Seizure 41 (2016) 112–115

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Seizure
journal homepage: www.elsevier.com/locate/yseiz

Comparison of the serum cytokine levels before and after

adrenocorticotropic hormone (ACTH) therapy in patients with
infantile spasm
Esra Türe a, Tülay Kamaşak b, Merve Cora c, Sevim Şahin b, Elif Acar Arslan b,
Neşe Kaklıkaya c, Ali Cansu b,*
a
Karadeniz Teknik University, Department of Pediatrics, Turkey
b
Karadeniz Teknik University, Department of Pediatric Neurology, Turkey
c
Karadeniz Teknik University, Department of Microbiology, Turkey

A R T I C L E I N F O A B S T R A C T

Article history: Purpose: Infantile spasm is an age-dependent epileptic syndrome seen in infancy or early childhood.
Received 28 January 2016 Although studies have investigated the epilepsy–cytokine relationship, there has been insufficient
Received in revised form 31 May 2016 research into the relation between cytokines and infantile spasm. The purpose of this study was to
Accepted 25 June 2016
examine the role of cytokines in the pathogenesis of infantile spasm by investigating cytokine levels
before and 1 month after adrenocorticotropic hormone (ACTH) therapy in patients diagnosed with the
Keywords: condition.
Infantile spasm
Method: Twenty patients aged between 1 month and 2 years and diagnosed with infantile spasm at the
Cytokines
IL-1beta
Karadeniz Technical University Medical Faculty Department of Child Health and Diseases Pediatric
IL-6 Neurology Clinic, Turkey, and 20 healthy children were included in the study. Patients received 11 doses
IL-17A of ACTH on 2 days a week. Levels of TNF-alpha and IL-2, the main cytokines involved in inflammation and
IL-2 recently associated with infantile spasm, and of IL-1beta, IL-6 and IL-17A, associated with epileptic
seizures, and serum levels of the IL-17A activator IL-23 were investigated in all patients at the start of
treatment and 1 month after completion of treatment.
Results: No statistically significant difference was observed between pre- and post-treatment patient
group and control group IL-1beta, IL-2, IL-23 or TNF-alpha levels. Pre-treatment IL-6 and IL-17A levels
were significantly higher in the untreated patient group compared to the healthy control group
(p < 0.001 and p = 0.002).
Conclusion: Our study supports the recent idea that IL-6 and IL-17A are cytokines involved in the
pathogenesis of infantile spasm.
ß 2016 Published by Elsevier Ltd on behalf of British Epilepsy Association.

[2_TD$IF]1. Introduction These include brain stem dysfunction due to pathology in

Infantile spasm, a childhood epilepsy with rather poor
prognosis, is characterized by myoclonic seizures, a hypsarrythmic
EEG pattern and developmental retardation. It was first described
by William West in his own son in 1841. Various hypotheses have
been proposed concerning the pathophysiology of infantile spasm.
* Corresponding author at: Karadeniz Teknik University, Pediatric Neurology,
Trabzon, Turkey. Tel.: +90 5055668944; fax: +90 4623250518.
E-mail addresses: esratopal05@hotmail.com (E. Türe), tkamasak@hotmail.com
(T. Kamaşak), merve.tokdemir@yahoo.com (M. Cora), sevimsahin1@yahoo.com
(S. Şahin), elifacararslan@yahoo.com.tr (E.A. Arslan), nkkaya@yahoo.com
(N. Kaklıkaya), acansu2011@hotmail.com (A. Cansu).
serotonergic neurons, interaction between the brain stem and a
focal or diffuse cortical abnormality, interaction between cortical–
subcortical abnormalities and immunological dysfunction [1–4].
Changes in the hypothalamus-pituitary-adrenal axis have also
been investigated, and it has been suggested that corticotropin
releasing hormone (CRH) increases neuronal stimulability and
seizures [5]. The hypothesis of a defect in the immune system in
association with infantile spasm is based on studies showing the
presence of antibodies forming against normal brain tissue in the
sera of patients with infantile spasm, increased numbers of B and T
cells in peripheral blood, and the presence of abnormal leukocyte
antigen in patients compared to control cases [6,7]. However, there
has been insufficient research into the relation between cytokines

http://dx.doi.org/10.1016/j.seizure.2016.06.021
1059-1311/ß 2016 Published by Elsevier Ltd on behalf of British Epilepsy Association.
 E. Türe et al. / Seizure 41 (2016) 112–115 113

and infantile spasm. The purpose of this study was to examine the to compare independent variables. The ‘Wilcoxon signed ranks test’
relation between infantile spasm-type seizures and cytokines and was used to compare non-normally distributed dependent constant
to determine whether, as predicted, cytokines are involved in the variables and the ‘Mann Whitney U test’ to compare independent
pathogenesis. constant variables. The ‘chi square test’ (x2) was used to test the
presence of relations between categoric variables. Relations between
2. Materials and methods constant variables were also examined using the ‘Spearman rank
correlation test.’ p values of less than 0.05 were considered significant
Twenty patients aged between 1 month and 2 years presenting for all test results.
to the Karadeniz Technical University Medical Faculty Department
of Child Health and Diseases Pediatric Neurology Clinic and
diagnosed with infantile spasm and 20 age- and sex-matched 3. Results
healthy children were included in the study. Decimal age, age at
onset of spasm, age at diagnosis, sex, characteristics of infantile The etiology of infantile spasm was determined to be
spasm, pre-, natal and postnatal histories, family history, type of symptomatic in 7 (35%) patients and cryptogenic in 13 (65%).
delivery, systemic and neurological examination findings, labora- Prematurity was present in 1 (14%) of the patients in the
tory findings, cerebral imaging (magnetic resonance imaging – MRI) symptomatic group, Hypoxic ischemic encephalopathy in 5
and electroencephalography (EEG) of all patients were investigated. (71%) and congenital CMV infection in 1 (14%). EEG was performed
Electroencephalography was performed using a 64-channel Nihon at time of diagnosis in all patients. A classic hypsarrhythmia
Kohden EEG device with an international electrode system. EEG was pattern was determined in 12 (60%) and a modified hypsar-
evaluated at the Department of Pediatric Neurology. Infantile spasm rhythmia pattern in 8 (40%). Cerebral imaging was performed in all
was diagnosed on the basis of flexor and extensor spasms recurring cases. No pathological finding was determined in 13 (65%) cases,
several times a day and lasting several seconds, hypsarrhythmia at while pathological MRI findings (multicystic encephalomalacia,
EEG, and interrupted psychomotor development and/or retardation cerebral atrophy, periventricular leukomalacia and hypoxic injury)
[8]. Hypsarrhythmia at EEG was defined as diffuse slow waves and were identified in 7 (35%).
suppression-burst findings in the form of spike-multiple spike No significant differences were determined at comparison of
activity or superimposed ‘spike, multiple spike slow wave and pre-and post-ACTH therapy serum IL-23, TNF-alpha, IL-2 and IL-
subsequent suppression periods’ [1]. The etiology of infantile spasm 1beta levels and those of the healthy control group (Table 1).
was classified under two main groups, symptomatic and crypto- Serum IL-6 levels decreased following ACTH therapy, although
genic, depending on the identification of a definable cause. Patients this was not statistically significant. However, comparison of
receiving corticosteroid derivative therapy in the previous patients’ pre-treatment IL-6 levels and those of the healthy control
3 months, with accompanying adrenal or pituitary insufficiency, group revealed significant elevation in the patient group
benign myoclonus of early infancy, tonic reflex seizures of early (p < 0.001) (Table 1). Additionally, a significant correlation was
infancy, benign neonatal sleep myoclonus or benign and severe determined between a decrease in IL-6 levels and type of infantile
myoclonic epilepsy were excluded from the study. Patients received spasm. The decrease in IL-6 levels in the cryptogenic group was
a total of 11 doses of ACTH (tetracosactide = Synacthen-Novartis significantly greater than that in the symptomatic group
depot vial) therapy on 2 days a week. Patients younger than (p = 0.044).
12 months received 0.5 mg (25 units) intramuscularly and those
Table 1
older than 12 months received 1 mg (50 units) intramuscularly. The Comparison of the pre-treatment and post-treatment groups and the pre-treatment
ACTH dose was interrupted if side-effects of ACTH therapy, and control groups.
hyperactivity, hypertension, allergic reactions, infection or hypo-
Pre-treatment Post-treatment p
glycemia, developed. Patients were divided into five groups based
IL-1b Patient group 16.82  22.08 13.78  10.66 0.985a
on their clinical response to treatment: complete response,
Control group 22.02  24.48
temporary complete response, <50% decrease in spasms, >50% in p 0.705b
spasms and no response.
IL-2 Patient group 24.86  15.55 24.49  14.32 0.946c
Venous blood specimens were taken from all patients at the
Control group 17.63  10.80
beginning and 1 month after completion of treatment in order to p 0.245b
measure IL-1beta, IL-2, IL-6, IL-17a, IL-23, TNF-alpha levels.
IL-6 Patient group 94.42  60.37 62.36  58.39 0.086a
Specimens were centrifuged at 3000 rpm for 15 min for serum
Control group 33.97  34.32
separation and were then stored at 800[1_TD$IF] C until analysis. Sera set p 0.001b
aside for IL-1beta, IL-2, IL-6, IL-17a, IL-23 and TNF-alpha
IL-17A Patient group 1.13  0.74 0.83  0.64 0.215a
measurements were thawed at room temperature and studied
Control group 0.61  0.54
using ELISA on the same day. The results were compared with p 0.002b
those of a healthy age- and sex-matched control group. The control
IL-23 Patient group 36.11  40.91 40.16  27.05 0.433a
group was established following permission for additional blood
Control group 42.63  26.15
collection from the families of healthy children of appropriate age p 0.140b
attending our hospital’s General Pediatric Department for check-
TNFa Patient group 14.68  8.06 12.62  6.73 0.313a
up purposes and scheduled for blood tests. Approval for this was Control group 15.62  11.50
granted by the Karadeniz Technical University Faculty of Medicine p 0.957b
ethical committee. a
Pretreatment group compared with postreatment group – Wilcoxon test.
The study data were analyzed on SPSS (Statistical Package for b
Patients compared with control group – Mann Whitney U test.
the Social Sciences, version 20, SSPS Inc., Chicago, Ill, USA) c
Pretreatment group compared with postreatment group for IL-2 – Paired t test.
software. Constant variables were expressed as ‘mean  standard Presence of differences in parameters between the patient and control groups was
deviation.’ The Shapiro–Wilk test was used to determine compatibil- tested using the Man Whitney U test (b) since the data were not normally
distributed. Since IL -2 was normally distributed the presence of a difference before
ity with normal distribution of constant variables in the data set. The and after treatment in the patient group was investigated using the Paired t test (c),
‘t test for dependent groups’ was used to compare normally while other parameters were compared using the Wilcoxon test (a) since these were
distributed constant variables and the ‘t test for independent groups’ not normally distributed.
114 E. Türe et al. / Seizure 41 (2016) 112–115
No significant difference was determined between pre- and
post ACTH therapy IL-17A levels. However, when patients’ pre-
ACTH therapy IL-17A levels were compared to those of the healthy
control group, these were significantly higher in the non-treated
group (p = 0.002) (Table 1).
In addition, no statistically significant correlations were
determined between age, sex, pre-, natal and post-natal char-
acteristics, type of infantile spasm, type of seizure, EEG character-
istics and cerebral pathology findings and IL-1beta, IL-2, IL-6, IL-
17a, IL-23, or TNF-alpha levels.
ACTH therapy was administered to all patients in 11 doses,
twice weekly. ACTH therapy-related side-effects developed in only
one patient. ACTH therapy was interrupted due to severe edema in
the patient’s face and extremities, inability to stand and inability to
make eye contact. Complete resolution of spasms was observed in
13 (65%) patients following ACTH therapy, relapse after complete
resolution in 2 (10%) and no response to medication in 5 (25%).
4. Discussion
The effectiveness of steroid therapy in infantile spasm and
seizures being triggered following viral infections suggests that
immunological and inflammatory mechanisms are involved in the
pathogenesis [9]. Our aim in this study was to investigate the levels
of IL-1beta, IL-2, IL-6, IL-17a, IL-23, and TNF-alpha, which are
known or thought to be associated with epilepsy, in patients with
infantile spasm. When we compared pre- and postnatal cytokine
levels in patients with infantile spasm with those of the healthy
control group, we determined significant elevation in IL-6 and IL-
17A levels in the pre-treatment group compared to the healthy
control group.
Inflammatory processes in the brain are known to play a role in
the pathophysiology of seizures and epilepsy [10]. IL-1beta, TNF-
alpha and IL-6 produced from monocytes and lymphocytes are also
secreted in small amounts in brain tissue, and their levels rise with
the occurrence of seizures [11]. In addition, IL-2 has been shown to
induce seizures and to lead to recurrent seizure activity in animal
models [12]. It also contributes to the inflammatory process by
increasing the release of IL-17A, IL-6 and IL-1beta, which have
recently begun attracting attention in the context of the
pathogenesis of epilepsy [13]. The release of IL-17A and IL-6 is
known to increase with IL-23 stimulation [14]. IL-2 levels of the
patients with infantile spasm in our study were higher than those
of the control group. However, no statistically significant differ-
ence was observed between IL-2 levels. Sousa et al. showed an
increase in IL-1b and IFN-g levels by investigating IL-1b, IL-5, IL-
10, TNF-b and IFN-g levels in CSF specimens from four patients
with infantile spasm, three symptomatic and one from the
cryptogenic group, aged 4–12 months [15]. Liu et al. reported
significantly higher IL-2 and TNF-alpha levels in patients with
West syndrome compared to a healthy control group. In another
study involving 23 patients with infantile spasm they reported
significant elevation in serum IL-2, TNF-alpha and IFN-a levels
compared to a healthy control group [16].
When patients’ pre-treatment IL-6 levels were compared with
the healthy control group in our study, these were significantly
higher in the pre-treatment patient group (p < 0.001). In addition,
a significant correlation was determined between a decrease in IL-
6 levels and type of infantile spasm. The decrease in IL-6 levels in
the cryptogenic group was significantly greater than that in the
symptomatic group (p = 0.044). IL-6 is one of the main proteins
regulating the organism’s immunological response [17]. Although
it is principally manufactured by Th2 cells, it is also secreted by
monocytes and macrophages, together with IL-1 and TNF-a [18]. It
is also released by astrocytes and microglia as a localized
inflammatory response in the brain and causes an inflammatory
effect in the CNS. High IL-6 levels are generally determined in
neurotropic viral infections in pathologies of the CNS [18]. Studies
have reported significant elevation in IL-6 levels in serum and CSF
in tonic clonic seizures, and this is thought to vary depending on
the seizure area of diffusion and duration [19]. In a study of 12
patients with infantile spasm, Tekgül et al. compared patients’ CSF
IL-6 levels with those of a control group with post-traumatic
seizure or intracranial infection, but determined no significant
findings in terms of these levels [20]. Shiihara et al. compared
peripheral lymphocyte and serum cytokine levels before and after
ACTH therapy in patients with infantile spasm. They determined
low pre-ACTH CD3, CD4 and CD4/CD8 levels and high pre-
treatment IL-1 beta, IL-5, IL-6, IL-12, IL-15, IFN-gamma, IL-1Ra,
exotoxin and macrophage inflammatory protein levels, decreasing
after treatment [21].
Comparison of pre-treatment and control group IL-17A levels in
our study revealed significantly higher levels in the untreated
patient group (p = 0.002). In addition to the previously known Th1
and Th2 cells, a Th17 cell subgroup largely producing IL-17 has
recently been described. Th17 cells are involved in defense against
extracellular pathogens and in inflammatory diseases. Like IL-17,
Th17 cells regulate inflammation by manufacturing cytokines and
diversify as a result of the effect of various antigens and cytokines
in T cells. IL-17 acts as a bridge between the natural and acquired
immune systems. Seizure production or recurring seizures are
thought to occur as a response to the process [22]. In a study of 16
patients with tuberous sclerosis, Jiao-Jiang at al. reported increased
IL-17 and IL-17R in the cortical lesions of patients with focal
cortical dysplasia. In addition to refractory seizures, infantile
spasm-type seizure was identified in 4 patients [23]. Mao et al.
reported significantly higher IL-17A levels in patients with
epilepsy compared to a healthy control group [24]. There is
insufficient information regarding the relation between infantile
spasm and IL-17A in the literature. In addition to contributing to
the literature, our study also suggests that further more detailed
studies concerning IL-17A are now needed to illuminate the
pathogenesis of infantile spasm.
Infantile spasm may cause severe neurological sequelae by
affecting patients’ mental, motor, psychological and emotional
development, in addition to refractory seizures, and may have fatal
consequences. We think that clinical and laboratory studies will
elucidate the mechanism by which infantile spasm develops and
assist with the development of etiology-specific treatment models,
and that only in this way can the morbidity and mortality of
infantile spasm be reduced to a minimum. Our study supports the
idea that immune and inflammatory processes are involved in the
pathophysiology of infantile spasm.
Limitations of this study include the low patient number and
the fact that several factors affect interleukin levels. Based on
clinical observation of an increased risk of seizure in periods of
infection, we think that cytokines occupy an important place in the
pathophysiology of epilepsy and hope that our study will assist
future wider studies with larger patient numbers.
Conflict of interest statement
None.
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