Thrombosis Research 136 (2015) 24–29
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Regular Article
The prognostic value of plasma soluble CD40 ligand levels following aneurysmal subarachnoid hemorrhage Xian-Dong Chen, Jun Sun, Chuan Lu, Hua-Jun Ba, Mao-Hua Chen, Jian-Hu Lin, Jian-Yong Cai ⁎ Department of Neurosurgery, The Central Hospital of Wenzhou City, 32 Dajian Lane, Wenzhou 325000, China
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Article history: Received 1 January 2015 Received in revised form 5 March 2015 Accepted 30 March 2015 Available online 9 April 2015 Keywords: Soluble CD40 ligand Intracranial aneurysmal Subarachnoid hemorrhage Prognosis Severity
a b s t r a c t Background: Increased circulating soluble CD40 ligand (sCD40L) levels have been reported to be associated with severity and mortality of severe traumatic brain injury. The current study tested the hypothesis that elevated plasma sCD40L levels are predictive of clinical outcomes of aneurysmal subarachnoid hemorrhage (aSAH). Methods: Plasma sCD40L concentrations of 120 aSAH patients and 120 healthy volunteers were measured using enzyme-linked immunosorbent assay. An unfavorable outcome was defined as Glasgow Outcome Scale score of 1-3. Results: Plasma sCD40L levels were significantly elevated in aSAH patients compared with healthy controls; plasma sCD40L levels were highly associated with clinical severity reflected by World Federation of Neurological Surgeons (WFNS) score and Fisher score; sCD40L emerged as an independent predictor of 6-month mortality and unfavorable outcome and 6-month overall survival; although a combined logistic-regression model did not demonstrate the additive benefit of sCD40L to WFNS score and Fisher score, sCD40L possessed similar predictive value to WFNS score and Fisher score based on receiver operating characteristic curves. Conclusions: Higher plasma sCD40L levels on presentation are associated with clinical severity and have potential to be a good prognostic biomarker of aSAH. © 2015 Elsevier Ltd. All rights reserved.
Introduction Aneurysmal subarachnoid hemorrhage (aSAH) carries the risk of high mortality and morbidity as well as has clinical and socioeconomic importance [1,2]. Accumulating evidence shows that the hemorrhaging of blood into the subarachnoid space induces further histological and inflammatory changes that contribute to the devastating effects of aSAH [3,4]. Nowadays, World Federation of Neurological Surgeons (WFNS) grade and Fisher grade are the important prediction systems of aSAH outcomes [5]. However, biomarkers are also attracting increasing attention as potential prognostic predictors of patients with aSAH [6,7]. CD 40 ligand (CD40L), formally known as CD154, is a 39 kD transmembrane glycoprotein and belongs to tumor necrosis factor-α family [8,9]. CD40L is expressed on the surface of monocytes, macrophages, T cells, B cells, platelets, and endothelial cells [10–12]. CD40L can be cleaved from the cell surface, releasing a soluble CD40L (sCD40L) which is biologically active [13]. Circulating sCD40L is mainly derived from platelets [14–16]. Multiple lines of recently collected data point to prothrombotic and proinflammatory properties of sCD40L on binding
to their cell surface receptor CD40 [17–22]. Circulating sCD40L levels are elevated in a great number of human disease including pelvic inflammatory disease, acute coronary syndrome, systemic autoimmune diseases, severe sepsis, chronic lymphocytic leukemia, psoriasis, inflammatory bowel disease and pancreatitis [23–31]. sCD40L is also associated with clinical outcomes and severity of influenza virus-associated encephalopathy, ST-segment elevation myocardial infarction, and severe sepsis [31–33]. In central nervous system, plasma sCD40L levels are obviously higher in ischemic stroke [34–36]; furthermore, elevated plasma sCD40L levels are highly associated with severity and mortality of traumatic brain injury [37]. However, there is a paucity of data available on the change of plasma sCD40L concentrations and sCD40L's relationship to clinical outcome after aSAH. Therefore, this study aimed to determine plasma sCD40L levels in a group of aSAH patients and meantime, assess the predictive value of sCD40L for long-term clinical outcomes after aSAH. Materials and Methods Study Population
Abbreviations: aSAH, aneurysmal subarachnoid hemorrhage; AUC, area under curve; CI, confidence interval; CT, computerized tomography; HR, hazard ratio; sCD40L, soluble CD40 ligand; WFNS, World Federation of Neurological Surgeons; OR, odds ratio; ROC, receiver operating characteristic. ⁎ Corresponding author. Tel./fax: +86 577 88070335. E-mail address: [email protected] (J.-Y. Cai).
http://dx.doi.org/10.1016/j.thromres.2015.03.025 0049-3848/© 2015 Elsevier Ltd. All rights reserved.
This prospective study was conducted during the period of January 2011 to May 2013 by the Department of Neurosurgery, Central Hospital of Wenzhou City, China. The inclusion criteria required that patients be admitted for the treatment of first-ever non-traumatic SAH, have clinical history of SAH within the last 24 hrs before admission, suffer from
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aSAH confirmed by computerized tomography (CT) angiography with or without digital subtraction angiography and receive the treatment by surgery or coiling within the 48 hrs after admission. Exclusion criteria included rebleeding after admission, less than 18 years of age, previous head trauma, previous neurological disease, previous use of antiplatelet or anticoagulant medication, other prior systemic diseases including uremia, liver cirrhosis, malignancy, chronic heart disease, chronic lung disease, diabetes mellitus and hypertension, unavailable biomarker measurements, refusal of participation and loss of followup. Control group included sex- and age-matched healthy individuals. Written informed consent to participate in the study was obtained from the subjects or their relatives. This protocol was approved by the Ethics Committee of The Central Hospital of Wenzhou City before implementation.
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sCD40L concentrations at admission. Meantime, area under curve (AUC) and 95% CI were calculated. AUC ranges from 0.5 to 1.0. An AUC closer to 1 indicates a higher predictive power. A combined logisticregression model was configured to estimate the additive benefit of plasma sCD40L levels to Fisher score and WFNS score. 6-month overall survival was estimated using the Kaplan–Meier method and the intergroup differences in survival time were tested using the log-rank test. Multivariate Cox’s proportional hazard analysis was carried out to identify independent prognostic factors for overall survival with identified hazard ratio (HR) and 95% CI. The variables, that univariate analyses revealed to be associated with poor prognosis, were incorporated into multivariate model. A P-value of less than 0.05 was considered statistically significant. Results
Clinical and Radiological Assessment Study Population Characteristics At admission, we collected information on demographic, clinical, radiological and outcome data for all patients. The radiological severity of SAH on admission was classified according to the Fisher grading system [38]. Clinical severity of SAH was classified according to the WFNS system [39]. Whenever clinical deterioration occurred, CT was performed to search for secondary complications such as hydrocephalus or ischemia. Clinical onset of cerebral vasospasm was defined as the acute onset of a focal neurologic deficit or a change in the Glasgow Coma Scale score of 2 or more points. All suspected cases of cerebral vasospasms were confirmed by CT angiography. All CT scans were performed according to the neuroradiology department protocol. Investigators who read them were blinded to clinical information. Clinical Endpoints For follow-up, structure telephone interviews were performed by 1 doctor, blinded to clinical information and biomarker levels. Participants were followed up until death or completion of 6 months after SAH. An unfavorable outcome was defined as a Glasgow outcome scale score 1-3 at 6 month. The end points were death and unfavorable outcome within 6 months after SAH. Immunoassay Methods All blood samples from the patients at admission and from the healthy controls at study entry were collected in plasma separator tubes and centrifuged within 30 minutes at 1,000*g for 15 minutes. The plasma was removed and frozen at -70 °C until measurement. Plasma sCD40L levels were assayed by specific enzyme-linked immunosorbent assay (R&D Systems, Abingdon, UK) according to the manufacturer’s instructions. All samples were assayed in duplicate. Statistical Analysis All data were analyzed using The Statistical Package for the Social Sciences version 19.0 (SPSS Inc., Chicago, IL, USA) and MedCalc 9.6.4.0 (MedCalc Software, Mariakerke, Belgium). The categorical variables are presented as percentages, and the continuous variables are presented as mean ± standard deviation. Comparisons were made by using chisquare test or Fisher exact test for categorical data as well as unpaired or paired Student t test for continuous variables. Bivariate correlations were analyzed by Spearman's correlation coefficient or Pearson’s correlation coefficient and then followed by a multivariate linear regression. The relationships between plasma sCD40L levels and 6-month mortality and unfavorable outcome were assessed using a binary logistic regression analyses with shown odds ratio (OR) and 95% confidence interval (CI). A receiver operating characteristic (ROC) curve analysis was performed to determine the cutoff point to differentiate patients with aSAH according to their death or unfavorable outcome based on their
Finally, One hundred and twenty aSAH patients and 120 age- and gender- matched healthy controls were enrolled in this study. The mean age of the patients with aSAH, a group consisting of 71 men and 49 women, was 41.1 ± 11.5 years (18 ~ 68 years). On admission, the mean WFNS score was 2.6 ± 1.1 (2 ~ 21) and the mean Fisher score was 2.8 ± 0.9 (2 ~ 5). Among these patients, 26 (21.7%) patients had acute hydrocephalus; 23 (19.2%) patients, intraventricular hemorrhage; 36 (30.0%) patients, vasospasm; 15 (12.5%) patients, computed tomography ischemia. The mean admission time was 6.4 ± 4.1 hours (0.5 ~ 22 hours); the mean plasma-sampling time, 8.2 ± 4.5 hours (2 ~ 23.5 hours); the mean systolic blood pressure, 130.7 ± 23.7 mmHg (83 ~ 187 mmHg); the mean diastolic blood pressure, 80.7 ± 13.6 mmHg (51 ~ 104 mmHg); the mean blood glucose level, 13.2 ± 4.8 mmol/L (3.3 ~ 24.0 mmol/L); the mean plasma Creactive protein level, 11.1 ± 3.3 mg/L (5.2 ~ 20.2 mg/L). 68 (56.7%) patients underwent surgery and 30 (25.0%) patients received external ventricular drain. The Change of Plasma sCD40L Level in aSAH Patients Fig. 1 showed that the admission sCD40L levels were significantly increased in all patients (3.2 ± 1.4 ng/mL), survivals (4.9 ± 1.4 ng/mL), non-survivals (3.0 ± 1.3 ng/mL), patients with unfavorable outcome (4.4 ± 1.3 ng/mL) and those with favorable outcome (2.7 ± 1.2 ng/mL) compared with healthy control individuals (1.1 ± 0.3 ng/mL, all P b 0.001). Correlation Analysis Table 1 showed that plasma sCD40L levels were highly associated with WFNS scores, Fisher scores, acute hydrocephalus, intraventricular hemorrhage, external ventricular drain, vasospasm, computed tomography ischemia, blood glucose levels and plasma C-reactive protein levels. When the above variables were introduced into the linear regression model, plasma sCD40L levels remained positively associated with WFNS scores (t = 5.465, P b 0.001) and Fisher scores (t = 4.537, P b 0.001). Fig. 2 showed their correlations. Mortality Prediction Sixteen patients (13.3%) died within 6 month after aSAH. Table 2 showed that the variables associated with death at 6 months included WFNS score, Fisher score, acute hydrocephalus, intraventricular hemorrhage, external ventricular drain, vasospasm, computed tomography ischemia, blood glucose levels, plasma C-reactive protein levels and plasma sCD40L levels. When the variables, that univariate analyses revealed to be associated with poor prognosis, were incorporated into a multivariate model, WFNS score (OR, 4.619; 95% CI, 1.739-11.870;
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Fig. 1. Comparison of plasma soluble CD40 ligand (sCD40L) levels between aneurysmal subarachnoid hemorrhage patients and healthy controls.
P = 0.001), Fisher score (OR, 3.903; 95% CI, 1.876-8.005; P = 0.003) and plasma sCD40L level (OR, 2.652; 95% CI, 1.279-5.501; P = 0.008) emerged as the independent predictors for 6-month mortality of patients. A ROC curve analysis showed that a plasma sCD40L level N3.6 ng/mL predicted 6-month mortality of patients with 81.2% sensitivity and 68.3% specificity (AUC, 0.843; 95%CI, 0.766-0.903) in Fig. 3A. Based on AUC, its predictive value was similar to WFNS score’s (AUC, 0.882; 95% CI, 0.810 - 0.934; P = 0.600) and Fisher score’s (AUC, 0.865; 95% CI, 0.791 - 0.921; P = 0.770). When a combined logistic-regression model was constructed, sCD40L did not enhance AUCs of WFNS score (AUC, 0.932; 95% CI, 0.871 – 0.970; P = 0.237) and Fisher score (AUC, 0.907; 95% CI, 0.840 – 0.952; P = 0.396).
A ROC curve analysis was performed to determine the cutoff point to differentiate patients with aSAH according to their unfavorable outcome based on their sCD40L concentrations at admission. This analysis showed that a plasma sCD40L level N 3.4 ng/mL predicted 6-month unfavorable outcome of patients with 77.1% sensitivity and 74.1% specificity (AUC, 0.825; 95%CI, 0.745-0.888) in Fig. 3B. Based on AUC, its predictive value was similar to WFNS score’s (AUC, 0.866; 95% CI, 0.792 - 0.921; P = 0.466) and Fisher score’s (AUC, 0.854; 95% CI, 0.778 - 0.912; P = 0.604). When a combined logistic-regression model was constructed, sCD40L numerically improved AUCs of WFNS score (AUC, 0.907; 95% CI, 0.840 – 0.952; P = 0.099) and Fisher score (AUC, 0.894; 95% CI, 0.825 – 0.943; P = 0.160). Analysis of Overall Survival
Poor Outcome Prediction Thirty-five patients (29.2%) suffered from unfavorable outcome within 6 months after aSAH. Table 2 showed that the variables associated with 6-month unfavorable outcome included WFNS score, Fisher score, acute hydrocephalus, intraventricular hemorrhage, external ventricular drain, vasospasm, computed tomography ischemia, blood glucose levels, plasma C-reactive protein levels and plasma sCD40L levels. When was configured a logistic-regression model which included the significant variables in the univariate analysis, it was demonstrated that WFNS score (OR, 4.276; 95% CI, 1.629-10.002; P = 0.001), Fisher score (OR, 3.861; 95% CI, 1.627-7.867; P = 0.002) and plasma sCD40L level (OR, 2.431; 95% CI, 1.223-4.998; P = 0.009) were the independent predictors for 6-month unfavorable outcome of patients. Table 1 The factors correlated with serum soluble CD40 ligand levels after aneurysmal subarachnoid hemorrhage. Characteristics
r value
P value
Gender (Male/Female) Age (y) WFNS score on admission Fisher score on admission Surgery Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Vasospasm Computed tomography ischemia Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L)
0.095 0.063 0.552 0.555 0.126 0.290 0.370 0.282 0.308 0.358 0.135 0.003 0.018 0.177 0.247 0.506
0.301 0.494 b0.001 b0.001 0.169 0.001 b0.001 0.002 0.001 b0.001 0.141 0.978 0.848 0.054 0.007 b0.001
Bivariate correlations were assessed by Spearman's or Pearson’s correlation coefficient. WFNS indicates World Federation of Neurological Surgeons.
The mean 6-month overall survival time was 161.4 days (95% CI: 152.6-170.3) in all patients. Table 3 showed that 6-month overall survival was correlated with WFNS score, Fisher score, acute hydrocephalus, intraventricular hemorrhage, external ventricular drain, vasospasm, computed tomography ischemia, blood glucose levels, plasma C-reactive protein levels and plasma sCD40L levels. A multivariate analyses selected WFNS score (HR, 3.894; 95% CI, 1.486-6.637; P = 0.001), Fisher score (HR, 3.651; 95% CI, 1.240-6.225; P = 0.001) and plasma sCD40L level (HR, 2.221; 95% CI, 1.197-4.716; P = 0.004) as the independent predictors for 6-month overall survival. Fig. 3C showed that, compared with patients with low sCD40L level (less than mean value), those with high sCD40L level (more than mean value) had significantly shorter 6-month overall survival time (the mean time, 146.2 days; 95% CI: 129.9-162.4 vs. the mean time, 175.7 days; 95% CI: 169.7-181.7; P b 0.001). Discussion Previous studies have found sCD40L levels are elevated in peripheral blood after brain injury including influenza virus-associated encephalopathy [32], ischemic stroke [34–36] and traumatic brain injury [37]. Recent study has also reported that there are an association between serum sCD40L levels and clinical severity and 30-day mortality of traumatic brain injury [37]. However, to the best of our knowledge, circulating sCD40L levels has not been studied in patients with aSAH. The current study determined sCD40L levels in plasma and further analyzed the association of plasma sCD40L levels with risk of long-term clinical outcomes and severity of aSAH. The most relevant and newer findings of our study were that serum sCD40L levels of patients were elevated compared with healthy controls, and that there were an association between serum sCD40L levels and aSAH severity and long-term prognosis including 6-month mortality and 6-month unfavorable outcome. The CD40–CD40L interaction is pivotal in the cellular immune response. CD40L was first described as an antigen, which is expressed
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Fig. 2. Correlative analysis of plasma soluble CD40 ligand (sCD40L) level with World Federation of Neurological Surgeons (WFNS) score and Fisher score.
on activated CD4 T cells, and CD40L interacts with CD40 expressed on B cells and induces the class switch. A broader role for CD40 signaling was revealed through the finding that CD40 is expressed on numerous cell types, including monocytes/macrophages, dendric cells, fibroblasts keratinocytes, endothelial cells, and vascular smooth muscle cells. Stimulation of these cell types through CD40 induces cell functions which contribute to inflammatory responses, including the expression of adhesion molecules, and also the release of proinflammatory cytokines, such as interleukin-6, interleukin-1beta, interleukin-8, interleukin-12, and tumor necrosis factor-alpha [8–12]. CD40L can be cleaved from the cell surface, releasing a sCD40L which is biologically active [13]. Therefore, sCD40L possesses proinflammatory effect [17–22]. Pathological findings has revealed that inflammatory cells were detectable in brain tissues after SAH and peripheral blood and cerebrospinal fluid concentrations of several proinflammatory cytokines such as interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha, are elevated and related to the clinical severity of aSAH. Accumulating evidence has demonstrated the crucial role of inflammatory process in the pathophysiological mechanism of aSAH [3,4]. The role of sCD40L in aSAH remains unclear but it is possible that its proinflamatory effects [13,40] could contribute to the pathophysiology of aSAH. 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 [41], with subsequent up regulation of proinflammatory factors. Ligation of CD40 on endothelial cells, smooth muscle cells, or mononuclear phagocytes triggers the expression of various proinflammatory mediators, such as the interleukin -1, interleukin-6, interleukin-12, tumor necrosis factor-alpha, and interferon-gamma [13] that have been demonstrated to be involved in the inflammatory process of brain injury after aSAH [3,4]. Thus, we hypothesized that CD40–sCD40L binding could facilitate the amplification of inflammatory response through the generation of cytokines and all these effects could facilitate the development of brain injury after aSAH. Although some researches have reported the elevated sCD40L levels in peripheral blood of patients with ischemic stroke [34–36], no study assesses the relationship between sCD40L and clinical severity and clinical outcome of ischemic stroke. Up to now, Leonardo et.al report serum sCD40L levels are associated with disease severity and 30-day mortality of brain trauma injury patients [37]. Our research, for the first time, demonstrated the close relationships between plasma sCD40L levels and disease severity and 6-month clinical outcome of aSAH. Importantly, the current study used a multivariate linear regression, but not a univariate analysis, to demonstrate the association of plasma sCD40L levels with the severity of aSAH reflected by WFNS scores and Fisher scores. In order to verify the relationship between sCD40L and long-term prognosis, we assessed both 6-month mortality and unfavorable outcome and 6-month overall survival using multivariate analysis and found their close associations. Interestingly, although a combined logisticregression model did not demonstrate the additive benefit of sCD40L to WFNS score and Fisher score, sCD40L possessed similar predictive value to WFNS score and Fisher score based on ROC curves. And,
Table 2 The factors associated with 6-month mortality and 6-month unfavorable outcome after aneurysmal subarachnoid hemorrhage. Mortality
Cases Male Age (y) WFNS score on admission Fisher score on admission Surgery Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Vasospasm Computed tomography ischemia Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Plasma sCD40L level (ng/mL)
Functional outcome
Non-survivors
Survivors
P value
Unfavorable outcome
Favorable outcome
P value
16 9 (56.3%) 41.9 ± 11.8 3.9 ± 0.7 3.9 ± 0.6 10 (62.5%) 8 (50.0%) 7 (43.8%) 8 (50.0%) 9 (56.3%) 6 (37.5%) 6.3 ± 6.0 8.0 ± 6.1 137.5 ± 18.8 85.4 ± 10.9 16.5 ± 4.7 13.7 ± 3.3 4.9 ± 1.4
104 62 (59.6%) 40.9 ± 11.5 2.4 ± 1.0 2.6 ± 0.8 58 (55.8%) 18 (17.3%) 16 (15.4%) 22 (21.2%) 27 (26.0%) 9 (8.7%) 6.4 ± 3.8 8.3 ± 4.3 129.7 ± 24.2 79.9 ± 13.8 12.8 ± 4.6 10.8 ± 3.1 3.0 ± 1.3
0.799 0.741 b0.001 b0.001 0.613 0.003 0.007 0.026 0.020 0.005 0.964 0.880 0.220 0.137 0.003 0.001 b0.001
35 20 (57.1%) 41.3 ± 10.7 3.6 ± 0.7 3.6 ± 0.8 22 (62.9%) 13 (37.1%) 12 (34.3%) 14 (40.0%) 16 (45.7%) 9 (25.7%) 5.6 ± 4.3 7.5 ± 4.5 134.7 ± 21.6 80.8 ± 12.3 15.0 ± 4.9 12.6 ± 3.3 4.4 ± 1.3
85 51 (60.0%) 40.9 ± 11.9 2.2 ± 1.0 2.5 ± 0.7 46 (54.1%) 13 (15.3%) 11 (12.9%) 16 (18.8%) 20 (23.5%) 6 (7.1%) 6.7 ± 4.0 8.5 ± 4.5 129.1 ± 24.4 80.6 ± 14.1 12.5 ± 4.5 10.5 ± 3.1 2.7 ± 1.2
0.772 0.858 b0.001 b0.001 0.380 0.008 0.007 0.015 0.016 0.012 0.170 0.238 0.244 0.945 0.009 0.002 b0.001
Numerical variables were presented as mean ± standard deviation. Categorical variables were expressed as counts (percentage). Numerical variables were analyzed by unpaired Student t test. Categorical variables were analyzed by chi-square test or Fisher exact test. WFNS indicates World Federation of Neurological Surgeons; sCD40L, soluble CD40 ligand.
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Fig. 3. Receiver operating characteristic curve analysis of plasma soluble CD40 ligand (sCD40L) levels for identifying aneurysmal subarachnoid hemorrhage patients with 6-month mortality (3A) and 6-month unfavorable outcome (3B). Survival curves (3C) for overall survival in patients with aneurysmal subarachnoid hemorrhage according to plasma soluble CD40 ligand (sCD40L) levels.
although sCD40L’s specificity and sensitivity values for prediction of mortality and unfavorable outcome were not very high, based on AUC, its predictive value was similar to WFNS score’s and Fisher score’s.
Therefore, sCD40L had shown high predictive performance for aSAH severity. Conclusions
Table 3 The factors associated with 6-month overall survival. Characteristics
Hazard ratio (95% confidence interval)
P value
Male Age (y) WFNS score on admission Fisher score on admission Surgery Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Vasospasm Computed tomography ischemia Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Plasma sCD40L level (ng/mL)
0.915 (0.341-2.457) 1.007 (0.965-1.051) 3.780 (2.215-6.453) 3.542 (2.097-5.981) 1.335 (0.485-3.675) 4.384 (1.642-11.702) 3.753 (1.395-10.092) 3.275 (1.228-8.736) 3.197 (1.190-8.590) 5.345 (1.936-14.754) 0.998 (0.881-1.131) 0.990 (0.883-1.109) 1.013 (0.993-1.033) 1.032 (0.992-1.074) 1.203 (1.064-1.361) 1.251 (1.089-1.437) 2.502 (1.687-3.712)
0.860 0.746 b0.001 b0.001 0.575 0.003 0.009 0.018 0.021 0.001 0.974 0.860 0.214 0.123 0.003 0.002 b0.001
Univariate logistic-regression analysis was used to calculate the hazard ratio and 95% confidence interval. WFNS indicates World Federation of Neurological Surgeons; sCD40L, soluble CD40 ligand.
This study demonstrates the there is an association between plasma sCD40L levels and aSAH severity and long-term prognosis. Our findings suggest that sCD40L may have potential to be a good prognostic biomarker of aSAH. Conflict of Interest Statement None Authors’ Contributions XDC and JYC were responsible of conceive, design and coordinate the study, made substantial contributions to acquisition of data, analysis and interpretation of data, and drafted the manuscript. JS and CL have made substantial contributions to acquisition of data and provided useful suggestions. HJB and MHC carried out the determination of sCD40L and have made substantial contributions to analysis and interpretation of data. JHL has made substantial contributions to analysis and interpretation of data.
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All authors revising the manuscript critically for important intellectual content and approved the final version of the manuscript. Funding This study was supported by a grant from Wenzhou Science and Technology Research Fund Project (No. Y20130263). Acknowledgement The authors thank all staffs in Department of Neurosurgery, The Central Hospital of Wenzhou City (Wenzhou, China) for their technical support. References [1] de Rooij NK, Linn FH, van der Plas JA, Algra A, Rinkel GJ. Incidence of subarachnoid haemorrhage: a systematic review with emphasis on region, age, gender and time trends. J Neurol Neurosurg Psychiatry 2007;78:1365–72. [2] Feigin VL, Lawes CM, Bennett DA, Barker-Collo SL, Parag V. Worldwide stroke incidence and early case fatality reported in 56 population-based studies: a systematic review. Lancet Neurol 2009;8:355–69. [3] Hanafy KA, Morgan Stuart R, Fernandez L, Schmidt JM, Claassen J, Lee K, et al. Cerebral inflammatory response and predictors of admission clinical grade after aneurysmal subarachnoid hemorrhage. J Clin Neurosci 2010;17:22–5. [4] Dhar R, Diringer MN. The burden of the systemic inflammatory response predicts vasospasm and outcome after subarachnoid hemorrhage. Neurocrit Care 2008;8: 404–12. [5] Le Roux PD, Elliott JP, Newell DW, Grady MS, Winn HR. Predicting outcome in poorgrade patients with subarachnoid hemorrhage: a retrospective review of 159 aggressively managed cases. J Neurosurg 1996;85:39–49. [6] Frijns CJ, Fijnheer R, Algra A, van Mourik JA, van Gijn J, Rinkel GJ. Early circulating levels of endothelial cell activation markers in aneurysmal subarachnoid haemorrhage: associations with cerebral ischaemic events and outcome. J Neurol Neurosurg Psychiatry 2006;77:77–83. [7] Khurana VG, Wijdicks EF, Heubline DM, McClellnd RL, Meyer FB, Piepgras DG, et al. A pilot study of Dendroaspis natriuretic peptide in aneursymal subarachnoid hemorrhage. Neurosurgery 2004;55:69–76. [8] Armitage RJ, Fanslow WC, Strockbine L, Sato TA, Clifford KN, Macduff BM, et al. Molecular and biological characterization of a murine ligand for CD40. Nature 1992; 357:80–2. [9] Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus G, et al. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 1998;391:591–4. [10] Foy TM, Aruffo A, Bajorath J, Buhlmann JE, Noelle RJ. Immune regulation by CD40 and its ligand GP39. Annu Rev Immunol 1996;14:591–617. [11] Burger D, Molnarfi N, Gruaz L, Dayer JM. Differential induction of IL-1beta and TNF by CD40 ligand or cellular contact with stimulated T cells depends on the maturation stage of human monocytes. J Immunol 2004;173:1292–7. [12] Suttles J, Milhorn DM, Miller RW, Poe JC, Wahl LM, Stout RD. CD40 signaling of monocyte inflammatory cytokine synthesis through an ERK1/2-dependent pathway. A target of interleukin (IL)-4 and IL-10 anti-inflammatory action. J Biol Chem 1999;274:5835–42. [13] Mach F, Schönbeck U, Sukhova GK, Bourcier T, Bonnefoy JY, Pober JS, et al. Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40-CD40 ligand signaling in atherosclerosis. Proc Natl Acad Sci U S A 1997;94:1931–6. [14] Andre P, Nannizzi-Alaimo L, Prasad SK, Phillips DR. Platelet-derived CD40L: the switch-hitting player of cardiovascular disease. Circulation 2002;106:896–9. [15] Danese S, Katz JA, Saibeni S, Papa A, Gasbarrini A, Vecchi M, et al. Activated platelets are the source of elevated levels of soluble CD40 ligand in the circulation of inflammatory bowel disease patients. Gut 2003;52:1435–41. [16] Nagasawa M, Zhu Y, Isoda T, Tomizawa D, Itoh S, Kajiwara M, et al. Analysis of serum soluble CD40 ligand (sCD40L) in the patients undergoing allogeneic stem cell transplantation: platelet is a major source of serum sCD40L. Eur J Haematol 2005;74: 54–60.
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[17] Zhou L, Stordeur P, de Lavareille A, Thielemans K, Capel P, Goldman M, et al. CD40 engagement on endothelial cells promotes tissue factor-dependent procoagulant activity. Thromb Haemost 1998;79:1025–8. [18] Hezi-Yamit A, Wong PW, Bien-Ly N, Komuves LG, Prasad KS, Phillips DR, et al. Synergistic induction of tissue factor by coagulation factor Xa and TNF: evidence for involvement of negative regulatory signaling cascades. Proc Natl Acad Sci U S A 2005; 102:12077–82. [19] Miller DL, Yaron R, Yellin MJ. CD40L-CD40 interactions regulate endothelial cell surface tissue factor and thrombomodulin expression. J Leukoc Biol 1998;63:373–9. [20] Slupsky JR, Kalbas M, Willuweit A, Henn V, Kroczek RA, Müller-Berghaus G. Activated platelets induce tissue factor expression on human umbilical vein endothelial cells by ligation of CD40. Thromb Haemost 1998;80:1008–14. [21] Prasad KS, Andre P, He M, Bao M, Manganello J, Phillips DR. Soluble CD40 ligand induces beta3 integrin tyrosine phosphorylation and triggers platelet activation by outside-in signaling. Proc Natl Acad Sci U S A 2003;100:12367–71. [22] Graf D, Muller S, Korthauer U, van Kooten C, Weise C, Kroczek RA. A soluble form of TRAP (CD40 ligand) is rapidly released after T cell activation. Eur J Immunol 1995; 25:1749–54. [23] Ho TC, Yang SF, Wang PH, Lin LY, Tee YT, Liao WC, et al. Increased serum soluble CD40 ligand concentration in pelvic inflammatory disease. Clin Chim Acta 2015; 438:236–40. [24] Aukrust P, Muller F, Ueland T, Berget T, Aaser E, Brunsvig A, et al. Enhanced levels of soluble and membrane-bound CD40 ligand in patients with unstable angina: possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes. Circulation 1999;100:614–20. [25] Varo N, de Lemos JA, Libby P, Morrow DA, Murphy SA, Nuzzo R, et al. Soluble CD40L: risk prediction after acute coronary syndromes. Circulation 2003;108:1049–52. [26] Vakkalanka RK, Woo C, Kirou KA, Koshy M, Berger D, Crow MK. Elevated levels and functional capacity of soluble CD40 ligand in systemic lupus erythematosus sera. Arthritis Rheum 1999;42:871–81. [27] Clodi K, Asgary Z, Zhao S, Kliche KO, Cabanillas F, Andreeff M, et al. Coexpression of CD40 and CD40 ligand in B-cell lymphoma cells. Br J Haematol 1998;103:270–5. [28] Erturan I, Köroğlu BK, Adiloğlu A, Ceyhan AM, Akkaya VB, Tamer N, et al. Evaluation of serum sCD40L and homocysteine levels with subclinical atherosclerosis indicators in patients with psoriasis: a pilot study. Int J Dermatol 2014;53:503–9. [29] Ludwiczek O, Kaser A, Tilg H. Serum levels of soluble CD40 ligand are elevated in inflammatory bowel diseases. Int J Colorectal Dis 2003;18:142–7. [30] Frossard JL, Morel P, Kwak B, Pastor C, Berney T, Buhler L, et al. Soluble CD40 ligand in prediction of acute severe pancreatitis. World J Gastroenterol 2006;12:1613–6. [31] Lorente L, Martín MM, Varo N, Borreguero-León JM, Solé-Violán J, Blanquer J, et al. Association between serum soluble CD40 ligand levels and mortality in patients with severe sepsis. Crit Care 2011;15:R97. [32] Ichiyama T, Morishima T, Suenaga N, Kajimoto M, Matsubara T, Furukawa S. Analysis of serum soluble CD40 ligand in patients with influenza virus-associated encephalopathy. J Neurol Sci 2005;239:53–7. [33] Pusuroglu H, Akgul O, Erturk M, Uyarel H, Bulut U, Akkaya E, et al. Predictive value of elevated soluble CD40 ligand in patients undergoing primary angioplasty for STsegment elevation myocardial infarction. Coron Artery Dis 2014;25:558–64. [34] Garlichs CD, Kozina S, Fateh-Moghadam S, Tomandl B, Stumpf C, Eskafi S, et al. Upregulation of CD40-CD40 Ligand (CD154) in patients with acute cerebral ischemia. Stroke 2003;34:1412–8. [35] Ferro D, Loffredo L, Polimeni L, Fimognari F, Villari P, Pignatelli P, et al. Soluble CD40 ligand predicts ischemic stroke and myocardial infarction in patients with nonvalvular atrial fibrillation. Arterioscler Thromb Vasc Biol 2007;27:2763–8. [36] Davì G, Tuttolomondo A, Santilli F, Basili S, Ferrante E, Di Raimondo D, et al. CD40 ligand and MCP-1 as predictors of cardiovascular events in diabetic patients with stroke. J Atheroscler Thromb 2009;16:707–13. [37] Lorente L, Martín MM, González-Rivero AF, Ramos L, Argueso M, Cáceres JJ, et al. Serum soluble CD40 Ligand levels are associated with severity and mortality of brain trauma injury patients. Thromb Res 2014;134:832–6. [38] Fisher CM, Kistler JP, Davis JM. Relation of cerebral vasospasm to subarachnoid hemorrhage visualized by computerized tomographic scanning. Neurosurgery 1980;6: 1–9. [39] Drake C. Report of World Federation of Neurological Surgeons Committee on a universal subarachnoid hemorrhage grading scale. J Neurosurg 1988;68:985–6. [40] Noelle RJ, Roy M, Shepherd DM, Stamenkovic I, Ledbetter JA, Aruffo A. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc Natl Acad Sci U S A 1992;9:6550–4. [41] Chen Y, Chen J, Xiong Y, Da Q, Xu Y, Jiang X, et al. Internalization of CD40 regulates its signal transduction in vascular endothelial cells. Biochem Biophys Res Commun 2006;345:106–17.
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Regular Article
The prognostic value of plasma soluble CD40 ligand levels following aneurysmal subarachnoid hemorrhage Xian-Dong Chen, Jun Sun, Chuan Lu, Hua-Jun Ba, Mao-Hua Chen, Jian-Hu Lin, Jian-Yong Cai ⁎ Department of Neurosurgery, The Central Hospital of Wenzhou City, 32 Dajian Lane, Wenzhou 325000, China
a r t i c l e
i n f o
Article history: Received 1 January 2015 Received in revised form 5 March 2015 Accepted 30 March 2015 Available online 9 April 2015 Keywords: Soluble CD40 ligand Intracranial aneurysmal Subarachnoid hemorrhage Prognosis Severity
a b s t r a c t Background: Increased circulating soluble CD40 ligand (sCD40L) levels have been reported to be associated with severity and mortality of severe traumatic brain injury. The current study tested the hypothesis that elevated plasma sCD40L levels are predictive of clinical outcomes of aneurysmal subarachnoid hemorrhage (aSAH). Methods: Plasma sCD40L concentrations of 120 aSAH patients and 120 healthy volunteers were measured using enzyme-linked immunosorbent assay. An unfavorable outcome was defined as Glasgow Outcome Scale score of 1-3. Results: Plasma sCD40L levels were significantly elevated in aSAH patients compared with healthy controls; plasma sCD40L levels were highly associated with clinical severity reflected by World Federation of Neurological Surgeons (WFNS) score and Fisher score; sCD40L emerged as an independent predictor of 6-month mortality and unfavorable outcome and 6-month overall survival; although a combined logistic-regression model did not demonstrate the additive benefit of sCD40L to WFNS score and Fisher score, sCD40L possessed similar predictive value to WFNS score and Fisher score based on receiver operating characteristic curves. Conclusions: Higher plasma sCD40L levels on presentation are associated with clinical severity and have potential to be a good prognostic biomarker of aSAH. © 2015 Elsevier Ltd. All rights reserved.
Introduction Aneurysmal subarachnoid hemorrhage (aSAH) carries the risk of high mortality and morbidity as well as has clinical and socioeconomic importance [1,2]. Accumulating evidence shows that the hemorrhaging of blood into the subarachnoid space induces further histological and inflammatory changes that contribute to the devastating effects of aSAH [3,4]. Nowadays, World Federation of Neurological Surgeons (WFNS) grade and Fisher grade are the important prediction systems of aSAH outcomes [5]. However, biomarkers are also attracting increasing attention as potential prognostic predictors of patients with aSAH [6,7]. CD 40 ligand (CD40L), formally known as CD154, is a 39 kD transmembrane glycoprotein and belongs to tumor necrosis factor-α family [8,9]. CD40L is expressed on the surface of monocytes, macrophages, T cells, B cells, platelets, and endothelial cells [10–12]. CD40L can be cleaved from the cell surface, releasing a soluble CD40L (sCD40L) which is biologically active [13]. Circulating sCD40L is mainly derived from platelets [14–16]. Multiple lines of recently collected data point to prothrombotic and proinflammatory properties of sCD40L on binding
to their cell surface receptor CD40 [17–22]. Circulating sCD40L levels are elevated in a great number of human disease including pelvic inflammatory disease, acute coronary syndrome, systemic autoimmune diseases, severe sepsis, chronic lymphocytic leukemia, psoriasis, inflammatory bowel disease and pancreatitis [23–31]. sCD40L is also associated with clinical outcomes and severity of influenza virus-associated encephalopathy, ST-segment elevation myocardial infarction, and severe sepsis [31–33]. In central nervous system, plasma sCD40L levels are obviously higher in ischemic stroke [34–36]; furthermore, elevated plasma sCD40L levels are highly associated with severity and mortality of traumatic brain injury [37]. However, there is a paucity of data available on the change of plasma sCD40L concentrations and sCD40L's relationship to clinical outcome after aSAH. Therefore, this study aimed to determine plasma sCD40L levels in a group of aSAH patients and meantime, assess the predictive value of sCD40L for long-term clinical outcomes after aSAH. Materials and Methods Study Population
Abbreviations: aSAH, aneurysmal subarachnoid hemorrhage; AUC, area under curve; CI, confidence interval; CT, computerized tomography; HR, hazard ratio; sCD40L, soluble CD40 ligand; WFNS, World Federation of Neurological Surgeons; OR, odds ratio; ROC, receiver operating characteristic. ⁎ Corresponding author. Tel./fax: +86 577 88070335. E-mail address: [email protected] (J.-Y. Cai).
http://dx.doi.org/10.1016/j.thromres.2015.03.025 0049-3848/© 2015 Elsevier Ltd. All rights reserved.
This prospective study was conducted during the period of January 2011 to May 2013 by the Department of Neurosurgery, Central Hospital of Wenzhou City, China. The inclusion criteria required that patients be admitted for the treatment of first-ever non-traumatic SAH, have clinical history of SAH within the last 24 hrs before admission, suffer from
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aSAH confirmed by computerized tomography (CT) angiography with or without digital subtraction angiography and receive the treatment by surgery or coiling within the 48 hrs after admission. Exclusion criteria included rebleeding after admission, less than 18 years of age, previous head trauma, previous neurological disease, previous use of antiplatelet or anticoagulant medication, other prior systemic diseases including uremia, liver cirrhosis, malignancy, chronic heart disease, chronic lung disease, diabetes mellitus and hypertension, unavailable biomarker measurements, refusal of participation and loss of followup. Control group included sex- and age-matched healthy individuals. Written informed consent to participate in the study was obtained from the subjects or their relatives. This protocol was approved by the Ethics Committee of The Central Hospital of Wenzhou City before implementation.
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sCD40L concentrations at admission. Meantime, area under curve (AUC) and 95% CI were calculated. AUC ranges from 0.5 to 1.0. An AUC closer to 1 indicates a higher predictive power. A combined logisticregression model was configured to estimate the additive benefit of plasma sCD40L levels to Fisher score and WFNS score. 6-month overall survival was estimated using the Kaplan–Meier method and the intergroup differences in survival time were tested using the log-rank test. Multivariate Cox’s proportional hazard analysis was carried out to identify independent prognostic factors for overall survival with identified hazard ratio (HR) and 95% CI. The variables, that univariate analyses revealed to be associated with poor prognosis, were incorporated into multivariate model. A P-value of less than 0.05 was considered statistically significant. Results
Clinical and Radiological Assessment Study Population Characteristics At admission, we collected information on demographic, clinical, radiological and outcome data for all patients. The radiological severity of SAH on admission was classified according to the Fisher grading system [38]. Clinical severity of SAH was classified according to the WFNS system [39]. Whenever clinical deterioration occurred, CT was performed to search for secondary complications such as hydrocephalus or ischemia. Clinical onset of cerebral vasospasm was defined as the acute onset of a focal neurologic deficit or a change in the Glasgow Coma Scale score of 2 or more points. All suspected cases of cerebral vasospasms were confirmed by CT angiography. All CT scans were performed according to the neuroradiology department protocol. Investigators who read them were blinded to clinical information. Clinical Endpoints For follow-up, structure telephone interviews were performed by 1 doctor, blinded to clinical information and biomarker levels. Participants were followed up until death or completion of 6 months after SAH. An unfavorable outcome was defined as a Glasgow outcome scale score 1-3 at 6 month. The end points were death and unfavorable outcome within 6 months after SAH. Immunoassay Methods All blood samples from the patients at admission and from the healthy controls at study entry were collected in plasma separator tubes and centrifuged within 30 minutes at 1,000*g for 15 minutes. The plasma was removed and frozen at -70 °C until measurement. Plasma sCD40L levels were assayed by specific enzyme-linked immunosorbent assay (R&D Systems, Abingdon, UK) according to the manufacturer’s instructions. All samples were assayed in duplicate. Statistical Analysis All data were analyzed using The Statistical Package for the Social Sciences version 19.0 (SPSS Inc., Chicago, IL, USA) and MedCalc 9.6.4.0 (MedCalc Software, Mariakerke, Belgium). The categorical variables are presented as percentages, and the continuous variables are presented as mean ± standard deviation. Comparisons were made by using chisquare test or Fisher exact test for categorical data as well as unpaired or paired Student t test for continuous variables. Bivariate correlations were analyzed by Spearman's correlation coefficient or Pearson’s correlation coefficient and then followed by a multivariate linear regression. The relationships between plasma sCD40L levels and 6-month mortality and unfavorable outcome were assessed using a binary logistic regression analyses with shown odds ratio (OR) and 95% confidence interval (CI). A receiver operating characteristic (ROC) curve analysis was performed to determine the cutoff point to differentiate patients with aSAH according to their death or unfavorable outcome based on their
Finally, One hundred and twenty aSAH patients and 120 age- and gender- matched healthy controls were enrolled in this study. The mean age of the patients with aSAH, a group consisting of 71 men and 49 women, was 41.1 ± 11.5 years (18 ~ 68 years). On admission, the mean WFNS score was 2.6 ± 1.1 (2 ~ 21) and the mean Fisher score was 2.8 ± 0.9 (2 ~ 5). Among these patients, 26 (21.7%) patients had acute hydrocephalus; 23 (19.2%) patients, intraventricular hemorrhage; 36 (30.0%) patients, vasospasm; 15 (12.5%) patients, computed tomography ischemia. The mean admission time was 6.4 ± 4.1 hours (0.5 ~ 22 hours); the mean plasma-sampling time, 8.2 ± 4.5 hours (2 ~ 23.5 hours); the mean systolic blood pressure, 130.7 ± 23.7 mmHg (83 ~ 187 mmHg); the mean diastolic blood pressure, 80.7 ± 13.6 mmHg (51 ~ 104 mmHg); the mean blood glucose level, 13.2 ± 4.8 mmol/L (3.3 ~ 24.0 mmol/L); the mean plasma Creactive protein level, 11.1 ± 3.3 mg/L (5.2 ~ 20.2 mg/L). 68 (56.7%) patients underwent surgery and 30 (25.0%) patients received external ventricular drain. The Change of Plasma sCD40L Level in aSAH Patients Fig. 1 showed that the admission sCD40L levels were significantly increased in all patients (3.2 ± 1.4 ng/mL), survivals (4.9 ± 1.4 ng/mL), non-survivals (3.0 ± 1.3 ng/mL), patients with unfavorable outcome (4.4 ± 1.3 ng/mL) and those with favorable outcome (2.7 ± 1.2 ng/mL) compared with healthy control individuals (1.1 ± 0.3 ng/mL, all P b 0.001). Correlation Analysis Table 1 showed that plasma sCD40L levels were highly associated with WFNS scores, Fisher scores, acute hydrocephalus, intraventricular hemorrhage, external ventricular drain, vasospasm, computed tomography ischemia, blood glucose levels and plasma C-reactive protein levels. When the above variables were introduced into the linear regression model, plasma sCD40L levels remained positively associated with WFNS scores (t = 5.465, P b 0.001) and Fisher scores (t = 4.537, P b 0.001). Fig. 2 showed their correlations. Mortality Prediction Sixteen patients (13.3%) died within 6 month after aSAH. Table 2 showed that the variables associated with death at 6 months included WFNS score, Fisher score, acute hydrocephalus, intraventricular hemorrhage, external ventricular drain, vasospasm, computed tomography ischemia, blood glucose levels, plasma C-reactive protein levels and plasma sCD40L levels. When the variables, that univariate analyses revealed to be associated with poor prognosis, were incorporated into a multivariate model, WFNS score (OR, 4.619; 95% CI, 1.739-11.870;
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Fig. 1. Comparison of plasma soluble CD40 ligand (sCD40L) levels between aneurysmal subarachnoid hemorrhage patients and healthy controls.
P = 0.001), Fisher score (OR, 3.903; 95% CI, 1.876-8.005; P = 0.003) and plasma sCD40L level (OR, 2.652; 95% CI, 1.279-5.501; P = 0.008) emerged as the independent predictors for 6-month mortality of patients. A ROC curve analysis showed that a plasma sCD40L level N3.6 ng/mL predicted 6-month mortality of patients with 81.2% sensitivity and 68.3% specificity (AUC, 0.843; 95%CI, 0.766-0.903) in Fig. 3A. Based on AUC, its predictive value was similar to WFNS score’s (AUC, 0.882; 95% CI, 0.810 - 0.934; P = 0.600) and Fisher score’s (AUC, 0.865; 95% CI, 0.791 - 0.921; P = 0.770). When a combined logistic-regression model was constructed, sCD40L did not enhance AUCs of WFNS score (AUC, 0.932; 95% CI, 0.871 – 0.970; P = 0.237) and Fisher score (AUC, 0.907; 95% CI, 0.840 – 0.952; P = 0.396).
A ROC curve analysis was performed to determine the cutoff point to differentiate patients with aSAH according to their unfavorable outcome based on their sCD40L concentrations at admission. This analysis showed that a plasma sCD40L level N 3.4 ng/mL predicted 6-month unfavorable outcome of patients with 77.1% sensitivity and 74.1% specificity (AUC, 0.825; 95%CI, 0.745-0.888) in Fig. 3B. Based on AUC, its predictive value was similar to WFNS score’s (AUC, 0.866; 95% CI, 0.792 - 0.921; P = 0.466) and Fisher score’s (AUC, 0.854; 95% CI, 0.778 - 0.912; P = 0.604). When a combined logistic-regression model was constructed, sCD40L numerically improved AUCs of WFNS score (AUC, 0.907; 95% CI, 0.840 – 0.952; P = 0.099) and Fisher score (AUC, 0.894; 95% CI, 0.825 – 0.943; P = 0.160). Analysis of Overall Survival
Poor Outcome Prediction Thirty-five patients (29.2%) suffered from unfavorable outcome within 6 months after aSAH. Table 2 showed that the variables associated with 6-month unfavorable outcome included WFNS score, Fisher score, acute hydrocephalus, intraventricular hemorrhage, external ventricular drain, vasospasm, computed tomography ischemia, blood glucose levels, plasma C-reactive protein levels and plasma sCD40L levels. When was configured a logistic-regression model which included the significant variables in the univariate analysis, it was demonstrated that WFNS score (OR, 4.276; 95% CI, 1.629-10.002; P = 0.001), Fisher score (OR, 3.861; 95% CI, 1.627-7.867; P = 0.002) and plasma sCD40L level (OR, 2.431; 95% CI, 1.223-4.998; P = 0.009) were the independent predictors for 6-month unfavorable outcome of patients. Table 1 The factors correlated with serum soluble CD40 ligand levels after aneurysmal subarachnoid hemorrhage. Characteristics
r value
P value
Gender (Male/Female) Age (y) WFNS score on admission Fisher score on admission Surgery Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Vasospasm Computed tomography ischemia Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L)
0.095 0.063 0.552 0.555 0.126 0.290 0.370 0.282 0.308 0.358 0.135 0.003 0.018 0.177 0.247 0.506
0.301 0.494 b0.001 b0.001 0.169 0.001 b0.001 0.002 0.001 b0.001 0.141 0.978 0.848 0.054 0.007 b0.001
Bivariate correlations were assessed by Spearman's or Pearson’s correlation coefficient. WFNS indicates World Federation of Neurological Surgeons.
The mean 6-month overall survival time was 161.4 days (95% CI: 152.6-170.3) in all patients. Table 3 showed that 6-month overall survival was correlated with WFNS score, Fisher score, acute hydrocephalus, intraventricular hemorrhage, external ventricular drain, vasospasm, computed tomography ischemia, blood glucose levels, plasma C-reactive protein levels and plasma sCD40L levels. A multivariate analyses selected WFNS score (HR, 3.894; 95% CI, 1.486-6.637; P = 0.001), Fisher score (HR, 3.651; 95% CI, 1.240-6.225; P = 0.001) and plasma sCD40L level (HR, 2.221; 95% CI, 1.197-4.716; P = 0.004) as the independent predictors for 6-month overall survival. Fig. 3C showed that, compared with patients with low sCD40L level (less than mean value), those with high sCD40L level (more than mean value) had significantly shorter 6-month overall survival time (the mean time, 146.2 days; 95% CI: 129.9-162.4 vs. the mean time, 175.7 days; 95% CI: 169.7-181.7; P b 0.001). Discussion Previous studies have found sCD40L levels are elevated in peripheral blood after brain injury including influenza virus-associated encephalopathy [32], ischemic stroke [34–36] and traumatic brain injury [37]. Recent study has also reported that there are an association between serum sCD40L levels and clinical severity and 30-day mortality of traumatic brain injury [37]. However, to the best of our knowledge, circulating sCD40L levels has not been studied in patients with aSAH. The current study determined sCD40L levels in plasma and further analyzed the association of plasma sCD40L levels with risk of long-term clinical outcomes and severity of aSAH. The most relevant and newer findings of our study were that serum sCD40L levels of patients were elevated compared with healthy controls, and that there were an association between serum sCD40L levels and aSAH severity and long-term prognosis including 6-month mortality and 6-month unfavorable outcome. The CD40–CD40L interaction is pivotal in the cellular immune response. CD40L was first described as an antigen, which is expressed
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Fig. 2. Correlative analysis of plasma soluble CD40 ligand (sCD40L) level with World Federation of Neurological Surgeons (WFNS) score and Fisher score.
on activated CD4 T cells, and CD40L interacts with CD40 expressed on B cells and induces the class switch. A broader role for CD40 signaling was revealed through the finding that CD40 is expressed on numerous cell types, including monocytes/macrophages, dendric cells, fibroblasts keratinocytes, endothelial cells, and vascular smooth muscle cells. Stimulation of these cell types through CD40 induces cell functions which contribute to inflammatory responses, including the expression of adhesion molecules, and also the release of proinflammatory cytokines, such as interleukin-6, interleukin-1beta, interleukin-8, interleukin-12, and tumor necrosis factor-alpha [8–12]. CD40L can be cleaved from the cell surface, releasing a sCD40L which is biologically active [13]. Therefore, sCD40L possesses proinflammatory effect [17–22]. Pathological findings has revealed that inflammatory cells were detectable in brain tissues after SAH and peripheral blood and cerebrospinal fluid concentrations of several proinflammatory cytokines such as interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha, are elevated and related to the clinical severity of aSAH. Accumulating evidence has demonstrated the crucial role of inflammatory process in the pathophysiological mechanism of aSAH [3,4]. The role of sCD40L in aSAH remains unclear but it is possible that its proinflamatory effects [13,40] could contribute to the pathophysiology of aSAH. 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 [41], with subsequent up regulation of proinflammatory factors. Ligation of CD40 on endothelial cells, smooth muscle cells, or mononuclear phagocytes triggers the expression of various proinflammatory mediators, such as the interleukin -1, interleukin-6, interleukin-12, tumor necrosis factor-alpha, and interferon-gamma [13] that have been demonstrated to be involved in the inflammatory process of brain injury after aSAH [3,4]. Thus, we hypothesized that CD40–sCD40L binding could facilitate the amplification of inflammatory response through the generation of cytokines and all these effects could facilitate the development of brain injury after aSAH. Although some researches have reported the elevated sCD40L levels in peripheral blood of patients with ischemic stroke [34–36], no study assesses the relationship between sCD40L and clinical severity and clinical outcome of ischemic stroke. Up to now, Leonardo et.al report serum sCD40L levels are associated with disease severity and 30-day mortality of brain trauma injury patients [37]. Our research, for the first time, demonstrated the close relationships between plasma sCD40L levels and disease severity and 6-month clinical outcome of aSAH. Importantly, the current study used a multivariate linear regression, but not a univariate analysis, to demonstrate the association of plasma sCD40L levels with the severity of aSAH reflected by WFNS scores and Fisher scores. In order to verify the relationship between sCD40L and long-term prognosis, we assessed both 6-month mortality and unfavorable outcome and 6-month overall survival using multivariate analysis and found their close associations. Interestingly, although a combined logisticregression model did not demonstrate the additive benefit of sCD40L to WFNS score and Fisher score, sCD40L possessed similar predictive value to WFNS score and Fisher score based on ROC curves. And,
Table 2 The factors associated with 6-month mortality and 6-month unfavorable outcome after aneurysmal subarachnoid hemorrhage. Mortality
Cases Male Age (y) WFNS score on admission Fisher score on admission Surgery Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Vasospasm Computed tomography ischemia Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Plasma sCD40L level (ng/mL)
Functional outcome
Non-survivors
Survivors
P value
Unfavorable outcome
Favorable outcome
P value
16 9 (56.3%) 41.9 ± 11.8 3.9 ± 0.7 3.9 ± 0.6 10 (62.5%) 8 (50.0%) 7 (43.8%) 8 (50.0%) 9 (56.3%) 6 (37.5%) 6.3 ± 6.0 8.0 ± 6.1 137.5 ± 18.8 85.4 ± 10.9 16.5 ± 4.7 13.7 ± 3.3 4.9 ± 1.4
104 62 (59.6%) 40.9 ± 11.5 2.4 ± 1.0 2.6 ± 0.8 58 (55.8%) 18 (17.3%) 16 (15.4%) 22 (21.2%) 27 (26.0%) 9 (8.7%) 6.4 ± 3.8 8.3 ± 4.3 129.7 ± 24.2 79.9 ± 13.8 12.8 ± 4.6 10.8 ± 3.1 3.0 ± 1.3
0.799 0.741 b0.001 b0.001 0.613 0.003 0.007 0.026 0.020 0.005 0.964 0.880 0.220 0.137 0.003 0.001 b0.001
35 20 (57.1%) 41.3 ± 10.7 3.6 ± 0.7 3.6 ± 0.8 22 (62.9%) 13 (37.1%) 12 (34.3%) 14 (40.0%) 16 (45.7%) 9 (25.7%) 5.6 ± 4.3 7.5 ± 4.5 134.7 ± 21.6 80.8 ± 12.3 15.0 ± 4.9 12.6 ± 3.3 4.4 ± 1.3
85 51 (60.0%) 40.9 ± 11.9 2.2 ± 1.0 2.5 ± 0.7 46 (54.1%) 13 (15.3%) 11 (12.9%) 16 (18.8%) 20 (23.5%) 6 (7.1%) 6.7 ± 4.0 8.5 ± 4.5 129.1 ± 24.4 80.6 ± 14.1 12.5 ± 4.5 10.5 ± 3.1 2.7 ± 1.2
0.772 0.858 b0.001 b0.001 0.380 0.008 0.007 0.015 0.016 0.012 0.170 0.238 0.244 0.945 0.009 0.002 b0.001
Numerical variables were presented as mean ± standard deviation. Categorical variables were expressed as counts (percentage). Numerical variables were analyzed by unpaired Student t test. Categorical variables were analyzed by chi-square test or Fisher exact test. WFNS indicates World Federation of Neurological Surgeons; sCD40L, soluble CD40 ligand.
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Fig. 3. Receiver operating characteristic curve analysis of plasma soluble CD40 ligand (sCD40L) levels for identifying aneurysmal subarachnoid hemorrhage patients with 6-month mortality (3A) and 6-month unfavorable outcome (3B). Survival curves (3C) for overall survival in patients with aneurysmal subarachnoid hemorrhage according to plasma soluble CD40 ligand (sCD40L) levels.
although sCD40L’s specificity and sensitivity values for prediction of mortality and unfavorable outcome were not very high, based on AUC, its predictive value was similar to WFNS score’s and Fisher score’s.
Therefore, sCD40L had shown high predictive performance for aSAH severity. Conclusions
Table 3 The factors associated with 6-month overall survival. Characteristics
Hazard ratio (95% confidence interval)
P value
Male Age (y) WFNS score on admission Fisher score on admission Surgery Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Vasospasm Computed tomography ischemia Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Plasma sCD40L level (ng/mL)
0.915 (0.341-2.457) 1.007 (0.965-1.051) 3.780 (2.215-6.453) 3.542 (2.097-5.981) 1.335 (0.485-3.675) 4.384 (1.642-11.702) 3.753 (1.395-10.092) 3.275 (1.228-8.736) 3.197 (1.190-8.590) 5.345 (1.936-14.754) 0.998 (0.881-1.131) 0.990 (0.883-1.109) 1.013 (0.993-1.033) 1.032 (0.992-1.074) 1.203 (1.064-1.361) 1.251 (1.089-1.437) 2.502 (1.687-3.712)
0.860 0.746 b0.001 b0.001 0.575 0.003 0.009 0.018 0.021 0.001 0.974 0.860 0.214 0.123 0.003 0.002 b0.001
Univariate logistic-regression analysis was used to calculate the hazard ratio and 95% confidence interval. WFNS indicates World Federation of Neurological Surgeons; sCD40L, soluble CD40 ligand.
This study demonstrates the there is an association between plasma sCD40L levels and aSAH severity and long-term prognosis. Our findings suggest that sCD40L may have potential to be a good prognostic biomarker of aSAH. Conflict of Interest Statement None Authors’ Contributions XDC and JYC were responsible of conceive, design and coordinate the study, made substantial contributions to acquisition of data, analysis and interpretation of data, and drafted the manuscript. JS and CL have made substantial contributions to acquisition of data and provided useful suggestions. HJB and MHC carried out the determination of sCD40L and have made substantial contributions to analysis and interpretation of data. JHL has made substantial contributions to analysis and interpretation of data.
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All authors revising the manuscript critically for important intellectual content and approved the final version of the manuscript. Funding This study was supported by a grant from Wenzhou Science and Technology Research Fund Project (No. Y20130263). Acknowledgement The authors thank all staffs in Department of Neurosurgery, The Central Hospital of Wenzhou City (Wenzhou, China) for their technical support. References [1] de Rooij NK, Linn FH, van der Plas JA, Algra A, Rinkel GJ. Incidence of subarachnoid haemorrhage: a systematic review with emphasis on region, age, gender and time trends. J Neurol Neurosurg Psychiatry 2007;78:1365–72. [2] Feigin VL, Lawes CM, Bennett DA, Barker-Collo SL, Parag V. Worldwide stroke incidence and early case fatality reported in 56 population-based studies: a systematic review. Lancet Neurol 2009;8:355–69. [3] Hanafy KA, Morgan Stuart R, Fernandez L, Schmidt JM, Claassen J, Lee K, et al. Cerebral inflammatory response and predictors of admission clinical grade after aneurysmal subarachnoid hemorrhage. J Clin Neurosci 2010;17:22–5. [4] Dhar R, Diringer MN. The burden of the systemic inflammatory response predicts vasospasm and outcome after subarachnoid hemorrhage. Neurocrit Care 2008;8: 404–12. [5] Le Roux PD, Elliott JP, Newell DW, Grady MS, Winn HR. Predicting outcome in poorgrade patients with subarachnoid hemorrhage: a retrospective review of 159 aggressively managed cases. J Neurosurg 1996;85:39–49. [6] Frijns CJ, Fijnheer R, Algra A, van Mourik JA, van Gijn J, Rinkel GJ. Early circulating levels of endothelial cell activation markers in aneurysmal subarachnoid haemorrhage: associations with cerebral ischaemic events and outcome. J Neurol Neurosurg Psychiatry 2006;77:77–83. [7] Khurana VG, Wijdicks EF, Heubline DM, McClellnd RL, Meyer FB, Piepgras DG, et al. A pilot study of Dendroaspis natriuretic peptide in aneursymal subarachnoid hemorrhage. Neurosurgery 2004;55:69–76. [8] Armitage RJ, Fanslow WC, Strockbine L, Sato TA, Clifford KN, Macduff BM, et al. Molecular and biological characterization of a murine ligand for CD40. Nature 1992; 357:80–2. [9] Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus G, et al. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 1998;391:591–4. [10] Foy TM, Aruffo A, Bajorath J, Buhlmann JE, Noelle RJ. Immune regulation by CD40 and its ligand GP39. Annu Rev Immunol 1996;14:591–617. [11] Burger D, Molnarfi N, Gruaz L, Dayer JM. Differential induction of IL-1beta and TNF by CD40 ligand or cellular contact with stimulated T cells depends on the maturation stage of human monocytes. J Immunol 2004;173:1292–7. [12] Suttles J, Milhorn DM, Miller RW, Poe JC, Wahl LM, Stout RD. CD40 signaling of monocyte inflammatory cytokine synthesis through an ERK1/2-dependent pathway. A target of interleukin (IL)-4 and IL-10 anti-inflammatory action. J Biol Chem 1999;274:5835–42. [13] Mach F, Schönbeck U, Sukhova GK, Bourcier T, Bonnefoy JY, Pober JS, et al. Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40-CD40 ligand signaling in atherosclerosis. Proc Natl Acad Sci U S A 1997;94:1931–6. [14] Andre P, Nannizzi-Alaimo L, Prasad SK, Phillips DR. Platelet-derived CD40L: the switch-hitting player of cardiovascular disease. Circulation 2002;106:896–9. [15] Danese S, Katz JA, Saibeni S, Papa A, Gasbarrini A, Vecchi M, et al. Activated platelets are the source of elevated levels of soluble CD40 ligand in the circulation of inflammatory bowel disease patients. Gut 2003;52:1435–41. [16] Nagasawa M, Zhu Y, Isoda T, Tomizawa D, Itoh S, Kajiwara M, et al. Analysis of serum soluble CD40 ligand (sCD40L) in the patients undergoing allogeneic stem cell transplantation: platelet is a major source of serum sCD40L. Eur J Haematol 2005;74: 54–60.
29
[17] Zhou L, Stordeur P, de Lavareille A, Thielemans K, Capel P, Goldman M, et al. CD40 engagement on endothelial cells promotes tissue factor-dependent procoagulant activity. Thromb Haemost 1998;79:1025–8. [18] Hezi-Yamit A, Wong PW, Bien-Ly N, Komuves LG, Prasad KS, Phillips DR, et al. Synergistic induction of tissue factor by coagulation factor Xa and TNF: evidence for involvement of negative regulatory signaling cascades. Proc Natl Acad Sci U S A 2005; 102:12077–82. [19] Miller DL, Yaron R, Yellin MJ. CD40L-CD40 interactions regulate endothelial cell surface tissue factor and thrombomodulin expression. J Leukoc Biol 1998;63:373–9. [20] Slupsky JR, Kalbas M, Willuweit A, Henn V, Kroczek RA, Müller-Berghaus G. Activated platelets induce tissue factor expression on human umbilical vein endothelial cells by ligation of CD40. Thromb Haemost 1998;80:1008–14. [21] Prasad KS, Andre P, He M, Bao M, Manganello J, Phillips DR. Soluble CD40 ligand induces beta3 integrin tyrosine phosphorylation and triggers platelet activation by outside-in signaling. Proc Natl Acad Sci U S A 2003;100:12367–71. [22] Graf D, Muller S, Korthauer U, van Kooten C, Weise C, Kroczek RA. A soluble form of TRAP (CD40 ligand) is rapidly released after T cell activation. Eur J Immunol 1995; 25:1749–54. [23] Ho TC, Yang SF, Wang PH, Lin LY, Tee YT, Liao WC, et al. Increased serum soluble CD40 ligand concentration in pelvic inflammatory disease. Clin Chim Acta 2015; 438:236–40. [24] Aukrust P, Muller F, Ueland T, Berget T, Aaser E, Brunsvig A, et al. Enhanced levels of soluble and membrane-bound CD40 ligand in patients with unstable angina: possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes. Circulation 1999;100:614–20. [25] Varo N, de Lemos JA, Libby P, Morrow DA, Murphy SA, Nuzzo R, et al. Soluble CD40L: risk prediction after acute coronary syndromes. Circulation 2003;108:1049–52. [26] Vakkalanka RK, Woo C, Kirou KA, Koshy M, Berger D, Crow MK. Elevated levels and functional capacity of soluble CD40 ligand in systemic lupus erythematosus sera. Arthritis Rheum 1999;42:871–81. [27] Clodi K, Asgary Z, Zhao S, Kliche KO, Cabanillas F, Andreeff M, et al. Coexpression of CD40 and CD40 ligand in B-cell lymphoma cells. Br J Haematol 1998;103:270–5. [28] Erturan I, Köroğlu BK, Adiloğlu A, Ceyhan AM, Akkaya VB, Tamer N, et al. Evaluation of serum sCD40L and homocysteine levels with subclinical atherosclerosis indicators in patients with psoriasis: a pilot study. Int J Dermatol 2014;53:503–9. [29] Ludwiczek O, Kaser A, Tilg H. Serum levels of soluble CD40 ligand are elevated in inflammatory bowel diseases. Int J Colorectal Dis 2003;18:142–7. [30] Frossard JL, Morel P, Kwak B, Pastor C, Berney T, Buhler L, et al. Soluble CD40 ligand in prediction of acute severe pancreatitis. World J Gastroenterol 2006;12:1613–6. [31] Lorente L, Martín MM, Varo N, Borreguero-León JM, Solé-Violán J, Blanquer J, et al. Association between serum soluble CD40 ligand levels and mortality in patients with severe sepsis. Crit Care 2011;15:R97. [32] Ichiyama T, Morishima T, Suenaga N, Kajimoto M, Matsubara T, Furukawa S. Analysis of serum soluble CD40 ligand in patients with influenza virus-associated encephalopathy. J Neurol Sci 2005;239:53–7. [33] Pusuroglu H, Akgul O, Erturk M, Uyarel H, Bulut U, Akkaya E, et al. Predictive value of elevated soluble CD40 ligand in patients undergoing primary angioplasty for STsegment elevation myocardial infarction. Coron Artery Dis 2014;25:558–64. [34] Garlichs CD, Kozina S, Fateh-Moghadam S, Tomandl B, Stumpf C, Eskafi S, et al. Upregulation of CD40-CD40 Ligand (CD154) in patients with acute cerebral ischemia. Stroke 2003;34:1412–8. [35] Ferro D, Loffredo L, Polimeni L, Fimognari F, Villari P, Pignatelli P, et al. Soluble CD40 ligand predicts ischemic stroke and myocardial infarction in patients with nonvalvular atrial fibrillation. Arterioscler Thromb Vasc Biol 2007;27:2763–8. [36] Davì G, Tuttolomondo A, Santilli F, Basili S, Ferrante E, Di Raimondo D, et al. CD40 ligand and MCP-1 as predictors of cardiovascular events in diabetic patients with stroke. J Atheroscler Thromb 2009;16:707–13. [37] Lorente L, Martín MM, González-Rivero AF, Ramos L, Argueso M, Cáceres JJ, et al. Serum soluble CD40 Ligand levels are associated with severity and mortality of brain trauma injury patients. Thromb Res 2014;134:832–6. [38] Fisher CM, Kistler JP, Davis JM. Relation of cerebral vasospasm to subarachnoid hemorrhage visualized by computerized tomographic scanning. Neurosurgery 1980;6: 1–9. [39] Drake C. Report of World Federation of Neurological Surgeons Committee on a universal subarachnoid hemorrhage grading scale. J Neurosurg 1988;68:985–6. [40] Noelle RJ, Roy M, Shepherd DM, Stamenkovic I, Ledbetter JA, Aruffo A. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc Natl Acad Sci U S A 1992;9:6550–4. [41] Chen Y, Chen J, Xiong Y, Da Q, Xu Y, Jiang X, et al. Internalization of CD40 regulates its signal transduction in vascular endothelial cells. Biochem Biophys Res Commun 2006;345:106–17.
