Ee

Ba

b

c

h

••••

a

ARRA

KIPCCG

1

boiet

Cf

h0

Neuroscience Letters 567 (2014) 6–10

Contents lists available at ScienceDirect

Neuroscience Letters

jo ur nal ho me p age: www.elsev ier .com/ locate /neule t

vidence for involvement of the CD40/CD40L system in post-strokepilepsy

ikui Zhanga, Min Chena,b, Heng Yangc, Tian Wua,b, Cuizhu Songa,b, Ren Guoa,∗

Department of Pharmacy, The Third Xiangya Hospital, Central South University, Changsha, Hunan, ChinaSchool of Pharmaceutical Sciences, Central South University, Changsha, Hunan, ChinaDepartment of Neurology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China

i g h l i g h t s

CD40/CD40L system is involved in the process of post-stroke epilepsy.T allele of CD40 −1C/T polymorphism is associated with post-stroke epilepsy.T allele carriers showed increased plasma sCD40L levels.sCD40L levels should be strictly controlled in stroke patients.

r t i c l e i n f o

rticle history:eceived 11 December 2013eceived in revised form 27 January 2014ccepted 1 March 2014

eywords:schemic stroke (IS)ost-stroke epilepsy (PSE)D40D40Lenetic polymorphism

a b s t r a c t

Post-stroke epilepsy (PSE) has a negative effect on stroke prognosis and quality of life. The CD40/CD40Lsystem is reported to be involved in the progression of multiple disease states. We investigated theassociation between functional polymorphism of CD40 and PSE susceptibility, and we also explored therole of the CD40/CD40L system in PSE. A case-control study was performed in 410 ischemic stroke (IS)patients and in 389 PSE patients. Genotyping was performed by polymerase chain reaction–restrictionfragment length polymorphism (PCR–RFLP). The CD40 mRNA and protein levels were determined byreal-time PCR and western blotting, respectively. The plasma sCD40L level was detected using an ELISAkit. The frequency of the T allele in PSE patients was significantly higher than in IS patients (P < 0.05).The plasma sCD40L level was significantly higher in the PSE patients than in the healthy controls andIS patients (P < 0.01, P < 0.05, respectively). The peripheral blood mononuclear cells (PBMCs) from PSE

patients showed significantly higher CD40 mRNA and protein expression than the healthy controls andIS patients (P < 0.01, P < 0.05, respectively). In the PSE patients, the T-allele carriers showed increasedplasma sCD40L levels and increased CD40 mRNA expression. Our study suggested that the T allele ofthe CD40 −1C/T polymorphism may be associated with PSE susceptibility. The CD40/CD40L system isinvolved in the process of PSE.

. Introduction

Epilepsy is a common and complex neurological disease causedy multiple factors and is characterized by an excessive dischargef certain neurons in the nervous system. Epilepsy affects approx-

mately 0.5–0.7% of the population worldwide [4]. According to anpidemiological survey, there are approximately 9 million epilep-ics in China. Ischemic stroke (IS) is one of the most frequent causes

∗ Corresponding author at: Department of Pharmacy, The Third Xiangya Hospital,entral South University, Changsha, Hunan, 410013, China. Tel.: +86 731 88618455;

ax: +86 731 88618455.E-mail address: grfw2012@aliyun.com (R. Guo).

ttp://dx.doi.org/10.1016/j.neulet.2014.03.003304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.

© 2014 Elsevier Ireland Ltd. All rights reserved.

of acquired epilepsy in the elderly population [13], and 2–14% ofelderly patients with stroke will suffer from epilepsy [11]. A Cana-dian research study suggests that post-stroke epilepsy (PSE) has anegative effect on stoke prognosis and quality of life [5]. To effec-tively prevent PSE, it is necessary to find some reliable predictorsthat can be easily detected in patients. It was reported that strokeseverity, occurrence of hemorrhagic stroke and cortical lesion sizewere independent predictors of acute symptomatic seizures [3,9],but these indices lack identified criteria for estimating their rela-tionship with the incidence of PSE.

Accumulating evidence indicates that oxidative stress inducedby increased reactive oxygen and/or nitrogen species is involvedin the pathogenesis and progression of epilepsy, and agentswith antioxidant effects have produced neuroprotection against

ience Letters 567 (2014) 6–10 7

oCnCflpdtnssC

swoTti

2

2

ac2lrmmbsMtsidh

so

2

wDumcmw5Poa

2

oP

Fig. 1. Genotyping for the CD40 −1C/T polymorphism using agarose gel elec-trophoresis. −1CC homozygotes showed two fragments (245 bp and 93 bp), −1CT

B. Zhang et al. / Neurosc

xidative stress-induced dysfunction in neurons [8,23]. TheD40/CD40L system serves as a link between inflammation, immu-ity, and tumorigenesis [2]. When combined with its ligand CD40L,D40 is activated and leads to the up-regulation of many proin-ammatory and proatherogenic genes through the NF-kappa Bathway. The CD40/CD40L interaction also produces ROS, whicheactivate endothelial nitric oxide and increase platelet aggrega-ion, causing endothelial and platelet dysfunction [18]. sCD40L isow considered to be a predictor for the progression and progno-is of patients with cardiovascular diseases [6,7]. However, limitedtudies have focused on the relationship between PSE and theD40/CD40L system.

In this study, we explored the association between a functionalingle-nucleotide polymorphism (SNP) in the CD40 Kozak sequenceith the susceptibility to PSE; furthermore, we detected the level

f the CD40/CD40L system in stroke patients with or without PSE.he aims of this study were to clarify the role of CD40/CD40L sys-em in PSE and to propose a more suitable index for predicting thencidence of PSE.

. Materials and methods

.1. Subjects

The study cohort consisted of 410 IS patients, 389 PSE patients,nd 160 healthy control subjects; all were enrolled at outpatientlinics in The Third Xiangya Hospital in Hunan from September010 to April 2013. All patients and controls are Han people and

ive in Changsha or nearby counties. Furthermore, all subjects expe-ienced no seizures before the stroke event, and all subjects wereatched for age and sex. There were no differences in pre-existingedical conditions between the IS and PSE patients. IS was defined

y focal neurological signs or symptoms of vascular origin that per-isted longer than 24 h and was confirmed by brain CT scan and/orRI under baseline conditions and brain CT scanning with con-

rast medium after 48–72 h. PSE patients were defined by clinicalymptoms and positive EEG performance. Subjects without fam-ly history of IS, epilepsy, coronary artery diseases, autoimmuneiseases and systemic inflammatory diseases were recruited asealthy controls.

Written informed consent was obtained from all subjects. Thetudy was performed with the approval of the Ethical Committeef the Third Xiangya Hospital of Central South University.

.2. Genotyping

Genomic DNA samples were extracted and purified from thehole blood with the standard phenol/chloroform protocols. AllNA samples were genotyped for the CD40 −1C/T polymorphismsing polymerase chain reaction–restriction fragment length poly-orphism (PCR–RFLP). PCR was performed on a personal thermal

ycler (Biometra®, Germany). To genotype the CD40 −1C/T poly-orphism, a 338 bp fragment was amplified from genomic DNAith the following primers: 5′-ACCATGCCTCCTCCCGTAC-3 (sense);

′-CCACTCCCAACTCCCGTCT-3′ (antisense). A 4 �L aliquot of theCR product was digested with 3U of Nco I endonuclease at 37 ◦Cvernight. The restriction digest products were analyzed using 2.0%garose gel electrophoresis (Fig. 1).

.3. Determination of sCD40L concentration in the plasma

5 mL venous blood sample was collected from each subjectn day 2 of disease onset for determination of sCD40L level.lasma levels of sCD40L were measured by an immunoassay

heterozygotes showed three fragments (338 bp, 245 bp, and 93 bp), and −1TThomozygotes showed only one fragment (338 bp).

(Quantikine CD40 ligand, R&D Systems). The intra-assay and inter-assay coefficients of variation were 3% and 5%, respectively.

2.4. Isolation of human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) were collectedfrom buffy coats, after centrifugation at 2000 × g for 30 min over5 mL Ficoll–Hypaque gradients (Sigma, USA), washed twice withice cold PBS, and then total RNA from the PBMCs was extracted forthe subsequent experiment. The PBMCs were collected at the sametime after the stroke event in each subject.

2.5. Real-time PCR analysis

To validate differentially expressed CD40 mRNA in isolatedPBMCs from subjects, real-time PCR was performed accordingto the manufacturer’s instructions using an ABI 7300 real-timePCR system with the SYBR Green method. The sequences ofprimers were as follows: CD40: 5′-GCAGGCACAAACAAGACTGA-3′

(sense) and 5′-TCGTCGG GAAATTGATCTC-3′ (antisense); GAPDH(endogenous control): 5′-CTGCACCACCAACTGCTTAG-3′(sense); 5′-AGGTCCACCACTGACACGTT -3′ (antisense). The relative abundanceof the CD40 mRNA from the PBMCs was normalized to the expres-sion level of GAPDH. All amplification reactions were performed intriplicate.

2.6. Western blotting

Protein was extracted from cultured PBMCs with RIPA lysisbuffer (containing 0.1% PMSF) (Beyotime Biotech, China) follow-ing the manufacturer’s instructions. An equal amount (100 �g)of the total protein was separated using SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) (Beyotime Biotech, China) and thentransferred onto a polyvinylidene difluoride (PVDF) (Pell, USA)membrane. The membranes were immunoblotted with antibodiesagainst CD40 (human monoclonal antibody, Abcam, UK) or GAPDH(Abcam, UK) followed by a horseradish peroxidase-conjugated sec-ondary antibody. The immunoblots were visualized using a Bio-RadCalibrated Densitometer.

2.7. Statistical analysis

The descriptive results of continuous variables are expressedas the mean ± SE. Statistical analysis of the data was carried outusing the SSPS 11.5 software. Differences in the genotype and allelefrequencies between groups were compared using the �2 test.Differences among groups were analyzed using one-way ANOVA

followed by the Student–Newman–Keuls test. The statistical powerof our study was assessed as described previously [12]. The signif-icance level was chosen as P < 0.05.

8 B. Zhang et al. / Neuroscience Letters 567 (2014) 6–10

Table 1General characteristics of the IS patients and PSE patients.

Parameter IS (n = 410) PSE (n = 389) P

Gender (male/female) 241/169 219/170 NSAge (year) 62.32 ± 8.06 64.46 ± 9.87 NSBMI (kg/m2) 22.34 ± 2.48 23.15 ± 1.77 NSSBP (mmHg) 140 ± 12 147 ± 14 NSDBP (mmHg) 82 ± 8 86 ± 10 NSCreatinine (Cr) (�mol/L) 84.42 ± 13.22 85.69 ± 16.47 NSHDL-C (mmol/L) 1.28 ± 0.49 1.32 ± 0.66 NSLDL-C (mmol/L) 2.84 ± 0.52 2.79 ± 0.82 NS

3

3P

tTd

3−

adHon3TtPp

3d

ohtstiwist

3d

afaCaCts

Fig. 2. The plasma sCD40L level in each group during the disease process. The plasmasCD40L concentration was detected using ELISA in each group. (A) The differencesin plasma sCD40L levels in healthy controls (n = 160), IS patients (n = 160) and PSEpatients (n = 160). The descriptive results are expressed as the mean ± SEM. ** P < 0.01compared with healthy controls, and # P < 0.05 compared with IS patients. (B) Theassociation between the sCD40L level and CD40 −1C/T genotype in PSE patients. The

Triglyceride (TG) (mmol/L) 1.92 ± 0.68 1.88 ± 1.02 NSTotal cholesterol (TC) (mmol/L) 4.66 ± 0.81 4.71 ± 0.95 NS

. Results

.1. Clinical and laboratory characteristics in the IS patients andSE patients

The demographic characteristics and distribution of risk fac-ors in both the IS patients and PSE patients are shown in Table 1.he clinical and laboratory characteristics were not significantlyifferent between the IS patients and PSE patients.

.2. Distribution of allele and genotype frequencies of CD401C/T genotype

The distribution and allele frequencies of the CD40 −1C/T vari-nt in IS patients and PSE patients are presented in Table 2. Theistribution of genotypes in both study groups complied with theardy–Weinberg equilibrium (P > 0.05). The results obtained fromur study demonstrated that the frequency of the T allele was sig-ificantly higher in the PSE group than in the IS group (50.5% vs.8.5%, �2 = 23.204, P = 0.0000017, OR = 1.628, 95% CI: 1.335–1.986,able 2), and the carriers of this allele were overrepresented inhe PSE group than in the IS group (70.4% vs. 58.8%, �2 = 11.838,

= 0.00058, OR = 1.671, 95% CI: 1.246–2.241, Table 2). The statisticalower of the present study was 96.2%.

.3. The association of the plasma sCD40L concentration withifferent groups

To evaluate the effect of the CD40/CD40L system on the processf IS and PSE, 480 blood samples from IS patients, PSE patients andealthy controls were collected, and the plasma sCD40L concen-ration was examined by ELISA. Our data revealed that the plasmaCD40L concentration was significantly higher in the PSE grouphan in the other groups (Fig. 2A). When we divided the PSE patientsnto three subgroups according to the CD40 −1C/T polymorphism,

e found that individuals carrying the TT genotype showed signif-cantly higher sCD40L levels (Fig. 2B). No differences in the plasmaCD40L concentrations were observed between the genotypes inhe IS patients and healthy controls (data not shown).

.4. Differences in PBMCs CD40 mRNA expression betweenifferent groups

To test the possibility that CD40 mRNA expression is associ-ted with the different disease states, total RNA was extractedrom the PBMCs in IS patients, PSE patients and healthy controls,nd real-time PCR was performed. Our data demonstrated thatD40 mRNA expression was significantly elevated in the IS patients

nd PSE patients, and the PSE groups showed the highest level ofD40 mRNA expression (Fig. 3A). We also explored the associa-ion between CD40 mRNA and the CD40 −1C/T polymorphism. Ashown in Fig. 3B, in PSE patients, carriers with the T allele showed

descriptive results are expressed as the mean ± SEM, n = 50 in each group. * P < 0.05,** P < 0.01 compared with the CC genotype, and # P < 0.05 compared with the CTgenotype.

an increased CD40 mRNA expression, and individuals with the TTgenotype showed the highest expression of CD40 mRNA (Fig. 3B).

3.5. Difference in CD40 protein level among different groups

Considering that the CD40 protein level also reflects the activityof the CD40/CD40L system, we examined CD40 protein expressionin the PBMCs from IS patients, PSE patients and healthy controls.We found that CD40 protein levels were significantly increased in ISpatients and PSE patients relative to the controls. When comparedwith the IS group, the PSE group showed a higher CD40 proteinlevel (Fig. 4).

4. Discussion

CD40 and CD40L belong to the tumor necrosis factor superfa-mily and the tumor necrosis factor family, respectively [1,17]. Afterstimulation with a wide range of platelet activators and cytokines,the CD40/CD40L system is activated and then induces the up-regulation of many pro-inflammatory genes and pro-coagulantgenes [2]. In the past several years, there have been numerous stud-ies focusing on the involvement of CD40 in cardiovascular disease[19,20]. Many studies have also been conducted to explore the rela-tionship between the genetic polymorphism of the CD40/CD40Lsystem and cardiovascular diseases [15,16,22]. Nonetheless, littleinformation is available regarding the effect of CD40 gene poly-

morphisms on PSE susceptibility. The data from our study haveprovided compelling evidence that the CD40 −1C/T polymorphismis associated with PSE susceptibility, and individuals with a T allelehave shown significantly higher incidence of epilepsy after stroke.

B. Zhang et al. / Neuroscience Letters 567 (2014) 6–10 9

Table 2Genotype distribution and allele frequency of the CD40 −1C/T polymorphism in IS and PSE patients.

n Genotype (%) Allele (%) CarriageCC CT TT C T T

IS patients 410 169 (41.2) 166 (40.5) 75 (18.3) 504 (61.5) 316 (38.5) 241 (58.8)PSE patients 389 115 (29.6) 155 (39.8) 119 (30.6) 385 (49.5) 393 (50.5) 274 (70.4)�2 15.396 23.204 11.838P-value 0.00045 0.0000017 0.00058

pIaetbppaaasttr

pCm

F−acaPsaa

OR (95% CI)

Furthermore, in our study, we observed an increased level oflasma sCD40L and increased CD40 expression in PSE patients.

ncreased sCD40L and CD40 levels reflect a pro-thrombotic statend an oxidative stress state; individuals with different sCD40L lev-ls showed different therapeutic effects and prognoses [15]. Alonghe same line, the up-regulation of CD40 and sCD40L appears toe responsible for the increased incidence of epilepsy in strokeatients. Monitoring the plasma sCD40L concentration in strokeatients may be a reliable method for forecasting stoke prognosesnd drug therapeutic effects. All patients in our study will receive

long period of follow-up. In this period, we will record the databout the frequency and severity of epilepsy in these patients andample a small amount of blood to measure the sCD40L levels. Athe end of the follow-up, these data will help us to assess the rela-ionship between the sCD40L levels and the epileptic recurrenceate.

Given that CD40 and CD40L may influence each other in the

rocess of diseases [24], we also explored the association betweenD40 and CD40L. Our results revealed that the CD40 −1C/T poly-orphism influences the plasma sCD40L level in PSE patients.

ig. 3. CD40 mRNA expression in each group and its association with the CD401C/T genotype. CD40 mRNA expression in PBMCs was analyzed by real-time PCRnd expressed as a ratio to the control gene. (A) CD40 mRNA expression in healthyontrols (n = 75), IS patients (n = 75) and PSE patients (n = 75). The descriptive resultsre expressed as the mean ± SEM. ** P < 0.01 compared with healthy controls, and #

< 0.05 compared with IS patients. (B) The association between CD40 mRNA expres-ion and CD40 −1C/T genotype in PSE patients. The descriptive results are expresseds the mean ± SEM, n = 50 in each group. ** P < 0.01 compared with the CC genotype,nd # P < 0.05 compared with the CT genotype.

1.628 (1.335–1.986) 1.671 (1.246–2.241)

Because this polymorphism exists in the Kozak sequence, whichcan influence the initiation of CD40 transcription [10], it may affectthe sCD40L level through feedback regulation during the stroke andinduce epilepsy. Based on these results, we can attain some enlight-enment for clinical treatment. Stroke patients with the T allele mayface a higher epilepsy risk, which would lead to a negative effecton the prognosis of stroke. Therefore, in stroke patients with the Tallele, it is crucial to control the plasma sCD40L and CD40 at a lowlevel.

In summary, all of these data convincingly support the hypoth-esis that an activated CD40/CD40L system may serve as a crucialpathogenic factor in the development of PSE. Our evidence suggestsan important therapeutic potential for controlling the CD40/CD40Lsystem in stroke patients. It should be noted that there are somelimitations to our study. For example, our sample size is relativelysmall, so more large-scale studies are needed to fully unveil the roleof the CD40/CD40L system in PSE. Some other gene polymorphismsassociated with atherosclerosis and epilepsy, such as ADAM 10 [14]and netrin G1 [21], may also have an effect on PSE. Further cellexperiments should also be conducted to explore the underlying

pathogenic mechanism of the CD40/CD40L system in PSE.

Fig. 4. The protein expression of CD40 in each group. PBMCs were harvested fromsubjects in healthy controls (n = 30), IS patients (n = 30) and PSE patients (n = 30).CD40 protein expression levels were assessed by western blotting. Data are pre-sented as the mean ± SEM. ** P < 0.01 compared with healthy controls, and # P < 0.05compared with IS patients.

1 ience L

S

d

C

A

Fd

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

during experimental temporal lobe status epilepticus, PLoS One 7 (2012)

0 B. Zhang et al. / Neurosc

ources of funding

This project was supported by Chinese National Science Foun-ation (No. 81000120).

onflicts of interest statement

There are no conflicts of interest.

cknowledgments

This project was supported by the Chinese National Scienceoundation (No. 81000120) to Ren Guo and Hunan Science foun-ation (No. 09JJ0651).

eferences

[1] S.X. Anand, J.F. Viles-Gonzalez, J.J. Badimon, E. Cavusoglu, J.D. Marmur,Membrane-associated CD40L and sCD40L in atherothrombotic disease,Thromb. Haemostasis 90 (2003) 377–384.

[2] C. Antoniades, C. Bakogiannis, D. Tousoulis, A.S. Antonopoulos, C. Stefanadis,The CD40/CD40 ligand system: linking inflammation with atherothrombosis,J. Am. Coll. Cardiol. 54 (2009) 669–677.

[3] E. Beghi, R. D’Alessandro, S. Beretta, D. Consoli, V. Crespi, L. Delaj, C. Gandolfo,G. Greco, A. La Neve, M. Manfredi, F. Mattana, R. Musolino, L. Provinciali, M.Santangelo, L.M. Specchio, G. Zaccara, Incidence and predictors of acute symp-tomatic seizures after stroke, Neurology 77 (2011) 1785–1793.

[4] G. Birbeck, E. Chomba, M. Atadzhanov, E. Mbewe, A. Haworth, The social andeconomic impact of epilepsy in Zambia: a cross-sectional study, Lancet Neurol.6 (2007) 39–44.

[5] J.G. Burneo, J. Fang, G. Saposnik, Impact of seizures on morbidity and mortalityafter stroke: a Canadian multi-centre cohort study, Eur. J. Neurol. 17 (2010)52–58.

[6] G. Davi, A. Tuttolomondo, F. Santilli, S. Basili, E. Ferrante, R.D. Di, A. Pinto, G.Licata, CD40 ligand and MCP-1 as predictors of cardiovascular events in diabeticpatients with stroke, J. Atheroscler. Thromb. 16 (2009) 707–713.

[7] D. Ferro, L. Loffredo, L. Polimeni, F. Fimognari, P. Villari, P. Pignatelli, V. Fuster, F.Violi, Soluble CD40 ligand predicts ischemic stroke and myocardial infarctionin patients with nonvalvular atrial fibrillation, Arterioscler., Thromb., Vasc. Biol.27 (2007) 2763–2768.

[8] J. Folbergrova, J. Otahal, R. Druga, Brain superoxide anion formation in imma-

ture rats during seizures: protection by selected compounds, Exp. Neurol. 233(2012) 421–429.

[9] G.J. Jungehulsing, P.U. Heuschmann, M. Holtkamp, S. Schwab, P.L. Kolominsky-Rabas, Incidence and predictors of post-stroke epilepsy, Acta Neurol. Scand.(2013), http://dx.doi.org/10.1111/ane.12070.

[

etters 567 (2014) 6–10

10] M. Kozak, Initiation of translation in prokaryotes and eukaryotes, Gene 234(1999) 187–208.

11] D.L. Labovitz, W.A. Hauser, R.L. Sacco, Prevalence and predictors of earlyseizure and status epilepticus after first stroke, Neurology 57 (2001)200–206.

12] J.M. Lalouel, A. Rohrwasser, Power and replication in case-control studies, Am.J. Hypertens. 15 (2002) 201–205.

13] X. Li, M.M. Breteler, M.C. de Bruyne, H. Meinardi, W.A. Hauser, A. Hofman,Vascular determinants of epilepsy: the Rotterdam Study, Epilepsia 38 (1997)1216–1220.

14] Y. Li, F. Liao, X.J. Yin, L.L. Cui, G.D. Ma, X.X. Nong, H.H. Zhou, Y.F. Chen, B. Zhao, K.S.Li, An association study on ADAM10 promoter polymorphisms and atheroscle-rotic cerebral infarction in a Chinese population, CNS Neurosci. Ther. 19 (2013)785–794.

15] A. Malarstig, B. Lindahl, L. Wallentin, A. Siegbahn, Soluble CD40L levels are regu-lated by the −3459 A > G polymorphism and predict myocardial infarction andthe efficacy of antithrombotic treatment in non-ST elevation acute coronarysyndrome, Arterioscler., Thromb., Vasc. Biol. 26 (2006) 1667–1673.

16] Y. Onouchi, S. Onoue, M. Tamari, K. Wakui, Y. Fukushima, M. Yashiro, Y. Naka-mura, H. Yanagawa, F. Kishi, K. Ouchi, M. Terai, K. Hamamoto, F. Kudo, H.Aotsuka, Y. Sato, A. Nariai, Y. Kaburagi, M. Miura, T. Saji, T. Kawasaki, Y. Naka-mura, A. Hata, CD40 ligand gene and Kawasaki disease, Eur. J. Hum. Genet. 12(2004) 1062–1068.

17] M.L. Prasad, M. Velickovic, S.A. Weston, E.M. Benson, Mutational screening ofthe CD40 ligand (CD40L) gene in patients with X linked hyper-IgM syndrome(XHIM) and determination of carrier status in female relatives, J. Clin. Pathol.58 (2005) 90–92.

18] M. Rizvi, D. Pathak, J.E. Freedman, S. Chakrabarti, CD40–CD40 ligand interac-tions in oxidative stress, inflammation and vascular disease, Trends Mol. Med.14 (2008) 530–538.

19] U. Schonbeck, N. Gerdes, N. Varo, R.S. Reynolds, D.B. Horton, U. Bavendiek,L. Robbie, P. Ganz, S. Kinlay, P. Libby, Oxidized low-density lipoprotein aug-ments and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors limitCD40 and CD40L expression in human vascular cells, Circulation 106 (2002)2888–2893.

20] U. Schonbeck, P. Libby, CD40 signaling and plaque instability, Circ. Res. 89(2001) 1092–1103.

21] A. Stepanyan, R. Zakharyan, A. Boyajyan, The netrin G1 gene rs628117 poly-morphism is associated with ischemic stroke, Neurosci. Lett. 549 (2013)74–77.

22] C. Tian, W. Qin, L. Li, W. Zheng, F. Qiu, A common polymorphism in CD40Kozak sequence (−1C/T) is associated with acute coronary syndrome, Biomed.Pharmacother. 64 (2010) 191–194.

23] C.Y. Tsai, J.Y. Chan, K.S. Hsu, A.Y. Chang, S.H. Chan, Brain-derivedneurotrophic factor ameliorates brain stem cardiovascular dysregulation

e33527.24] B. Zhang, T. Wu, C. Song, M. Chen, H. Li, R. Guo, Association of CD40 −1C/T

polymorphism with cerebral infarction susceptibility and its effect on sCD40Lin Chinese population, Int. Immunopharmacol. 16 (2013) 461–465.