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1 These authors contributed equally to this study.
Byung Ok Kwak
Footnotes
1 These authors contributed equally to this study.
Affiliations
Biopharmaceuticals & Herbal Medicine Evaluation Department, Biologics Division, National Institute of Food and Drug Safety Evaluation, Chungcheongbuk-do, Republic of Korea
Corresponding author at: Department of Pediatrics, Konkuk University Medical Center, 120−1 Neungdong−ro (Hwayang−dong), Gwangjin−gu, Seoul, 05030, Republic of Korea.
There are many studies about cytokine levels and febrile seizures(FS).
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The association between cytokine levels and FSs is inconclusive.
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This study shows FS patients have significantly higher CSF IL-1β and serum IL-6 levels.
Abstract
Purpose
Febrile seizures (FSs) are the most common form of childhood seizures. During infection, both pro-inflammatory and anti-inflammatory cytokines are produced. Complex interactions among immune-inflammatory process, cytokine activation, and genetic factors are involved in the pathogenesis of FSs. The association between cytokines and FSs during childhood is inconclusive due to inconsistent results reported in different studies. We performed a systematic review and meta-analysis to determine an association between cytokines and FS in children.
Methods
We searched PubMed, EMBASE, and Cochrane databases for studies published up to January 2017 using the following key words: [“cytokine” OR “interleukin” OR “tumor necrosis factor alpha” OR “interferon-gamma” OR “single nucleotide polymorphism”] AND [“febrile seizure” OR “febrile convulsion”] AND [“pediatric” OR “infant” OR “child”]. Standardized mead difference (SMD) and 95% confidence intervals (CI) were calculated using standard meta-analysis techniques.
Results
A total of 6 studies enrolling 243 children with FS and 234 controls were included in the meta-analysis. A total of 4 different inflammatory mediators were. The results indicated that CSF IL-1β level and serum IL-6 level were significantly associated with FS (CSF IL-1β: SMD, 1.064; 95% CI, 0.217–1.611; P < 0.01, serum IL-6 SMD, 2.654; 95% CI, 2.332–2.975; P < 0.01).
Conclusion
The results of this meta-analysis suggest that CSF IL-1β level and serum IL-6 level are associated with an increased risk of FSs in children. Based on these results, it is expected that a therapeutic agent for specific cytokines could be developed in the future to prevent FS.
]. FSs are defined by the International League Against Epilepsy as an elevated or rapidly rising fever of short duration associated with uncomplicated seizure that does not predispose to epilepsy and is not accompanied by any neurologic abnormality, no previous neonatal seizures or a previous unprovoked seizure, and not meeting the criteria for other acute symptomatic seizures in children between 6 months and 5 years of age [
FS can be divided into 2 categories. Simple FS is a seizure that only occurs once in 24 h, is generalized, has a duration of less than 15 min, while complex FS is a seizure recurs within 24 h, is focal, and has a duration of more than 15 min [
Pro-inflammatory and anti-inflammatory cytokines regulate immune response. During infection, both pro-inflammatory and anti-inflammatory cytokines are produced [
]. IL-1β, TNF- α and IL-6 are pro-inflammatory cytokines that participate in the induction of acute-phase inflammation reactions, including fever. IL-1 receptor antagonist (IL-1RA) and IL-10 are anti-inflammatory cytokines and have a negative feedback effect during fever [
]. The balance between these two cytokine groups influences the severity of the fever. Complex interactions among immune-inflammatory process, cytokine activation, and genetic factors are involved in the pathogenesis of FSs [
]. Experimental studies demonstrate that inflammation and inflammatory mediators are the main causes and propagators of both febrile and epileptic seizures [
Several case-control studies have been performed to measure the concentration of cytokines in the serum or cerebrospinal fluid (CSF) of seizure patients compared with that of healthy controls without seizures [
]. Additionally, case-control genetic association studies have been conducted to establish a potential correlation between genetic polymorphisms and susceptibility to common diseases [
Meta-analyses are required to pool the existing inconsistent data. A systematic literature review of case-control studies that measured cytokine concentrations was also performed.
2. Materials and methods
We conducted a systematic review and meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.
2.1 Method for searching and identifying studies
A systematic literature search was conducted in PubMed, EMBASE, Cochrane Trial (CENTRAL) by using various synonyms for epilepsy and cytokines, such as “febrile seizure”, “febrile convulsion”, “cytokine”, “interleukin”, “tumor necrosis factor alpha”, “TNF-alpha”, “interferon-gamma”, and “single nucleotide polymorphism”, with “pediatric”, “infant”, or “child”. There were no restrictions on language, population, or publication year. The last search was performed on January 17, 2017.
We included studies of human epilepsy or FSs concerning cytokine measurement in serum and cerebrospinal fluid (CSF). Animal studies, articles that were not included studies, reviews, comments, case reports, and studies with inadequate data were excluded. All titles and abstracts were independently screened by two investigators (A and BO). Both authors independently checked the remaining articles for full-text eligibility.
2.2 Study selection and data extraction
The following inclusion criteria were applied: 1) the study was designed as a case-control study, 2) it diagnosed patients with FS/epilepsy without any other neurologic complications 3) it enrolled healthy controls, 4) it measured plasma cytokine concentrations, and 5) it provided adequate data, including genotype/allele frequency in both the case and control groups to allow calculation of the pooled odds ratio. Reviews, comments, animal studies, case reports and studies with inadequate data were excluded from the meta-analysis. Additionally, we excluded studies that used a definition of FS other than that used in our study, those that stimulated seizure by using lipopolysaccharide, and studies that did not include a healthy comparison group (i.e., the control group had epilepsy, encephalopathy or CNS disorder).
The titles and abstracts of the identified articles were checked and independently reviewed by two of the authors (A and BO), and discrepancies were resolved by discussion. The following data were extracted: the first author, publication year, study design, study location, ethnicity, study population, sample size, sample material, investigated cytokine gene, and the cytokine levels of the case and control groups. Any discrepancies in the interpretations of the data were resolved via discussion with a third reviewer.
2.3 Quality assessment
The two authors separately assessed the quality of the included studies. Any disagreement was resolved via discussion with a third reviewer, after which the study was reevaluated. We evaluated case control studies by using the Newcastle-Ottawa Scale. Nine points were given to studies of the highest quality, which were considered to have sufficiently “high quality” for inclusion in the meta-analysis. A total score ≤3 was considered to represent “low quality,” a score of 4 or 5 was considered to represent “moderate quality,” and a score ≥6 was considered to represent “high quality.”
2.4 Data synthesis and analysis
The mean differences and 95% confidence intervals (CIs) were calculated from the extracted data. We assessed interstudy heterogeneity by using I2 statistics. The I2 value was expressed as a percentage of the total variation across studies; when I2 > 50%, the assumption of homogeneity was deemed invalid, and the random effects model (DerSimonian-Laird method) was applied; otherwise, the fixed model (Mantel-Haenszel method) was used for the meta-analysis. A sensitivity analysis was performed by removing each study sequentially to evaluate the robustness of the combined estimates and to examine its contribution to the pooled odds ratio (OR). Publication bias was evaluated by using funnel plots, Egger’s test and the Begg-Mazumdar rank correlation test. P < 0.05 was considered statistically significant. The meta-analysis was performed using Comprehensive Meta-Analysis version 2.0 (Biostat, Englewood, NJ, USA).
3. Result
3.1 Literature search and selection
Fig. 1 shows how relevant studies were identified for this meta-analysis and why studies were excluded. Among a total of 206 studies that were identified from the initial search, 3 additional studies were added; 27 duplicates were then removed. After the abstracts and titles of 182 studies were reviewed, 31 articles remained. Through the full text review, 25 studies were excluded (the reasons are described in Fig. 1), and finally, 6 reports were included in this meta-analysis.
Fig. 1Flow diagram of the literature search and study selection process. The values in parentheses indicate the number of documents corresponding to each category.
A total of 6 studies were selected for this meta-analysis. All the studies were prospective case-controlled. Quality was evaluated with the Newcastle-Ottawa Scale, and all the studies were awarded 7 to 8 stars, indicating high quality (Table 1).
Table 1Assessment of the quality of the included studies by using the Newcastle-Ottawa scale.
Overall, 243 FS patients and 234 controls were enrolled in all the included studies. The characteristics of the included studies are summarized in Table 2. A total of 4 different inflammatory mediators were investigated in these studies. The studies reported protein levels in serum or CSF and compared them with those of controls. We conducted meta-analyses of serum IL-1β levels based on five studies [
]. The cytokine levels of patients and controls were expressed in pg/ml and significant difference was shown in P-value. Consistent significant differences in certain cytokines were not detected.
Table 2Characteristics of the studies included in the meta-analysis.
Serum IL-1β levels were investigated in five case-control studies with 143 patients with FS and 134 healthy controls. There were two investigations in Asian populations, two in Caucasians and one in Egyptians.
Compared with the control group, the FS patients’ serum IL-1β level did not differ significantly (standardized mean difference = 0.487; 95% CI = −0.480 to +1.455, P > 0.05). The included studies were statistically heterogeneous (I2 = 92.7%); thus, the random effects model was used for the meta-analysis (Fig. 2).
Fig. 2Meta-analysis of serum IL-1β levels using the random effects model.
In the subgroup analysis, there were two studies of participants with Asian ethnicity; they included 71 patients with FS and 71 healthy controls. Compared with the control group’s serum IL-1β levels, the Asian patient group’s standardized mean difference was 1.403, the 95% CI was from −0.086 to 2.892, and the P-value was 0.065. There were two studies of participants with Caucasian ethnicity; they included 39 patients with FS and 25 healthy controls. Compared with the control group’s IL-1β levels, the Caucasian patient group’s standardized mean difference was −0.122, the 95% CI was from −1.657 to 1.414, and the P-value was 0.877. One investigation examined Egyptian participants and included 33 patients with FS and 38 healthy controls. Compared with the controls, the Egyptian patients’ standardized mean difference was −0.186, the 95% CI was from −2.275 to 1.902, and the P-value was 0.861 (Fig. 3). No significant differences were detected according to ethnicity (Fig. 3).
Fig. 3Subgroup meta-analysis of serum IL-1β levels according to ethnicity.
Publication bias was found using a funnel plot (Fig. 4). However, the Begg-Mazumdar rank correlation test and Egger’s regression test did not show evidence of publication bias.
There were 70 patients with FS and 56 healthy controls extracted from 2 studies. The serum TNF-α levels of the FS patients and the healthy controls did not differ significantly (standardized mean difference = −0.006; 95% CI = −0.565 to 0.554, P > 0.05). The included studies were statistically heterogeneous (I2 = 54.653%); thus, the random effects model was used for the meta-analysis (Fig. 5).
Two studies were included in the serum IL-6 meta-analysis. The studies included 141 patients with FS and 141 healthy controls. The FS group’s serum IL-6 level was significantly higher than that of the control group. The standardized mean difference was 2.654, the 95% CI was from 2.332 to 2.975, and the P-value was <0.01. The included studies were statistically heterogeneous (I2 = 78.248%); thus, the random effects model was used for the meta-analysis (Fig. 6).
Thirty-nine patients with FS and 25 healthy controls were extracted from 2 studies. Regarding CSF IL-1β, the FS group’s levels were significantly higher than those of the control group. The standardized mean difference was 1.064, 95% CI was from 0.217 to 1.611, and the P-value was <0.01. The included studies were statistically heterogeneous (I2 = 73.1%); thus, the random effects model was used for the meta-analysis (Fig. 7).
In the present study, we reviewed and comprehensively summarized case–control studies and found potentially important information. First, the IL-1β level differed according to whether it originated from the serum or the CSF. The serum IL-1β level of the patient group did not significantly differ from that of the control group. However, the CSF IL- 1β level of the patient group was significantly higher than that of the control group. Second, the serum IL-6 level was significantly higher in the patient group.
Although numerous studies have investigated FS, the exact pathophysiology remains unclear. Since FSs occur during a high body temperature or a rapidly rising fever, several factors associated with fever generation could be involved in the mechanism of FS. Cytokines are among the factors that may be involved in the pathogenesis of FS [
]. IL-1β has various activities, including fibroblast proliferation, cartilage breakdown, and initiation of the host response to infection. IL-1β is a pluripotent pro-inflammatory cytokine that binds to IL-1R1, a Toll receptor family member; via an NFkB-dependent mechanism, it induces the transcription of various genes that encode several downstream mediators of inflammation, including IL-6 and TNF-α. The time scale of these effects is between 30 and 90 min [
In a previous study, Tütüncüoğlu S. et al. reported increased levels of plasma IL-1β in FS patients. The report suggested that in comparison with the control group, the patients’ IL-1β level during the acute phase were significantly increased, but the IL-1β levels during the delayed phase were not significantly different. The direct relationship between plasma IL-1β concentration and fever level indicated that IL-1β has a more active role than other cytokines in fever development [
Conflicting results regarding the IL-1β levels in FS patients have been reported for plasma and CSF. Tütüncüoğlu S. et al. reported that IL-1β levels were significantly high in plasma but not in CSF [
]. Lahat et al. reported that both plasma and CSF IL-1β levels were not significantly higher compared with the control group. These results imply difficulties with obtaining clinical samples and measuring free IL-1β [
In this study, the IL-1β level was significantly higher among patients than controls when measured in the CSF but not when measured in the serum. The suspected reason for this inconsistent finding compared with previous studies is that the sampling time of the included studies differed. As mentioned above, IL-1β increases significantly during the acute phase of fever. Therefore, if samples are obtained within 30 to 90 min, IL-1β levels might appear significantly high. However, under different circumstances, we can assume that blood samples and CSF samples were obtained during different phases of fever. Consequently, the IL-1β levels varied among the studies, and this meta-analysis only found significantly high levels in the CSF. To perform a more sophisticated study, sampling times should be documented, and the sample should be divided into acute and delayed phases before the IL-1β level is assessed.
IL-6 is a pro-inflammatory cytokine that is pleiotropic and is secreted by T-lymphocytes, macrophages, endothelial cells and epithelial cells. It has a wide range of biological activities in immune regulation, hematopoiesis, inflammation, and neoplasia. IL-6 also has a strong correlation with fever [
Several studies reported that plasma IL-6 levels were significantly higher in FS patient groups compared with control groups. Azab S. et al. showed significant positive associations between FSs and the IL-6 level and reported the related genotype (G allele at the −174 position and the −174 GG or −597 GG). Their findings revealed that the patient group was more susceptible to FS. In addition, they reported a negative association between FSs and the C allele at the −174 position, indicating that this association could represent a protective effect against FSs [
Choi J. et al. (2011) reported that serum IL-6 was higher in children with FS than in healthy controls who had only fever. In addition, the serum IL-6 levels in FS patients were much higher than those in patients who had undergone an afebrile seizure attack. These findings suggest that IL-6 has a pro-convulsant action in FSs [
In this meta study, as expected, we found that serum IL-6 levels in patients with FS were significantly elevated compared with those of the control group. This result was consistent with the findings of previous studies. Based on these results, it is expected that a therapeutic agent for specific cytokines could be developed in the future to prevent FS. However, in this meta-analysis, we did not include genotyping results; therefore, in future studies, cytokines and genotypes should be studied after sampling, and their correlation, whether positive or negative, should be evaluated.
This study had some limitations. First, among these was its small sample size. We suggest that multicenter approaches may be necessary to attain larger sample sizes. Second, only 3 different cytokines were included in this meta-analysis. For better understanding of pathogenesis of FS, we need to evaluate more cytokines. Additional high-quality studies are required for further verification. In addition, the genotypes of cytokines were not evaluated. There was also publication bias indicated by funnel plot. This may affect the interpretation of our results.
5. Conclusion
In conclusion, this meta-analysis systematically revealed that the FS patients had significantly higher CSF IL-1β and serum IL-6 levels compared with the control group. Further studies, specifically well-designed, large-scale, case-controlled trials, are needed to evaluate the precise concentrations of certain cytokines in FS patients and to determine the cytokines’ various activities during FS. Based on these results, it is expected that a therapeutic agent for specific cytokines could be developed in the future to prevent FS.
Conflicts of interest
Conflict of interest relevant to this article was not reported.
Funding
This research did not receive grant support from funding agencies in the public, commercial, or not-for-profit sectors.
Author contributions
AK, BOK, KK, JH, JSS, SJK, SWB, SNK, and RL contributed to the study design, data collection and analysis, manuscript preparation, and manuscript approval.
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