mesial temporal lobe epilepsy with hippocampal sclerosis ( mtle - hs ) is the most common type of partial or localization - related seizure disorder in humans .
mesial temporal sclerosis ( mts ) with prominent neuronal loss and gliosis in the hippocampus is the most common pathology in temporal lobe epilepsy ( tle ) .
although the pathogenesis and epileptogenesis of hippocampal sclerosis ( hs ) has been studied for years , the information available so far still does not fully explain the situation .
patients with mtle - hs usually have a history of febrile seizures , status epilepticus , trauma or , in some cases , a mixture of all these symptoms .
recently , some studies have implicated inflammatory mechanisms that contribute or predispose to the occurrence of seizures in mtle - hs .
cytokines such as interleukins ( il ) are involved in inflammation , immune activation , cell differentiation and apoptosis . in seizures ,
there is increasing evidence to support the role of il-1 in reducing seizure threshold and epileptogenesis in the pilocarpine status epilepticus model of epilepsy .
found a strong association between the homozygotes for allele t at position -511 of the il-1 gene promoter region ( il-1-511 t / t ) and genetic predisposition to the development of hs in japanese tle patients .
stated that increased il-1-511 t - allele frequency proved to be a potent determinant of prolonged febrile seizure , thus a discrepancy in prolonged febrile seizure incidence could explain recent conflicting results .
three polymorphisms within the il-1 gene promoter region located at positions -1470 , -511 and -31 basepairs from the transcription start site were found to be in strong linkage disequilibrium . additionally , when analyzed in combination ,
interestingly , however , these two publications have conflicting results in terms of transcriptional activity of il-1 promoter .
while chen et al . concluded that the il-1-511 t allele was associated with higher il-1 promoter activity , wen et al . demonstrated that haplotype combinations carrying -511 c allele cause higher levels of secretion of il-1 from the lipopolysaccharide ( lps)-stimulated lymphocytes . in both of these studies ,
therefore , we intended to analyze the effect of -511 c / t polymorphism on il - i secretion from lps - induced lymphocytes in an attempt to reveal the possible role of this particular polymorhism in the etiopathogenesis of mtle - hs .
thirty patients with mtle - hs admitted to the akdeniz university hospital neurology department , turkey , were enrolled in this study .
a control group of 32 healthy volunteers with matching age , gender ratio and ethnic origins was also included .
diagnosis of mtlehs was based on clinical history , electroencephalography , magnetic resonance imaging , and , in some patients , on histopathological findings after the selective amgydalahippocampectomy and anterior temporal lobectomy .
the control subjects had no history of seizures or neurological disorders and no central nervous system or other infections at the time of blood sample collection .
peripheral venous blood samples ( 3 ml ) were collected from each subject into vacutainer tubes containing edta .
dna was extracted from blood samples using the robotic system magnapure ( roche ) and the amount of dna was measured by spectrophotometer .
the -511 region of il-1 gene has been genotyped with polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) using 2 mg of genomic dna as template . by using the primers for -511 c / t polymorphism described below
, a 304 bp of dna region was amplified by pcr ( total 35 cycles , each cycle 30 s at 95c , 45 s at 50c , and 45 s at 72c ) .
each pcr product was then cleaved by ava i enzyme and the digested pcr products were subjected to agarose gel electrophoresis .
the gels were stained with ethidium bromide and the dna fragments were visualized by an uv transluminator .
pcr products acquired from those with a cc genotype were cleaved into two fragments of 190 and 114 bp by ava i enzyme , whereas the pcr products of people with tt genotype was not digested at all , yielding a product of 304 bp .
oligonucleotides used for pcr : forward primer : 5-tggcattgatctggttcatc reverse primer : 5-gtttaggaatcttcccactt peripheral blood of patients and healthy individuals was drawn into heparinized tubes , and lymphocytes were isolated with a density gradient method and pipetted into plates ( 210 cells each well ) and incubated for 24 h ( 5% co2 , 37c ) in an rpmi 1640 culture medium including 10% fcs with ( 0.5 ng / ml ) or without lps . the cells were precipitated by centrifuge at 1600 rpm for 10 min after 4 and 24 h. the supernatants were transferred to another tube and stored at 80c until elisa analyses .
il1 levels in cell culture supernatants were studied according to the manufacturer s protocol ( ebioscience , cat . n. 88 - 7010 ) .
all statistical analysis was carried out using statistical package for social sciences ( spss ) windows version 15.0 ( spss inc . ,
tests for differences in allelic and genotypic frequencies were performed using 2 test and fisher s exact test .
thirty patients with mtle - hs admitted to the akdeniz university hospital neurology department , turkey , were enrolled in this study .
a control group of 32 healthy volunteers with matching age , gender ratio and ethnic origins was also included .
diagnosis of mtlehs was based on clinical history , electroencephalography , magnetic resonance imaging , and , in some patients , on histopathological findings after the selective amgydalahippocampectomy and anterior temporal lobectomy .
the control subjects had no history of seizures or neurological disorders and no central nervous system or other infections at the time of blood sample collection .
peripheral venous blood samples ( 3 ml ) were collected from each subject into vacutainer tubes containing edta .
dna was extracted from blood samples using the robotic system magnapure ( roche ) and the amount of dna was measured by spectrophotometer .
the -511 region of il-1 gene has been genotyped with polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) using 2 mg of genomic dna as template . by using the primers for -511 c / t polymorphism described below ,
a 304 bp of dna region was amplified by pcr ( total 35 cycles , each cycle 30 s at 95c , 45 s at 50c , and 45 s at 72c ) .
each pcr product was then cleaved by ava i enzyme and the digested pcr products were subjected to agarose gel electrophoresis .
the gels were stained with ethidium bromide and the dna fragments were visualized by an uv transluminator .
pcr products acquired from those with a cc genotype were cleaved into two fragments of 190 and 114 bp by ava i enzyme , whereas the pcr products of people with tt genotype was not digested at all , yielding a product of 304 bp .
peripheral blood of patients and healthy individuals was drawn into heparinized tubes , and lymphocytes were isolated with a density gradient method and pipetted into plates ( 210 cells each well ) and incubated for 24 h ( 5% co2 , 37c ) in an rpmi 1640 culture medium including 10% fcs with ( 0.5 ng / ml ) or without lps . the cells were precipitated by centrifuge at 1600 rpm for 10 min after 4 and 24 h. the supernatants were transferred to another tube and stored at 80c until elisa analyses .
il1 levels in cell culture supernatants were studied according to the manufacturer s protocol ( ebioscience , cat . n. 88 - 7010 ) .
all statistical analysis was carried out using statistical package for social sciences ( spss ) windows version 15.0 ( spss inc . ,
tests for differences in allelic and genotypic frequencies were performed using 2 test and fisher s exact test .
there was no significant difference between the mtle - hs patients and controls according to genotype and allele frequencies of il-1-511 .
levels of secreted il-1 from the cultured lymphocytes incubated either with or without lps were measured at three different time frames during the culture period ( basal , 4 h and 24 h ) .
however , no statistically significant difference was observed between the groups ( table 3 ) .
since il-1-511 t allele has been suggested to be associated with increased in vitro production of il-1 and that il-1 is involved in hs pathogenesis , we also analyzed the il-1 secretion between the patients and control individuals who carry the t allele at position -511 of the il-1 gene .
however , although spontaneous secretion levels of il-1 were higher in mtle - hs patients , there was no significant difference between the groups ( figure 1 ) .
in addition , despite the fact that both spontaneous and stimulated secretion levels of il-1 were higher in mtle - hs patients who carry the t allele with respect to those patients who do not , the difference was not statistically significant ( figure 2 ) .
in this study , we found that neither the il-1 -511 c / t polymorphism nor the secreted il-1 from the cultured lymphocytes were associated with mtle - hs in a group of turkish patients . in our study , the average age of onset of seizures and gender were similar to those presented in other studies . on the other hand ,
the incidence of history of febrile seizures was found to be 76.7% , which was higher than the values mentioned in several previous reports .
cytokine proteins have both pro - inflammatory and anti - inflammatory forms . among proinflammatory cytokines ,
three biallelic polymorphisms in il-1 , all of which result from c to t transitions at positions -511 , -31 or + 3954 from the transcriptional start site , have been most frequently evaluated for their association with diverse conditions besides epilepsy .
chen et al . demonstrated that the il-1-511 t allele strongly enhanced the transcription of the il-1 gene in the context of the il-1-31 c allele .
il-1-511 t single nucleotide polymorphism has been thought to be associated with mtlehs and febrile seizures .
previously showed no association between il-1 gene polymorphism and tle with hs in turkish ancestry in a larger group .
recently , a meta - analysis and a review was published by kauffman et al . demonstrating a modest association between the il-1-511 t polymorphism and tle with hs . in the current study
, we did not find any significant difference between mtlehs patients and control group with respect to the genotype and allele frequencies of il-1-511 ( table 2 ) . in this study ,
the frequency of t / t genotype was found to be 15.6% whereas , it was reported to be 18% and 20.5% in european and japanese populations , respectively . during the acute phases of status epilepticus , upregulation of il-1 occurred in activated microglia and astrocytes and sustained inflammation was only evident in astrocytes during epileptogenesis . in chronic epileptic tissue ,
il-1 was still expressed by astrocytes , and it resumed in microglia when rats displayed a high frequency of spontaneous seizures .
these findings demonstrated that il-1 is chronically over - expressed in astrocytes after status epilepticus , and il-1 expression in microglia is related to ongoing and severe epileptic activity .
together with il-1 , tnf- and il-6 levels increase rapidly after the induction of seizures , then decline to basal levels within 48 - 72 h of the onset of seizures . but il-1 is still up - regulated in the brain 60 days after status epilepticus in rats with spontaneous seizures .
the recent data suggested that lipopolysaccharide mimics the actions of an endogenously released protein called high mobility group box 1 that interacts with tolllike receptor 4 to promote seizures .
il-1 and high mobility group box 1 activate pathophysiological cascade of inflammatory events in epilepsy through binding to il-1r1 and tolllike receptor 4 , respectively .
besides these experimental studies , some clinical studies have measured il-1 secretion from the lymphocytes and serum in febrile seizure and epilepsy patients .
showed increased plasma levels of il-6 and levels of il-1 unchanged after seizures in chronic localization - related epilepsy patients .
found no difference between febrile seizure patients and controls with respect to serum il-1 levels .
reported increased serum levels of il-6 in tle that supported chronic overproduction of cytokines in refractory focal epilepsy . on
the other hand , measurement of the secreted il-1 levels from stimulated lymphocytes can differ from serum levels .
demonstrated that il-1 secretion from the lymphocytes was significantly higher in febrile seizure patients . in another study ( haspolat , unpublished data , 2008 ) , significantly increased levels of il-1 from lps - stimulated mononuclear cells at 24 h were observed in complicated febrile seizure patients compared to simple ones .
although these findings suggest that the increased levels of il-1 in mtle - hs patients may lead to hs and explain epileptogenesis and ictogenesis , in this study we were not able to detect any statistically significant differences in secreted il-1 levels from cultured lymphocytes between the patient and control groups .
in addition , comparison of il-1 levels secreted from lymphocytes obtained from individuals carrying a t allele at the -511 position of the il-1 gene to those obtained from individuals who do not carry the t allele did not reveal any significant difference either .
interestingly , however , the spontaneous and stimulated levels of il-1 secretion were higher in mtle - hs patients carrying the t allele with respect to the patients who do not .
the relatively small number of patients and controls could explain the reason why a statistically significant difference was not observed in this study .
in addition , it should be kept in mind that promoter regulation is a complex mechanism involving a large variety of transcription factors acting together .
thus , besides ( -511 ) polymorphism , a combination of different haplotypes and many other transcription factors binding to different sites should impinge on the transcriptional activity . in conclusion , in experimental studies , activation of cytokines has been observed especially in areas of onset of seizures and spreading of discharge . in clinical studies ,
cytokine levels in peripheral blood seem to reflect the central nervous system s production of cytokines , but these are not always correlated .
 mesial temporal lobe epilepsy with hippocampal sclerosis ( mtle - hs ) is a common medically intractable epilepsy syndrome . 
 although pathogenesis of hs still remains highly controversial , genetics may play a role as a predisposing factor . 
 previous evidence in a japanese population revealed that the homozygotes for allele t at position -511 of the interleukin ( il)-1 gene promoter region ( il-1-511 t / t ) confers susceptibility to the development of hs . 
 however , whether this polymorphism has an effect on il-1 levels in mtlehs patients was not demonstrated . 
 this study aimed to analyze the distribution of this particular polymorphism in a group of turkish hs patients and correlate the polymorphism with il-1 secretion from the lymphocytes , thus revealing a functional role for il-1 in the etiopathogenesis of hs . 
 a single base pair polymorphism at position 511 in the promoter region of the il-1 gene was analyzed . 
 the spontaneous and 1 ng / ml lipopolysaccharidestimulated production of il-1 by peripheral blood mononuclear cells after 4 and 24 h of incubation were measured by elisa method . the heterozygous type ( 511 c / t ) was the most common genotype . 
 there was no difference in frequency of allele 511 t between patients and controls . 
 analysis of il-1 levels , genotype and allele distributions showed no significant difference among the groups ( p>0.05 ) . 
 nevertheless , it was seen that patients who carry a t allele at position -511 of the il-1 gene had increased il-1 levels . 
 t - allele carriage may be important . 
 only il-1 secretion from the lymphocytes has been assessed in this study . 
 considering the importance of il-1 in the etiopathogenesis of hs , further studies are needed to evaluate locally produced il-1 levels . 
Introduction
Materials and Methods
Subjects
Genotyping using polymerase chain reaction-restriction fragment length polymorphism
Measurement of IL1 levels using ELISA
Statistical analysis
Results
Discussion and Conclusions