombotic therapy, the rate varies from fewer than 2 to more than 10 strokes per 100 patientyears. Thus, quantifying the risk of stroke is crucial for determining which AF patients would benefit most from warfarin therapy.

Several schemes exist to risk stratify subjects with NVAF to help select appropriate candidates for anticoagulant therapy, but their predictive abilities in patients receiving anticoagulant or antiplatelet treatment are unknown. Predicting which patients with atrial fibrillation will have a stroke or systemic embolic event is not an easy task. Baruch So, in the future, a more accurate prediction rule for stroke may be possible by incorporating additional factors such as immunoinflammatory variables at baseline. These immuno-inflammatory biomarkers could be one possible way to address the anticoagulant treatment or for a dose adjustment towards more "aggressive" doses or therapeutic targets in patients with higher levels of these variables potentially predictive of cerebrovascular events occurrence at follow-up Our findings could suggest that, in patients with chronic NVAF, a combination of biomarkers such as TNF-α, IL-6 and vWF at admission, could carry prognostic information towards new cerebrovascular events at three year follow-up. Furthermore, these results could reinforce the need to assess whether, in clinical practice, inflammatory biomarkers can be used to tailor optimal medical management of this subset of patients by addressing more intensive anticoagulation treatment in patients with higher plasma levels of immuno-inflammatory variables. A possible limitation of this study is that we cannot completely exclude the possibility that incidence of new onset stroke at follow-up may have been influenced by the use of antithrombotic therapy (most of the enrolled patients were receiving some form of antithrombotic therapy during the study). Nevertheless on multivariate analysis, adjusting for antithrombotic therapy, we showed no significant effect of antithrombotic treatment on the observed HR for each cytokine.

Only point measurement could be another limitation of the study since repeated measures may be necessary to define the variance as well as predictive nature of a biomarker [24], but in support of our study, several studies previously reported the predictive value of only point measurement of inflammatory biomarkers in atrial fibrillation [START_REF] Lip | Fibrin d-dimer and beta-thromboglobulin as markers of thrombogenesis and platelet activation in atrial fibrillation: effects of introducing ultra-low-dose warfarin and aspirin[END_REF][START_REF] Lip | Fibrinogen and fibrin d-dimer levels in paroxysmal atrial fibrillation: evidence for intermediate elevated levels of intravascular thrombogenesis[END_REF][START_REF] Chung | C-reactive protein elevation in patients with atrial arrhythmias: inflammatory mechanisms and persistence of atrial fibrillation[END_REF][START_REF] Conway | Prognostic significance of raised plasma levels of interleukin-6 and C-reactive protein in atrial fibrillation[END_REF] Moreover with our sample size it is possible to detect a minimal relative prognostic risk of 2.6 for patients with different levels of cytokines with an alfa error of 0.05 and a power of 0.80.

Nevertheless this is a prospective longitudinal study with a pathophysiologic background and on this basis our sample size would be plausible even without a prior evaluation of study size.

Finally, a possible question is how many of the observed strokes at follow-up are of cardioembolic origin. The predictive value of these cytokines was calculated for the simple endpoint ischemic stroke. Thus, it is unclear from our study whether TNF-α, IL-6 and vWF are sensitive to ischemic cerebrovascular events of thromboembolic or also atherotrombotic origin. Larger sample size is, therefore, necessary to analyze the relationship between these immuno-inflammatory variables and each diagnostic subtype of ischemic stroke. Assuming that the patients were adequately anticoagulated the majority of "ischemic stroke" events could be related to atherosclerotic disease and not to embolism due to atrial fibrillation. Thus, the association of high cytokine levels with ischemic stroke could probably reflect more extensive cerebrovascular atherosclerotic disease in these patients in addition to indicating a possible link between inflammation and cardiac embolism. However, even in adequately anticoagulated patients embolic events are commonly registered. Evans A. et al. [25] in 392 patients with NVAF considered for adjusted-dose warfarin and followed up for 2 years reported a rate of stroke incidence respectively of 9.5% and 4.9% showing that stroke rate was due predominantly to cardioembolic recurrence in patients presenting initially with cardioembolic stroke. So it's plausible that most of the observed events in our enrolled subjects could be of cardioembolic origin.

In conclusion, our study showed that baseline plasma levels of TNF-α, IL-6 and vWF are associated with new onset ischemic stroke at follow-up in patients with chronic non valvular atrial fibrillation so encompassing three markers of inflammation, endothelial dysfunction and thrombosis as predictive markers of future cerebral ischemic events.

Medications at discharge

  Patients with chronic non valvular AF consecutively admitted to the Division of Internal Medicine and Cardioangiology of the Policlinico "P.Giaccone" of Palermo from 1998 to 2003 were enrolled. Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080372 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080372

Clinical

  Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080372 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080372 A c c e p t e d M a n u s c r i p t 12-lead ECG, M-mode, and 2-dimensional echocardiography with echocolor-doppler, echocolordoppler of the supra-aortic trunks, and chest roentgenography. Left ventricular hypertrophy (LVH)

Clinical

  Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080372 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080372

  IL-1β, TNF-α, IL-6 and IL-10 and VWF antigen were measured using a sandwich ELISA (Human IL-1β, TNF-α, IL-6 and IL-10 Quantikine, R&D Systems) (VWF ELISA kit durian, Instrumentation Laboratory, Milano, Italy); VCAM-1, ICAM-1, E-selectin, P-selectin, were measured by commercial bioimmunoassay (Human sICAM-1, sVCAM-I, sE-selectin and sP-selectin Parameter, Quantikine, R&D Systems ELISA Kit).

Clinical

  Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080372 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080372 A c c e p t e d M a n u s c r i p t

  Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080372 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080372 A c c e p t e d M a n u s c r i p t All statistical tests used were two-sided. Values of P < 0.05 were considered statistically significant. SPSS version 10.0 for Windows software (SPSS, Inc., Chicago, IL) was used for data analysis.

Clinical

  Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080372 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080372 A c c e p t e d M a n u s c r i p t

Clinical

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Clinical

  Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080372 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080372

  schemes in a large clinical trial-based program of patients with atrial fibrillation showing that all

Clinical

  Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080372 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080372 A c c e p t e d M a n u s c r i p t

Clinical

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  21. Roldán V, Marín F, García-Herola A, Lip GY. Correlation of plasma von Willebrand factor levels, an index of endothelial damage/dysfunction, with two point-based stroke risk stratification scores in atrial fibrillation. Thromb Res. 2005;116(4):321-5. 22. Kerr R, Stirling D, Ludlam CA. Interleukin 6 and haemostasis. Br J Hematol 2001;115:3-12. 11. 23. Baruch L, Gage BF, Horrow J, Juul-Moller S, Labovitz A, Persson M, Zabalgoitia M. Can patients at elevated risk of stroke treated with anticoagulants be further risk stratified? A comparison of seven stroke prediction schemes. Stroke 2007;38:2459 -2463 . 24. MacFadyen RJ Intraindividual temporal variance of biomarkers and the definition of individualized prognosis in cardiovascular patients. J Hum Hypertens.2006 Jun;20(6):383-6. 25. Evans A, Perez I, Yu G, Kalra L Should Stroke subtype influence anticoagulation decisions to prevent recurrence in stroke patients with atrial fibrillation Stroke 2001; 32:2828-32. Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080372 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080372 A c c e p t e d M a n u s c r i p t

A c c e

  p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Figure 3 :Quartile 1 2 Clinical

 312 Figure 3 :Kaplan-Meier analysis. Ischemic stroke-free survival by vWF quartiles

Table 1 : Demographic and clinical variables in NVAF patients Dichotomic variables are expressed as frequence (n/%); Continous variables are expressed as mean±ds. CAD: Coronary Artery Disease; LVH: left ventricular Hypertrophy; CHF: congestive heart failure SBP: Systolic blood pressure; DBP: diastolic blood pressure; EF: Ejection Fraction

 1

	Sex (M/W)	237/136 (63.5/34.6)
	Age (years)	66.08 ±7.35
	Hypertension (n/%)	270 (72)
	Diabetes (n/ %)	164 (43.9)
	CAD (n/ %)	208 (55.7)
	LVH (n/%)	119 (31.9)
	CHF (n/%)	88 (23.5)
	Smoking habit (n/%)	124 (33.15)
	Carotid plaque (n/ %)	90 ( 24.1)
	Previous stroke (n/ %)	34 (9.1)

Medications at at the time of blood sampling

	Antiplatelet treatment	88 (23.5)
	Vitamin-K-antagonists	187 (50,1)
	Heparine	10 (2.6)
	Statins	80 (21.45)
	ACE inhibitors	171 (45.84)
	Angiotensin type-1 receptor blockers	57 (15.28)
	Calcium antagonists	187 (50.73)
	Beta blockers	101 (27.07)
	Diuretics	181 (48.52)
	Digitalis	202 (54.11)

mm/Hg) (mean ±ds) (n: 373) DBP (mm/Hg) (mean ±ds) (n: 373)

	Antiplatelet treatment	122 (33.8)
	Vitamin-K-antagonists	251 (67.2)
	Heparine	0 (0)
	Statins	95 (25.46)
	ACE inhibitors	154 (41.28)
	Angiotensin type-1 receptor blockers	89 (23.86)
	Calcium antagonists	201 (53.8)
	Beta blockers	122 (32.7)
	Diuretics	193 (51.74)
	Digitalis	134 (35.92)
	SBP ( 136±6.8
		87±4.5
	EF (%)

(mean ±ds) (n: 373) 48±3 New onset ischemic stroke at follow-up ( fatal and not fatal) ( n/%)

	Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080372
	Fatal stroke (n/%) A c c e p t e d M a n u s c r i p t 51 (13.6) 5 (9.8 )	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080372

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Table 3 -Univariate and multivariate Cox analysis adjusted for age (with 95% CI) about the relative contribution of each clinical and laboratory variable at baseline to occurrence of the end point stroke during follow- up and hazard ratios for quartiles with the analyse of p for trend. CHF: congestive heart failure; CAD: Coronary Artery Disease; LVH: left ventricular Hypertrophy; CRP: C-reactive protein; TNF-α: Tumor Necrosis Factor α; IL-1 β: Interleukin-1-β; IL-6: Interleukin-6; IL-10: Interleukin 10; ICAM-1: intercellular adhesion molecule-1; V-CAM-1: vascular cell adhesion molecule-1; vWF: von Willebrand Factor. *p for trend for quartiles are given for immunoinflammatory variables significantly associated with stroke at univariate analysis.

 3 Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080372 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080372

		Univariate analysis
		HR (95% CI	p
	Age	1.87 (1.10 -2.98)	0.0001
	Hypertension	1.33 (1.12 -1.85)	0.03
	Diabetes	1.37 (1.52 -3.11)	0.04
	CHF	1.38 (1.13-1.94	0.032
	Previous stroke	2.60 (1.49 -3.49)	0.0001
	Smoking habit	0.85 (0.50 -1.02)	0.55
	CAD	0.75 (0.50 -1.22)	0.25
	LVH	0.35(0.26 -1.02)	0.65
	CRP	1.55 ( 1,21-2.35)	0.039
	Il-10	0.79 (0.20 -1.02)	0.35
	ICAM-1	0.45 (0.36 -1.03)	0.21
	VCAM-1	0.95 (0.40 -1.12)	0.66
	P-selectin	0.59(0.25 -1.11)	0.38
	E-selectin	0.55(0.26 -1.02)	0.65
				HR for	p for
				quartiles	trend *
	IL1-β	1.21 (1.00 -1.54)	0.036 Quartile 1	1.0
			Quartile 2	2.16	0.056
			Quartile 3	0.63
			Quartile 4	4.43
	IL6	1.93 (1.02 -2.05)	0.0001 Quartile 1	1.0
			Quartile 2	0.26
			Quartile 3	2.11	<0.0005
			Quartile 4	9.37
	TNF-α	2.07 (1.72 -2.98)	0.0001 Quartile 1	1.0
			Quartile 2	2.67
			Quartile 3	18.18	<0.0005
			Quartile 4	14.29
	vWF	2.86 (1.73-3.04)	0.0001 Quartile 1	1.0
			Quartile 2	1.42
			Quartile 3	7.14	<0.0005
			Quartile 4	308.00
	Multivariate analysis
		HR (95% CI)	p
	Age	1.94 (0.76-2.69)	0.005
	Hypertension	1.83 (1.01-2.55)	0.005
	Diabetes	0.81 (0.20-1.32)	0.06
	CHF	0.69 (0.26-1.53)	0.43
	Previous stroke	0.55 (0.38-1.25)	0.26
	CRP	0.89 ( 0.56-1.21)	0.059
	IL-1 β	0.85 (0.63-1.33)	0.06
	IL6	2.43 (1.78-3.05)	0.0001
	TNF-α	2.81 (1.80-3.70)	0.0001
	vWF	3.69 (1.96-4.5)	0.0001

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society