Thrombosis Research 134 (2014) 832–836
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Serum soluble CD40 Ligand levels are associated with severity and mortality of brain trauma injury patients Leonardo Lorente a,⁎, María M. Martín b, Agustín F. González-Rivero c, Luis Ramos d, Mónica Argueso e, Juan J. Cáceres f, Jordi Solé-Violán g, Nicolás Serrano a, Sergio T. Rodríguez b, Alejandro Jiménez h, Juan M. Borreguero-León c a
Intensive Care Unit, Hospital Universitario de Canarias, Ofra, s/n. La Laguna - 38320, Santa Cruz de Tenerife, Spain Intensive Care Unit, Hospital Universitario Nuestra Señora de Candelaria, Crta del Rosario s/n. Santa Cruz de Tenerife - 38010, Spain c Laboratory Deparment, Hospital Universitario de Canarias, Ofra, s/n. La Laguna - 38320, Santa Cruz de Tenerife, Spain d Intensive Care Unit, Hospital General La Palma, Buenavista de Arriba s/n, Breña Alta, La Palma- 38713, Spain e Intensive Care Unit, Hospital Clínico Universitario de Valencia, Avda, Blasco Ibáñez n°17-19, Valencia - 46004, Spain f Intensive Care Unit, Hospital Insular, Plaza Dr. Pasteur s/n. Las Palmas de Gran Canaria - 35016, Spain g Intensive Care Unit, Hospital Universitario Dr. Negrín, Barranco de la Ballena s/n. Las Palmas de Gran Canaria - 35010, Spain h Research Unit, Hospital Universitario de Canarias, Ofra, s/n. La Laguna - 38320, Santa Cruz de Tenerife, Spain b
a r t i c l e
i n f o
Article history: Received 22 May 2014 Received in revised form 24 June 2014 Accepted 30 July 2014 Available online 2 August 2014 Keywords: sCD40L Brain trauma Patients Mortality Injury
a b s t r a c t Background: Serum soluble CD40 Ligand (sCD40L) levels, which exhibit prothrombotic and proinflammatory properties, have not been studied in patients with traumatic brain injury (TBI). Thus, the objective of this study was to determine whether serum sCD40L levels are associated with severity and mortality in patients with severe TBI. Methods: This was a prospective, observational and multicenter study carried out in six Spanish Intensive Care Units. Patients with severe TBI defined as Glasgow Coma Scale (GCS) lower than 9 were included, while those with Injury Severity Score (ISS) in non-cranial aspects higher than 9 were excluded. Serum levels of sCD40L were measured on the day of TBI. Endpoint was established in 30-day mortality. Results: We found higher serum sCD40L levels (P b 0.001) in non-surviving TBI patients (N = 27) than in survivor ones (N = 73). Logistic regression analysis showed that serum sCD40L levels were associated with 30-day mortality (OR = 1.58; 95% CI = 1.12-2.21; P = 0.008) controlling for APACHE-II score and computer tomography findings. The area under the curve (AUC) for serum sCD40L levels as predictor of 30-day mortality was 0.79 (95% CI = 0.70-0.86; P b 0.001). Survival analysis showed that patients with serum sCD40L levels higher than 2.11 ng/mL presented increased 30-day mortality than patients with lower levels (Hazard ratio = 9.0; 95% CI = 4.25-19.27; P b 0.001). We found an association between serum sCD40L levels and APACHE-II (rho = 0.33; P = 0.001), and GCS score (rho = -0.21; P = 0.04). Conclusions: To our knowledge, this is the first study reporting data on serum sCD40L levels in patients with severe TBI. The most relevant and newer findings of our study are that serum sCD40L levels in non-surviving patients with severe TBI are higher than in surviving ones, and that there are an association between serum sCD40L levels and TBI severity and mortality. © 2014 Elsevier Ltd. All rights reserved.
Introduction Traumatic brain injury (TBI) is an important cause of death, disability and resources consumption [1]. Initial primary injury is referred to the physical forces applied to brain during the impact. Secondary injury occurs later over a period of hours or days after the initial traumatic Abbreviations: sCD40L, soluble CD40 Ligand; TNF, tumour necrosis factor; TF, tissue factor; ICU, Intensive Care Unit; SOFA, Sepsis-related Organ Failure Assessment score. ⁎ Corresponding author. E-mail address: [email protected] (L. Lorente).
http://dx.doi.org/10.1016/j.thromres.2014.07.034 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
injury and is due to different mechanisms [2,3]. Among them, inflammation and thrombosis are mechanisms that play a pivotal role in this delayed response. CD40 Ligand (CD40L) and its soluble counterpart (sCD40L) are proteins that exhibit proinflamatory [4,5] and procoagulant [6–11] properties on binding to their cell surface receptor CD40 [12,13]. CD40L is a member of the tumor necrosis factor (TNF) family and is expressed as a transmembrane protein in activated platelets [14,15]. Raised levels of sCD40L have been found in patients with acute coronary syndrome [16,17], stroke [18–22] and sepsis [23,24]. However, circulating sCD40L levels has not been studied in patients with TBI.
L. Lorente et al. / Thrombosis Research 134 (2014) 832–836
Thus, the objective of this study was to determine whether serum sCD40L levels are associated with mortality, severity, and inflammatory and prothrombotic markers in patients with severe TBI. Methods Design This was a prospective, observational, multicenter study carried out in 6 Intensive Care Units of Spain belonging to the following hospitals: Hospital Universitario de Canarias (La Laguna, Tenerife, Spain), Hospital Universitario Nuestra Señora de Candelaria (Tenerife, Spain), Hospital General de La Palma (La Palma, Tenerife, Spain), Hospital Clínico Universitario de Valencia (Valencia, Spain), Hospital Insular (Las Palmas de Gran Canaria, Spain), Hospital Universitario Dr. Negrín (Las Palmas de Gran Canaria, Spain). The study was approved by the Institutional Ethic Review Boards of the 6 participant hospitals. A written informed consent from the patient or from the legal next of kin was obtained. We included 100 patients with severe TBI. Severity of brain trauma injury was classified according to Glasgow Coma Scale (GCS) [25], and severe TBI was defined as GCS lower than 9 points. Exclusion criteria were: age less than 18 years, pregnancy, inflammatory or malignant disease, and Injury Severity Score (ISS) [26] in non-cranial aspects higher than 9 points. The following variables were recorded for each patient: sex, age, ISS, GCS, blood lactate, platelets, international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen, Acute Physiology and Chronic Health Evaluation II (APACHE II) score [27] and brain lesion according to the Marshall computer tomography (CT) classification [28]. The end-point was 30-day mortality. Laboratory Determinations Blood samples were collected on the day of TBI to measure serum sCD40L levels. We also measured serum TNF-alpha levels, and plasma tissue factor (TF) levels. Venous blood samples were collected in serum separator tubes (SST) for determination of serum sCD40L and TNF-alpha levels; and in citrate collected plasma tubes to determination of plasma TF levels. Blood samples were centrifuged within 30 minutes at 1000*g for 15 min. The serum and plasma were removed and frozen at -80 °C until measurement. The determination of serum sCD40L and TNFalpha levels, and plasma TF levels were centralized in the Laboratory Department of the Hospital Universitario de Canarias (La Laguna, Santa Cruz de Tenerife, Spain). Serum sCD40L levels were assayed by specific ELISA (Bender MedSystems GmbH, Vienna, Austria). The intra-assay and inter-assay coefficients of variation (CV) were 4% (n = 8) and 6.8% (n = 8) respectively; and detection limits for the assays was 0.06 ng/mL. Serum TNF-alpha levels were measured by a solid-phase, chemiluminiscents immunometrics assays kit (Immulite®, Siemens Healthcare Diagnostics Products, Llanberis, United Kingdom). The intra-assay and inter-assay CV were b3.6 % (n = 20) and b6.5% (n = 20) respectively; and detection limits for the assays was 1.7 pg/mL. Plasma TF levels were assayed by specific ELISA (Imubind® Tissue Factor ELISA, American Diagnostica, Inc, Stanford, CT, USA). The intraassay and inter-assay CV were b7.2% (n = 20) and b 8% (n = 20) respectively; and detection limits for the assays was 10 pg/mL.
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on categorical variables were carried out with chi-square test. Multiple binomial logistic regression analysis was applied to predict 30-day mortality. As number of events was 27 exitus, we constructed two multiple binomial logistic regression models with only three predictor variables in each to avoid an over fitting effect that may lead to choose a final model of order slightly higher than required [29]. In the first regression model were included serum sCD40L, APACHE-II score and CT classification. Previously to include the variable CT classification in the regression analysis, it was recoded according with the risk of death observed in the bivariated analysis as low (CT types 2 and 5) and high risk (CT types 3, 4 and 6) of death. In the second regression model were included serum sCD40L levels, GCS and age. Odds Ratio and 95% confidence intervals were calculated as measurement of the clinical impact of the predictor variables. Receiver operating characteristic (ROC) analysis was carried out to determine the goodness-of-fit of the of serum sCD40L levels to predict 30-day mortality. Kaplan-Meier analysis of survival at 30 days and comparisons by log-rank test were carried out using serum sCD40L levels lower/higher than 2.11 ng/mL as the independent variable and survival at 30 days as the dependent variable. The association between continuous variables was carried out using Spearman´s rank correlation coefficient. A P value of less than 0.05 was considered statistically significant. Statistical analyses were performed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and NCSS 2000 (Kaysville, Utah) and LogXact 4.1, (Cytel Co., Cambridge, MA). Results From the 100 TBI patients in the study, non-survivors (n = 27) showed lower GCS, and higher age, female rate and APACHE-II score than survivors (n = 73). CT classification also showed statistically significant differences between non-surviving and surviving patients. In addition, non-survivors showed higher serum sCD40L levels than survivors. However, no significant differences were found between nonsurviving and surviving patients in serum TNFalpha and plasma TF levels (Table 1). As shown in Table 1, mortality rate varied with CT classification types: thus 3/24 patients (12.5%) in type 2, 5/18 (27.8%) in type 3, 6/ 16 (37.5%) in type 4, 5/31 (16.1%) in type 5 and 8/11 (72.7%) in type 6. Once the CT classification variable was recoded for the regression analysis the new variable considered CT classification types 2 and 5 as low death risk, with a observed mortality rate of 8/55 (14.5%), and CT classification types 3, 4 and 6 as high death risk, with a observed mortality rate of 19/45 (42.2%). Multiple binomial logistic regression analysis showed that serum sCD40L levels could predict 30-day mortality (OR = 1.58; 95% CI = 1.12-2.21; P = 0.008) controlling for APACHE-II and CT classification (Table 2). Multiple binomial logistic regression analysis also showed that serum sCD40L levels could predict 30- day mortality (OR = 1.43; 95% CI = 1.05-1.95; P = 0.02) controlling for GCS and age (Table 2). The area under the curve (AUC) for serum sCD40L levels as predictor of 30-day mortality was 0.79 (95% CI = 0.70-0.86; P b 0.001) (Fig. 1). Survival analysis showed that patients with serum sCD40L levels higher than 2.11 ng/mL presented higher 30-day mortality than patients with lower levels (Chi-square: 19.8; Hazard ratio = 9.0 (95% IC = 4.2519.27); P b 0.001) (Fig. 2). We found an association between serum sCD40L levels and APACHE-II (rho = 0.33; P = 0.001), GCS score (rho = -0.21; P = 0.04) and age (rho = 0.30; P = 0.003) (Table 3).
Statistical Methods
Discussion
Continuous variables are reported as medians and interquartile ranges. Categorical variables are reported as frequencies and percentages. Comparisons of continuous variables between groups were carried out using Wilcoxon-Mann-Whitney test. Comparisons between groups
Raised levels of sCD40L related to impaired prognosis have been previously reported in patients with acute coronary artery syndrome and [30] and sepsis [23,24]; however, to our knowledge, this is the first study reporting data on serum sCD40L levels in patients with severe TBI.
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Table 1 Baseline clinical and biochemical characteristics of survivor and non-survivor patients.
Gender female – n (%) Age (years) - median (p 25-75) Computer tomography classification - n (%) · Type 1 · Type 2 · Type 3 · Type 4 · Type 5 · Type 6 Temperature (°C) - median (p 25-75) Sodium (mEq/L)- median (p 25-75) Glycemia (g/dL) - median (p 25-75) Leukocytes - median*103/mm3 (p 25-75) PaO2 (mmHg) - median (p 25-75) PaO2/FI02 ratio - median (p 25-75) Bilirubin (mg/dl) - median (p 25-75) Creatinine (mg/dl) - median (p 25-75) Hemoglobin (g/dL) - median (p 25-75) Glasgow Coma Scale score - median (p 25-75) Lactic acid (mmol/L) median (p 25-75) Platelets - median*103/mm3 (p 25-75) INR - median (p 25-75) aPTT (seconds) - median (p 25-75) Fibrinogen (mg/dl) - median (p 25-75) APACHE-II score - median (p 25-75) ISS - median (ppe 25-75) ICP (mmHg) - median (p 25-75) CPP (mmHg) - median (p 25-75) sCD40L (ng/mL) - median (p 25-75) TNF-alpha (pg/mL) - median (p 25-75) Tissue Factor (pg/mL) - median (p 25-75)
Survivors (n = 73)
Non-survivors (n = 27)
P value
12 (16.4) 47 (32-67)
11 (40.7) 66 (45-76)
0.02 b0.001 0.002
0 21 (28.8) 13 (17.8) 10 (13.7) 26 (35.6) 3 (4.1) 37. (35.6-37.3) 139 (138-142) 139 (120-163) 14.7 (10.2-19.3) 151 (116-217) 336 (242-407) 0.50 (0.40-0.87) 0.80 (0.70-0.90) 11.4 (10.4-13.0) 7 (6-8) 1.70 (1.23-2.50) 182 (143-252) 1.03 (0.92-1.15) 28 (25-32) 350 (282-444) 19 (17-23) 25 (25-32) 15 (14-20) 68 (57-70) 1.80 (0.60-2.79) 9.72 (7.88-13.40) 189 (129-247)
0 3 (11.1) 5 (18.5) 6 (22.2) 5 (18.5) 8 (29.6) 36.0 (35.0-37.0) 141 (135-149) 161 (142-189) 18.3 (10.7-23.9) 141 (104-186) 190 (154-316) 0.75 (0.53-1.05) 0.95 (0.70-1.10) 11.1 (9.4-12.3) 3 (3-6) 1.90 (1.15-4.55) 215 (139-264) 1.22 (1.01-1.67) 26 (25-31) 376 (246-560) 26 (25-32) 25 (25-27) 20 (12-30) 60 (54-69) 4.00 (2.36-5.46) 13.65 (8.35-22.75) 194 (169-295)
0.12 0.19 0.08 0.46 0.34 0.11 0.045 0.44 0.87 b0.001 0.16 0.48 0.15 0.86 0.32 b0.001 0.24 0.27 0.46 b0.001 0.12 0.18
P 25-75 = percentile 25th-75th; PaO2 = pressure of arterial oxygen/fraction inspired oxygen; FIO2 = pressure of arterial oxygen/fraction inspired oxygen; ISS = Injury Severity Score; INR = international normalized ratio; aPTT = activated partial thromboplastin time; APACHE II = Acute Physiology and Chronic Health Evaluation; ICP = intracranial pressure; CPP = cerebral perfusion pressure; TNF = tumor necrosis factor.
The role of sCD40L in TBI remains unclear but it is possible that its proinflamatory [4,5] and procoagulant [6–11] effects could contribute to the pathophysiology of TBI. CD40L is stored in α-granules in unstimulated platelets but it rapidly translocates to the surface when platelets become activated. Once in the platelet surface, CD40L is cleaved and released into circulation as sCD40L. The sCD40L binds to circulating monocytes through its receptor CD40, promoting their adhesion to vascular endothelium. The sCD40L also binds to CD40 on endothelial cell surfaces. Activated endothelial cells produce the overexpression of transcriptional factors such as nuclear factor-kappa B [NFkß] [31], with subsequent up regulation of proinflammatory and prothrombotic factors. Ligation of CD40 on endothelial cells, smooth muscle cells, or mononuclear phagocytes triggers the expression of various proinflammatory mediators, such as the interleukin (IL)-1, IL-6, IL-12, TNF-alpha, and interferon-gamma [5]. In addition, sCD40L could have prothrombotic effects via induction of TF [6–9],
diminishing thrombomodulin expression [8,9], and binding to the glycoprotein IIb/IIIa platelet receptor [10,11]. All these effects could facilitate the development of vascular thrombosis, brain ischemia and death. The most relevant and newer findings of our study are: a) nonsurviving TBI patients had higher serum sCD40L levels than surviving
Table 2 Multiple binomial logistic regression analysis of variables to predict 30-day mortality. Variable First Model Serum sCD40L levels APACHE-II score Computer tomography classification (reference category: low risk of death) Second Model Serum sCD40L levels GCS score Age
Odds Ratio
95% Confidence Interval
P
1.58 1.38 5.94
1.12–2.21 1.18–1.62 1.39–25.30
0.008 b0.001 0.02
1.43 0.59 1.06
1.05–1.95 0.43–0.80 1.02–1.10
0.02 0.001 0.003
APACHE II = Acute Physiology and Chronic Health Evaluation; GCS = Glasgow Coma Scale.
Fig. 1. Receiver operation characteristic analysis using serum sCD40L levels as predictor of mortality at 30 days. For cut-off serum sCD40L levels higher than 2.11 ng/mL, we found sensitivity = 89% (95% CI = 71-98%), specificity = 70% (95% CI = 48-72%), positive predicted value = 45% (95% CI = 32-60%), negative predicted value = 94% (95% CI = 8299%), positive likelihood ratio = 2.2 (95% CI = 1.3-3.1), negative likelihood ratio = 0.2 (95% CI = 0.01-0.05).
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However, some limitations of our study should be recognized. First, we did not perform the analysis of serum sCD40L levels during followup to describe the evolution in nonsurviving and surviving TBI patients. Second, we have determined sCD40 levels only in serum and not in plasma samples to evaluate possible differences due to there has been reported higher sCD40L levels in serum than in plasma levels [38]. Third, it is known that functional TF is bound to the outer leaflet of cell membrane including intact monocytes as well as microparticles shed from monocytes [39]; thus, it is possible that only a certain portion of TF could be detected by the method used for the detection of TF in the present study. Four, we have determined TNF-alpha due to that ligation of CD40 to receptor triggers the expression of various proinflammatory mediators, such as TNF-alpha [5]; and we have determined TF due to that sCD40L could have prothrombotic effects via induction of TF [6–9]. However, we did not found an association between serum sCD40L, TNF-alpha, and TF levels. Thus, the analysis of other coagulation factors, cytokines or microparticles could be interesting. Finally, additional studies are needed to confirm the findings of our study. Fig. 2. Survival curves at 30 days using 2.11 ng/mL of serum sCD40L levels as cut-off. Patients with serum sCD40L levels higher than 2.11 ng/mL presented higher 30-day mortality (Log rank = 19.8; Hazard ratio = 9.0 (95% IC = 4.25-19.27); P b 0.001).
ones, b) there was an association between serum sCD40L levels and TBI severity and mortality, and c) serum sCD40L levels could be used as a prognostic biomarker of mortality in TBI patients. We chose mortality at 30 days as end point because this is an endpoint used frequently in research studies. We found for the first time an association between serum sCD40L levels and patient severity assess by APACHE II and GCS scores. However, we did not found an association between serum sCD40L and TNFalpha. Neither, we found an association between serum sCD40L and TF levels, which has been described in culture of vascular endothelial cells [6–9]. It is possible that other reported prothrombotic effects of sCD40L, such as reduced thrombomodulin expression [8,9] and binding to the glycoprotein IIb/IIIa platelet receptor [10,11] could lead to vascular thrombosis, brain ischemia and, finally, death in these patients with TBI. We found for the first time an association between serum sCD40L levels and age in TBI patients; this association was previously described in healthy subjects [32] and patients with coronary disease [33]. We have not found an association between serum sCD40L levels and platelet count and coagulation, although platelets are the major source of sCD40L in circulation [14,15]. From a therapeutic perspective, the use of sCD40L modulators could be used as a new class of drugs for the treatment of TBI [34–37]. In some studies including patients with coronary artery disease, the use of statins decreased circulating sCD40L levels [34–36]. Besides, in animal TBI models have been found the benefit of statins [37]. It is possible that TBI patients, over all those with high serum sCD40L levels, could benefit of the early administration of statins.
Table 3 Correlation between serum sCD40L levels and other baseline clinical and biochemical characteristics. APACHE-II score GCS score Serum TNF-alpha (pg/mL) levels Plasma tissue factor (pg/mL) levels Age (years) Bilirubin (mg/dl) Platelets count INR aPTT (seconds)
rho = 0.33; P = 0.001 rho = -0.21; P = 0.04 rho = 0.16; P = 0.40 rho = 0.02; P = 0.87 rho = 0.30; P = 0.003 rho = 0.16; P = 0.13 rho = -0.02; P = 0.87 rho = 0.03; P = 0.79 rho = 0.05; P = 0.66
APACHE II = Acute Physiology and Chronic Health Evaluation; GCS = Glasgow Coma Scale; TNF = tumor necrosis factor; INR = international normalized ratio; aPTT = activated partial thromboplastin time.
Conclusions To our knowledge, this is the first study reporting data on serum sCD40L levels in patients with severe TBI. The most relevant and newer findings of our study are: a) Serum sCD40L levels in nonsurviving patients with severe TBI are higher than in surviving ones, b) There are an association between serum sCD40L levels and TBI severity and mortality, and c) Serum sCD40L levels could have a role as a prognostic biomarker of mortality in TBI patients. Competing Interests None Authors´Contributions LLo was responsible of conceive, design and coordinate the study, made substantial contributions to acquisition of data, analysis and interpretation of data, and drafted the manuscript. MMM, LR, MA, JJC, JSV, NS, STR have made substantial contributions to acquisition of data and provided useful suggestions. AGR and JMBL carried out the determination of sCD40L, TNF-α and TF determination and have made substantial contributions to analysis and interpretation of data. AJ has made substantial contributions to analysis and interpretation of data All authors revising the manuscript critically for important intellectual content and approved the final version of the manuscript Funding This study was supported, in part, by grants from Instituto de Salud Carlos III (I3SNS-INT-11-063 and I3SNS-INT-12-087) (Madrid, Spain) and co-financed with Fondo Europeo de Desarrollo Regional (FEDER). Acknowledgments This study was supported, in part, by grants from Instituto de Salud Carlos III (I3SNSINT-11-063 and I3SNS-INT-12-087) (Madrid, Spain) and co-financed with Fondo Europeo de Desarrollo Regional (FEDER). References [1] Brain Trauma Foundation, American Association of Neurological Surgeons, Congress of Neurological Surgeons. Guidelines for the management of severe traumatic brain injury. J Neurotrauma 2007;24(Suppl. 1):S1–S106. [2] Rovegno M, Soto PA, Sáez JC, von Bernhardi R. Biological mechanisms involved in the spread of traumatic brain damage. Med Intensiva 2012;36:37–44.
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Regular Article
Serum soluble CD40 Ligand levels are associated with severity and mortality of brain trauma injury patients Leonardo Lorente a,⁎, María M. Martín b, Agustín F. González-Rivero c, Luis Ramos d, Mónica Argueso e, Juan J. Cáceres f, Jordi Solé-Violán g, Nicolás Serrano a, Sergio T. Rodríguez b, Alejandro Jiménez h, Juan M. Borreguero-León c a
Intensive Care Unit, Hospital Universitario de Canarias, Ofra, s/n. La Laguna - 38320, Santa Cruz de Tenerife, Spain Intensive Care Unit, Hospital Universitario Nuestra Señora de Candelaria, Crta del Rosario s/n. Santa Cruz de Tenerife - 38010, Spain c Laboratory Deparment, Hospital Universitario de Canarias, Ofra, s/n. La Laguna - 38320, Santa Cruz de Tenerife, Spain d Intensive Care Unit, Hospital General La Palma, Buenavista de Arriba s/n, Breña Alta, La Palma- 38713, Spain e Intensive Care Unit, Hospital Clínico Universitario de Valencia, Avda, Blasco Ibáñez n°17-19, Valencia - 46004, Spain f Intensive Care Unit, Hospital Insular, Plaza Dr. Pasteur s/n. Las Palmas de Gran Canaria - 35016, Spain g Intensive Care Unit, Hospital Universitario Dr. Negrín, Barranco de la Ballena s/n. Las Palmas de Gran Canaria - 35010, Spain h Research Unit, Hospital Universitario de Canarias, Ofra, s/n. La Laguna - 38320, Santa Cruz de Tenerife, Spain b
a r t i c l e
i n f o
Article history: Received 22 May 2014 Received in revised form 24 June 2014 Accepted 30 July 2014 Available online 2 August 2014 Keywords: sCD40L Brain trauma Patients Mortality Injury
a b s t r a c t Background: Serum soluble CD40 Ligand (sCD40L) levels, which exhibit prothrombotic and proinflammatory properties, have not been studied in patients with traumatic brain injury (TBI). Thus, the objective of this study was to determine whether serum sCD40L levels are associated with severity and mortality in patients with severe TBI. Methods: This was a prospective, observational and multicenter study carried out in six Spanish Intensive Care Units. Patients with severe TBI defined as Glasgow Coma Scale (GCS) lower than 9 were included, while those with Injury Severity Score (ISS) in non-cranial aspects higher than 9 were excluded. Serum levels of sCD40L were measured on the day of TBI. Endpoint was established in 30-day mortality. Results: We found higher serum sCD40L levels (P b 0.001) in non-surviving TBI patients (N = 27) than in survivor ones (N = 73). Logistic regression analysis showed that serum sCD40L levels were associated with 30-day mortality (OR = 1.58; 95% CI = 1.12-2.21; P = 0.008) controlling for APACHE-II score and computer tomography findings. The area under the curve (AUC) for serum sCD40L levels as predictor of 30-day mortality was 0.79 (95% CI = 0.70-0.86; P b 0.001). Survival analysis showed that patients with serum sCD40L levels higher than 2.11 ng/mL presented increased 30-day mortality than patients with lower levels (Hazard ratio = 9.0; 95% CI = 4.25-19.27; P b 0.001). We found an association between serum sCD40L levels and APACHE-II (rho = 0.33; P = 0.001), and GCS score (rho = -0.21; P = 0.04). Conclusions: To our knowledge, this is the first study reporting data on serum sCD40L levels in patients with severe TBI. The most relevant and newer findings of our study are that serum sCD40L levels in non-surviving patients with severe TBI are higher than in surviving ones, and that there are an association between serum sCD40L levels and TBI severity and mortality. © 2014 Elsevier Ltd. All rights reserved.
Introduction Traumatic brain injury (TBI) is an important cause of death, disability and resources consumption [1]. Initial primary injury is referred to the physical forces applied to brain during the impact. Secondary injury occurs later over a period of hours or days after the initial traumatic Abbreviations: sCD40L, soluble CD40 Ligand; TNF, tumour necrosis factor; TF, tissue factor; ICU, Intensive Care Unit; SOFA, Sepsis-related Organ Failure Assessment score. ⁎ Corresponding author. E-mail address: [email protected] (L. Lorente).
http://dx.doi.org/10.1016/j.thromres.2014.07.034 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
injury and is due to different mechanisms [2,3]. Among them, inflammation and thrombosis are mechanisms that play a pivotal role in this delayed response. CD40 Ligand (CD40L) and its soluble counterpart (sCD40L) are proteins that exhibit proinflamatory [4,5] and procoagulant [6–11] properties on binding to their cell surface receptor CD40 [12,13]. CD40L is a member of the tumor necrosis factor (TNF) family and is expressed as a transmembrane protein in activated platelets [14,15]. Raised levels of sCD40L have been found in patients with acute coronary syndrome [16,17], stroke [18–22] and sepsis [23,24]. However, circulating sCD40L levels has not been studied in patients with TBI.
L. Lorente et al. / Thrombosis Research 134 (2014) 832–836
Thus, the objective of this study was to determine whether serum sCD40L levels are associated with mortality, severity, and inflammatory and prothrombotic markers in patients with severe TBI. Methods Design This was a prospective, observational, multicenter study carried out in 6 Intensive Care Units of Spain belonging to the following hospitals: Hospital Universitario de Canarias (La Laguna, Tenerife, Spain), Hospital Universitario Nuestra Señora de Candelaria (Tenerife, Spain), Hospital General de La Palma (La Palma, Tenerife, Spain), Hospital Clínico Universitario de Valencia (Valencia, Spain), Hospital Insular (Las Palmas de Gran Canaria, Spain), Hospital Universitario Dr. Negrín (Las Palmas de Gran Canaria, Spain). The study was approved by the Institutional Ethic Review Boards of the 6 participant hospitals. A written informed consent from the patient or from the legal next of kin was obtained. We included 100 patients with severe TBI. Severity of brain trauma injury was classified according to Glasgow Coma Scale (GCS) [25], and severe TBI was defined as GCS lower than 9 points. Exclusion criteria were: age less than 18 years, pregnancy, inflammatory or malignant disease, and Injury Severity Score (ISS) [26] in non-cranial aspects higher than 9 points. The following variables were recorded for each patient: sex, age, ISS, GCS, blood lactate, platelets, international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen, Acute Physiology and Chronic Health Evaluation II (APACHE II) score [27] and brain lesion according to the Marshall computer tomography (CT) classification [28]. The end-point was 30-day mortality. Laboratory Determinations Blood samples were collected on the day of TBI to measure serum sCD40L levels. We also measured serum TNF-alpha levels, and plasma tissue factor (TF) levels. Venous blood samples were collected in serum separator tubes (SST) for determination of serum sCD40L and TNF-alpha levels; and in citrate collected plasma tubes to determination of plasma TF levels. Blood samples were centrifuged within 30 minutes at 1000*g for 15 min. The serum and plasma were removed and frozen at -80 °C until measurement. The determination of serum sCD40L and TNFalpha levels, and plasma TF levels were centralized in the Laboratory Department of the Hospital Universitario de Canarias (La Laguna, Santa Cruz de Tenerife, Spain). Serum sCD40L levels were assayed by specific ELISA (Bender MedSystems GmbH, Vienna, Austria). The intra-assay and inter-assay coefficients of variation (CV) were 4% (n = 8) and 6.8% (n = 8) respectively; and detection limits for the assays was 0.06 ng/mL. Serum TNF-alpha levels were measured by a solid-phase, chemiluminiscents immunometrics assays kit (Immulite®, Siemens Healthcare Diagnostics Products, Llanberis, United Kingdom). The intra-assay and inter-assay CV were b3.6 % (n = 20) and b6.5% (n = 20) respectively; and detection limits for the assays was 1.7 pg/mL. Plasma TF levels were assayed by specific ELISA (Imubind® Tissue Factor ELISA, American Diagnostica, Inc, Stanford, CT, USA). The intraassay and inter-assay CV were b7.2% (n = 20) and b 8% (n = 20) respectively; and detection limits for the assays was 10 pg/mL.
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on categorical variables were carried out with chi-square test. Multiple binomial logistic regression analysis was applied to predict 30-day mortality. As number of events was 27 exitus, we constructed two multiple binomial logistic regression models with only three predictor variables in each to avoid an over fitting effect that may lead to choose a final model of order slightly higher than required [29]. In the first regression model were included serum sCD40L, APACHE-II score and CT classification. Previously to include the variable CT classification in the regression analysis, it was recoded according with the risk of death observed in the bivariated analysis as low (CT types 2 and 5) and high risk (CT types 3, 4 and 6) of death. In the second regression model were included serum sCD40L levels, GCS and age. Odds Ratio and 95% confidence intervals were calculated as measurement of the clinical impact of the predictor variables. Receiver operating characteristic (ROC) analysis was carried out to determine the goodness-of-fit of the of serum sCD40L levels to predict 30-day mortality. Kaplan-Meier analysis of survival at 30 days and comparisons by log-rank test were carried out using serum sCD40L levels lower/higher than 2.11 ng/mL as the independent variable and survival at 30 days as the dependent variable. The association between continuous variables was carried out using Spearman´s rank correlation coefficient. A P value of less than 0.05 was considered statistically significant. Statistical analyses were performed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and NCSS 2000 (Kaysville, Utah) and LogXact 4.1, (Cytel Co., Cambridge, MA). Results From the 100 TBI patients in the study, non-survivors (n = 27) showed lower GCS, and higher age, female rate and APACHE-II score than survivors (n = 73). CT classification also showed statistically significant differences between non-surviving and surviving patients. In addition, non-survivors showed higher serum sCD40L levels than survivors. However, no significant differences were found between nonsurviving and surviving patients in serum TNFalpha and plasma TF levels (Table 1). As shown in Table 1, mortality rate varied with CT classification types: thus 3/24 patients (12.5%) in type 2, 5/18 (27.8%) in type 3, 6/ 16 (37.5%) in type 4, 5/31 (16.1%) in type 5 and 8/11 (72.7%) in type 6. Once the CT classification variable was recoded for the regression analysis the new variable considered CT classification types 2 and 5 as low death risk, with a observed mortality rate of 8/55 (14.5%), and CT classification types 3, 4 and 6 as high death risk, with a observed mortality rate of 19/45 (42.2%). Multiple binomial logistic regression analysis showed that serum sCD40L levels could predict 30-day mortality (OR = 1.58; 95% CI = 1.12-2.21; P = 0.008) controlling for APACHE-II and CT classification (Table 2). Multiple binomial logistic regression analysis also showed that serum sCD40L levels could predict 30- day mortality (OR = 1.43; 95% CI = 1.05-1.95; P = 0.02) controlling for GCS and age (Table 2). The area under the curve (AUC) for serum sCD40L levels as predictor of 30-day mortality was 0.79 (95% CI = 0.70-0.86; P b 0.001) (Fig. 1). Survival analysis showed that patients with serum sCD40L levels higher than 2.11 ng/mL presented higher 30-day mortality than patients with lower levels (Chi-square: 19.8; Hazard ratio = 9.0 (95% IC = 4.2519.27); P b 0.001) (Fig. 2). We found an association between serum sCD40L levels and APACHE-II (rho = 0.33; P = 0.001), GCS score (rho = -0.21; P = 0.04) and age (rho = 0.30; P = 0.003) (Table 3).
Statistical Methods
Discussion
Continuous variables are reported as medians and interquartile ranges. Categorical variables are reported as frequencies and percentages. Comparisons of continuous variables between groups were carried out using Wilcoxon-Mann-Whitney test. Comparisons between groups
Raised levels of sCD40L related to impaired prognosis have been previously reported in patients with acute coronary artery syndrome and [30] and sepsis [23,24]; however, to our knowledge, this is the first study reporting data on serum sCD40L levels in patients with severe TBI.
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Table 1 Baseline clinical and biochemical characteristics of survivor and non-survivor patients.
Gender female – n (%) Age (years) - median (p 25-75) Computer tomography classification - n (%) · Type 1 · Type 2 · Type 3 · Type 4 · Type 5 · Type 6 Temperature (°C) - median (p 25-75) Sodium (mEq/L)- median (p 25-75) Glycemia (g/dL) - median (p 25-75) Leukocytes - median*103/mm3 (p 25-75) PaO2 (mmHg) - median (p 25-75) PaO2/FI02 ratio - median (p 25-75) Bilirubin (mg/dl) - median (p 25-75) Creatinine (mg/dl) - median (p 25-75) Hemoglobin (g/dL) - median (p 25-75) Glasgow Coma Scale score - median (p 25-75) Lactic acid (mmol/L) median (p 25-75) Platelets - median*103/mm3 (p 25-75) INR - median (p 25-75) aPTT (seconds) - median (p 25-75) Fibrinogen (mg/dl) - median (p 25-75) APACHE-II score - median (p 25-75) ISS - median (ppe 25-75) ICP (mmHg) - median (p 25-75) CPP (mmHg) - median (p 25-75) sCD40L (ng/mL) - median (p 25-75) TNF-alpha (pg/mL) - median (p 25-75) Tissue Factor (pg/mL) - median (p 25-75)
Survivors (n = 73)
Non-survivors (n = 27)
P value
12 (16.4) 47 (32-67)
11 (40.7) 66 (45-76)
0.02 b0.001 0.002
0 21 (28.8) 13 (17.8) 10 (13.7) 26 (35.6) 3 (4.1) 37. (35.6-37.3) 139 (138-142) 139 (120-163) 14.7 (10.2-19.3) 151 (116-217) 336 (242-407) 0.50 (0.40-0.87) 0.80 (0.70-0.90) 11.4 (10.4-13.0) 7 (6-8) 1.70 (1.23-2.50) 182 (143-252) 1.03 (0.92-1.15) 28 (25-32) 350 (282-444) 19 (17-23) 25 (25-32) 15 (14-20) 68 (57-70) 1.80 (0.60-2.79) 9.72 (7.88-13.40) 189 (129-247)
0 3 (11.1) 5 (18.5) 6 (22.2) 5 (18.5) 8 (29.6) 36.0 (35.0-37.0) 141 (135-149) 161 (142-189) 18.3 (10.7-23.9) 141 (104-186) 190 (154-316) 0.75 (0.53-1.05) 0.95 (0.70-1.10) 11.1 (9.4-12.3) 3 (3-6) 1.90 (1.15-4.55) 215 (139-264) 1.22 (1.01-1.67) 26 (25-31) 376 (246-560) 26 (25-32) 25 (25-27) 20 (12-30) 60 (54-69) 4.00 (2.36-5.46) 13.65 (8.35-22.75) 194 (169-295)
0.12 0.19 0.08 0.46 0.34 0.11 0.045 0.44 0.87 b0.001 0.16 0.48 0.15 0.86 0.32 b0.001 0.24 0.27 0.46 b0.001 0.12 0.18
P 25-75 = percentile 25th-75th; PaO2 = pressure of arterial oxygen/fraction inspired oxygen; FIO2 = pressure of arterial oxygen/fraction inspired oxygen; ISS = Injury Severity Score; INR = international normalized ratio; aPTT = activated partial thromboplastin time; APACHE II = Acute Physiology and Chronic Health Evaluation; ICP = intracranial pressure; CPP = cerebral perfusion pressure; TNF = tumor necrosis factor.
The role of sCD40L in TBI remains unclear but it is possible that its proinflamatory [4,5] and procoagulant [6–11] effects could contribute to the pathophysiology of TBI. CD40L is stored in α-granules in unstimulated platelets but it rapidly translocates to the surface when platelets become activated. Once in the platelet surface, CD40L is cleaved and released into circulation as sCD40L. The sCD40L binds to circulating monocytes through its receptor CD40, promoting their adhesion to vascular endothelium. The sCD40L also binds to CD40 on endothelial cell surfaces. Activated endothelial cells produce the overexpression of transcriptional factors such as nuclear factor-kappa B [NFkß] [31], with subsequent up regulation of proinflammatory and prothrombotic factors. Ligation of CD40 on endothelial cells, smooth muscle cells, or mononuclear phagocytes triggers the expression of various proinflammatory mediators, such as the interleukin (IL)-1, IL-6, IL-12, TNF-alpha, and interferon-gamma [5]. In addition, sCD40L could have prothrombotic effects via induction of TF [6–9],
diminishing thrombomodulin expression [8,9], and binding to the glycoprotein IIb/IIIa platelet receptor [10,11]. All these effects could facilitate the development of vascular thrombosis, brain ischemia and death. The most relevant and newer findings of our study are: a) nonsurviving TBI patients had higher serum sCD40L levels than surviving
Table 2 Multiple binomial logistic regression analysis of variables to predict 30-day mortality. Variable First Model Serum sCD40L levels APACHE-II score Computer tomography classification (reference category: low risk of death) Second Model Serum sCD40L levels GCS score Age
Odds Ratio
95% Confidence Interval
P
1.58 1.38 5.94
1.12–2.21 1.18–1.62 1.39–25.30
0.008 b0.001 0.02
1.43 0.59 1.06
1.05–1.95 0.43–0.80 1.02–1.10
0.02 0.001 0.003
APACHE II = Acute Physiology and Chronic Health Evaluation; GCS = Glasgow Coma Scale.
Fig. 1. Receiver operation characteristic analysis using serum sCD40L levels as predictor of mortality at 30 days. For cut-off serum sCD40L levels higher than 2.11 ng/mL, we found sensitivity = 89% (95% CI = 71-98%), specificity = 70% (95% CI = 48-72%), positive predicted value = 45% (95% CI = 32-60%), negative predicted value = 94% (95% CI = 8299%), positive likelihood ratio = 2.2 (95% CI = 1.3-3.1), negative likelihood ratio = 0.2 (95% CI = 0.01-0.05).
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However, some limitations of our study should be recognized. First, we did not perform the analysis of serum sCD40L levels during followup to describe the evolution in nonsurviving and surviving TBI patients. Second, we have determined sCD40 levels only in serum and not in plasma samples to evaluate possible differences due to there has been reported higher sCD40L levels in serum than in plasma levels [38]. Third, it is known that functional TF is bound to the outer leaflet of cell membrane including intact monocytes as well as microparticles shed from monocytes [39]; thus, it is possible that only a certain portion of TF could be detected by the method used for the detection of TF in the present study. Four, we have determined TNF-alpha due to that ligation of CD40 to receptor triggers the expression of various proinflammatory mediators, such as TNF-alpha [5]; and we have determined TF due to that sCD40L could have prothrombotic effects via induction of TF [6–9]. However, we did not found an association between serum sCD40L, TNF-alpha, and TF levels. Thus, the analysis of other coagulation factors, cytokines or microparticles could be interesting. Finally, additional studies are needed to confirm the findings of our study. Fig. 2. Survival curves at 30 days using 2.11 ng/mL of serum sCD40L levels as cut-off. Patients with serum sCD40L levels higher than 2.11 ng/mL presented higher 30-day mortality (Log rank = 19.8; Hazard ratio = 9.0 (95% IC = 4.25-19.27); P b 0.001).
ones, b) there was an association between serum sCD40L levels and TBI severity and mortality, and c) serum sCD40L levels could be used as a prognostic biomarker of mortality in TBI patients. We chose mortality at 30 days as end point because this is an endpoint used frequently in research studies. We found for the first time an association between serum sCD40L levels and patient severity assess by APACHE II and GCS scores. However, we did not found an association between serum sCD40L and TNFalpha. Neither, we found an association between serum sCD40L and TF levels, which has been described in culture of vascular endothelial cells [6–9]. It is possible that other reported prothrombotic effects of sCD40L, such as reduced thrombomodulin expression [8,9] and binding to the glycoprotein IIb/IIIa platelet receptor [10,11] could lead to vascular thrombosis, brain ischemia and, finally, death in these patients with TBI. We found for the first time an association between serum sCD40L levels and age in TBI patients; this association was previously described in healthy subjects [32] and patients with coronary disease [33]. We have not found an association between serum sCD40L levels and platelet count and coagulation, although platelets are the major source of sCD40L in circulation [14,15]. From a therapeutic perspective, the use of sCD40L modulators could be used as a new class of drugs for the treatment of TBI [34–37]. In some studies including patients with coronary artery disease, the use of statins decreased circulating sCD40L levels [34–36]. Besides, in animal TBI models have been found the benefit of statins [37]. It is possible that TBI patients, over all those with high serum sCD40L levels, could benefit of the early administration of statins.
Table 3 Correlation between serum sCD40L levels and other baseline clinical and biochemical characteristics. APACHE-II score GCS score Serum TNF-alpha (pg/mL) levels Plasma tissue factor (pg/mL) levels Age (years) Bilirubin (mg/dl) Platelets count INR aPTT (seconds)
rho = 0.33; P = 0.001 rho = -0.21; P = 0.04 rho = 0.16; P = 0.40 rho = 0.02; P = 0.87 rho = 0.30; P = 0.003 rho = 0.16; P = 0.13 rho = -0.02; P = 0.87 rho = 0.03; P = 0.79 rho = 0.05; P = 0.66
APACHE II = Acute Physiology and Chronic Health Evaluation; GCS = Glasgow Coma Scale; TNF = tumor necrosis factor; INR = international normalized ratio; aPTT = activated partial thromboplastin time.
Conclusions To our knowledge, this is the first study reporting data on serum sCD40L levels in patients with severe TBI. The most relevant and newer findings of our study are: a) Serum sCD40L levels in nonsurviving patients with severe TBI are higher than in surviving ones, b) There are an association between serum sCD40L levels and TBI severity and mortality, and c) Serum sCD40L levels could have a role as a prognostic biomarker of mortality in TBI patients. Competing Interests None Authors´Contributions LLo was responsible of conceive, design and coordinate the study, made substantial contributions to acquisition of data, analysis and interpretation of data, and drafted the manuscript. MMM, LR, MA, JJC, JSV, NS, STR have made substantial contributions to acquisition of data and provided useful suggestions. AGR and JMBL carried out the determination of sCD40L, TNF-α and TF determination and have made substantial contributions to analysis and interpretation of data. AJ has made substantial contributions to analysis and interpretation of data All authors revising the manuscript critically for important intellectual content and approved the final version of the manuscript Funding This study was supported, in part, by grants from Instituto de Salud Carlos III (I3SNS-INT-11-063 and I3SNS-INT-12-087) (Madrid, Spain) and co-financed with Fondo Europeo de Desarrollo Regional (FEDER). Acknowledgments This study was supported, in part, by grants from Instituto de Salud Carlos III (I3SNSINT-11-063 and I3SNS-INT-12-087) (Madrid, Spain) and co-financed with Fondo Europeo de Desarrollo Regional (FEDER). References [1] Brain Trauma Foundation, American Association of Neurological Surgeons, Congress of Neurological Surgeons. Guidelines for the management of severe traumatic brain injury. J Neurotrauma 2007;24(Suppl. 1):S1–S106. [2] Rovegno M, Soto PA, Sáez JC, von Bernhardi R. Biological mechanisms involved in the spread of traumatic brain damage. Med Intensiva 2012;36:37–44.
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