il-18 is a pleiotropic proinflammatory cytokine 
 that stimulates production of ifn- , tnf- , il-1 , il-2 , adhesion molecules and apoptosis 
 factors , increasing t - lymphocyte proliferation , and enhancing the lytic 
 activity of nk - cells .
it participates in the cellular and humoral immune 
 response , both innate and adaptive .
it has been shown that il-18 is 
 involved in the pathogenesis of various diseases including type - i diabetes , 
 rheumatoid arthritis , crohn 's disease , and liver cirrhosis .
allelic 
 variants of cytokine genes associated with promoter gene region polymorphisms 
 do not influence the protein amino acid sequence but can result in changes in 
 cytokine production . in consequence , they may alter the immune responses mediated 
 by the cytokine in question and could be associated with various immunological 
 diseases .
three single nucleotide polymorphisms ( snps ) are found in the il-18 
 gene promoter in positions 656 gt , 607 ca , and 137 gc .
two of these , 607 ca and 137 gc , are located at the binding sites for creb 
 transcriptional factors ( camp response - element binding proteins ) and the h4tf-1 nuclear factor , 
 respectively , therefore mutation at these two sites could influence il-18 
 expression and change the production of the cytokine .
recent studies have 
 investigated the relationship between il-18 gene polymorphisms and 
 predispositions to various diseases .
it has been shown that individuals 
 carrying the 607c 137c haplotype 
 are prevalent in rheumatoid arthritis cases among the populations of germany and scotland .
women carrying the 
 c - allele at 137 have a 
 heightened risk of developing ovarian carcinoma .
the presence of such a 
 genotype in patients with nasopharyngeal carcinoma of an undifferentiated type 
 worsens the prognosis for the disease and is associated with earlier metastasis 
 .
the g - allele at 137 is associated 
 with the development of bronchial asthma and dermal allergic reactions . in 
 patients with hepatitis b from the chinese population ,
the 137gg and 607ca allele 
 variants of the il-18 gene promoter are consistently more frequent ; conversely , 
 hepatitis b is encountered consistently more rarely in persons with the 
 c - allele at 137 . 

nevertheless , the impact of one or other il-18 allele variant on the level of 
 protein production has not been adequately studied .
the aim of our study was therefore 
 as follows : to investigate the distribution of il-18 allele variants at 
 positions 607ca and 137gc within a population of healthy donors from 
 southwestern siberia , and the influence of these allele variants on the level 
 of il-18 production by their peripheral blood mononuclear cells ( mncs ) .
dna was obtained from pbmc from adult ( 2050 years of age ) 
 healthy donors ( n = 146 ) to study polymorphisms in the promoter region of the 
 il-18 gene and the production il-18 protein .
all subjects gave written informed 
 consent for enrolment in the study , which was approved by the local ethics 
 committee .
genomic dna was extracted from pbmc by a set 
 test - na ( dna - technology , russia ) .
allele - specific 
 amplification was assessed by fluorescent detection of the products of real - time 
 pcr .
four primers were used for amplification , two specific for each polymorphism 
 at positions 607 and 137 . 

products were detected using the intercalating dye sybr - green i. 
 pcr was performed using an iq4 cycler ( bio - rad , usa ) .
the reaction 
 mixture ( 15 l final volume ) contained 0.3 m specific and control primers ; pcr 
 buffer ( 16 mm tris - hcl , ph 8.9 , 2.4 mm mgcl2 , 65 mm ( nh4)2so4 ) ; 
 0.2 mm dntp ; 1   sybr - green i ; 1 unit taq - polymerase ; and 5 ng dna .
the pcr 
 conditions were as follows : denaturation ( 95c , 3 minutes ) followed 
 by 5 cycles of amplification at 95c for 10 seconds , 57c 
 for 10 seconds , and 72c for 10 seconds ; then 30 cycles at 95c 
 for 3 seconds , 59c for 3 seconds , 72c for 3 seconds , 78c 
 for 10 seconds
( plate read ) , and 82c for 10 seconds ( plate read ) . a 
 melting curve analysis
was then performed : 60 cycles starting at 65c 
 with temperature increments of 0.5c , the plate being read at each 
 cycle .
the data obtained were interpreted on the basis with the increase in 
 fluorescence , and the melting curve was used to assess specificity .
the 
 specific primers used were 
 137 c 5-taatgtaatatcactattttcatgaaatc-3137 g 5-aatgtaatatcactattttcatgaaatg-3607 c 5-gttgcagaaagtgtaaaaattattac-3607 a 5-gttgcagaaagtgtaaaaattattaa-3. 137 c 5-taatgtaatatcactattttcatgaaatc-3 137 g 5-aatgtaatatcactattttcatgaaatg-3 607 c 5-gttgcagaaagtgtaaaaattattac-3 607 a 5-gttgcagaaagtgtaaaaattattaa-3. the internal control primers wereltm 1 tgggtgctagaggtataatcgltm 2 ttagaggaagctgggtaagag .
as we used two pairs of allele - specific primers , we studied the 
 reactions in four test tubes simultaneously for each sample to detect any of 
 the four possible haplotypes .
pbmc were isolated from heparinized blood 
 using ficoll - urografin density gradients and were cultured ( 1  10 cells / ml ) for 48 hours in a 48-well plate ( beckton 
  dikkinson , usa ) 
 in the presence or absence of lps ( 055/b5 , sigma , usa ) 
 at a final concentration of 10 g / ml . the level of il-18 protein in the conditioned 
 medium was determined by elisa ( bender med systems , austria ) .
all snps were in hardy - weinberg equilibrium 
 in investigated population.for statistical analysis of the data , we used the 
 nonparametric mann - uitni 's test employing the statistica 6.0 program . to 
 identify quantitative differences in il-18 production levels , we used quantile 
 rank classification according to the mosteller - tewkee algorithm . to 
 analyze the il-18 concentrations
, we 
 determined the probability limits of 
 the empirical distribution quantiles(x(qi ) ) .
a number 
 of quantile ranges ( k ) , into which the total amplitude of variation of the 
 il-18 level was split , were calculated starting from a minimal sample size 
 ( nmin ) by sturges 's formula .
-criterion ( p ) and fisher 's precise method for small samples 
 ( ptmf ) were used to judge the reliability ofoccurrence for qualitative 
 features and frequencies of events within different ranges of il-18 variation 
 .
the distribution of the 
 susceptibility / resistance gene alleles responsible for resistance to or risk of 
 developing immunopathological diseases is unique to each population .
this could 
 be one reason for the diversity of immune responses to an antigen .
this study 
 of two snps in the il-18 gene was conducted on a group of healthy persons 
 ( n = 146 ) .
the relative frequencies of the c / c , c / a , and a / a variants in the 607ca polymorphism were 39.2% , 44.7% , and 16.1% , 
 respectively .
the c and a alleles occurred at frequencies of 64.5% and 35.5% , 
 respectively .
analysis of the il-18 gene polymorphism at the 137g position gave the following results : the 
 frequency of the homozygous variant ( g / g ) was 45.5% , and that of the 
 heterozygous variant g / c was 42.7% ; the c / c genotype was found in 11.8% of 
 cases , and the g and c allele frequencies were 66.8% and 33.2% , respectively . 
 the frequencies of the ac ( 607/137 ) haplotype were 22% , 
 ag 14.6% , cg 55.9% , and cc 7.5% .
we next investigated the il-18 concentration in 
 media conditioned by the pbmc from healthy donors .
the median levels of il-18 
 protein were 21.86 pg / ml [ 9.83 ; 57.60 ( quartiles ) ] for spontaneous production and 72.44 pg / ml [ 32.96 ; 103.07 
 ( quartiles ) ] for lps - stimulated production .
we further studied the correlation 
 between il-18 production and the polymorphic variants 607ca in the il-18 gene promoter ( see figure 2 ) .
il-18 production by lps - stimulated pbmc was 
 significantly greater in healthy donors carrying the ca genotype than in those 
 with the cc genotype at the 607 position of 
 the promoter .
we analyzed the relationship between genotype frequency 
 distributions and il-18 production , dividing the cytokine concentrations into 
  low ,   medium ,  and  high  classes .
concentrations in the 1585% quantile 
 range ( 3.593.5 pg / ml ) for 
 spontaneous production were classed as  medium . 
production levels outside 
 these limits were classed as  low  or  high 
we found a higher 
 frequency of a - allele carriers among persons with a high level of spontaneous 
 il-18 production .
we studied the correlation between il-18 production 
 by pbmc from healthy donors and variants of the il-18 genotype 137 at the 
 position , and we obtained the following data ( see figure 1 ) .
il-18 production by lps - stimulated 
 pbmc was significantly less in persons carrying the cc genotype than in those 
 with the gg genotype at the 137 position . to 
 investigate this further
, we analyzed the relationship between the genotype 
 frequency distribution and levels of il-18 production .
the limits of three 
 primary ranges of variation in il-18 production levels were determined by quantile 
 rank analysis which is tolerant of random statistical  surges  and critical 
 deviations of a sign from normal distribution . in accordance with common 
 practice ,
 concentrations in the range 1585% ( 25.6114.3 pg / ml ) for 
 lps - stimulated production were classed as  medium .
 concentrations outside the 
 limits of these quantiles , that is , less than 25.6 pg / ml and more than 114.3 pg / ml , were classed as 
the frequency of the c allele at 
 the 137 position was 
 significantly higher in donors with low lps - stimulated il-18 production than in 
 those with medium or high production .
thus , lps - stimulated production of il-18 
 is lower in persons with the cc genotype than in donors with the gg genotype at 
 the 137 position of 
 the promoter .
the c allele frequency is greater in the group with a low level 
 of stimulated il-18 production .
as these two polymorphisms , 137 g / c 
 and 607 c / a , were in strong linkage disequilibrium , all possible 
 combinations of alleles 607 ca and 137 gc have been investigated .
there has been shown 
 increased spontaneous production of il-18 associated with haplogenotypes 607cn/137gn in comparison with 607aa/137gn and 607cn/137cc .
thus alleles 
 607c and 137 g are associated with increase of spontaneous production of 
 il-18 . increased lps - stimulated production of il-18
is associated with a haplogenotype 607cn/137gn in comparison with 607cn/137cc ( see figure 3 ) .
this study showed that the 
 frequencies of il-18 genotypes in healthy donors from novosibirsk city 
 ( southwestern siberia ) were as follows : 607cc , 32.2% ; 
 607ac , 44.7% ; 607aa , 16.1% ; 137gg , 45.5% ; 137gc , 42.7% ; and 137cc , 11.8% . for 
 the haplotypes , the values were as follows : ac
( 607/137 ) , 22% ; ag , 
 14.6% ; cg , 55.9% ; and cc , 7.5% .
lps - stimulated production of il-18 was lower in 
 persons with the cc genotype than in donors with the gg genotype at the 137 position of 
 the gene promoter .
il-18 production by lps - stimulated pbmc from healthy donors 
 carrying the ca genotype was significantly higher than from those carrying the 
 cc genotype at the 607 position . 

alleles 607c and 137 g are associated with increase of spontaneous production 
 of il-18 , allele 137 g increases spontaneous and 
 stimulated production .
genetic factors 
 are important in the pathogenesis of all major human diseases and it is known 
 that human populations vary genetically because of polymorphisms , which can 
 affect aspects of the immune response .
diverse polymorphic variants of cytokine 
 genes may induce high or low levels of protein production .
single nucleotide 
 polymorphisms in the il-18 gene promoter at 607 ca and 137 gc affect functionally active parts of this 
 promoter , that is , binding sites for creb transcriptional factors and the 
 h4tf-1 nuclear factor , respectively .
therefore , mutation at these two sites 
 could influence il-18 expression and alter the level of il-18 production . considering the distributions of 
 il-18 gene alleles in healthy donors from different populations
, we found 
 various frequency rates . in the japanese population , the genotypes gg , gc , and 
 cc were found at the 137 position of 
 the promoter with frequencies of 75.4% , 23.1% , and 1.2% , respectively ; the 
 genotypes aa , ac , and cc at the 607 position had 
 the frequencies 43.9% , 39.2% , and 16.9% , respectively .
genotype 
 frequencies in the chinese population were 137gg , 67.3% ; 137gc , 30% ; 137cc , 2.7% ; 607aa , 24.7% ; 607ac , 53.3% ; and 607cc , 22% . 
 for european populations from switzerland   and 
 italy , the characteristic frequencies of the genotypes 137gg , 137gc , and 137cc were 54.1% 
 and 48.3% , 38.7% and 43.8% , and 7.2% and 7.9% , respectively .
il-18 genotypes at 
 the 607 position of 
 the promoter were found in the italian population at the following rates : aa , 
 23.6% ; ac , 47.2% ; cc , 29.2% .
we found the following il-18 
 genotype frequencies in healthy donors from novosibirsk ( southwestern siberia ) : gg , gc , and cc at the 137 position of 
 the promoter were found in 45.5% , 42.7% , and 11.8% of cases , respectively ; aa , 
 ac , and cc at the 607 position were 
 found in 16.1% , 44.7% , and 39.2% , respectively .
thus , the genotype frequencies 
 within the population of southwestern siberia are similar to those of european 
 countries ( italy and switzerland ) . 
 there are , however , some differences in genotype distribution between the 
 inhabitants of southwestern siberia and the populations of china and japan .
it is well known that gene promoter polymorphisms can affect the level 
 of protein production .
our study demonstrated a relationship between two il-18 
 gene promoter polymorphisms and the level of lps - stimulated il-18 production . 

pbmc from persons carrying the c allele at the 137 position of 
 the promoter show low lps - stimulated production of the cytokine .
also , the 
 level of il-18 production by pbmc from donors with the 607ac genotype is 
 higher than that from donors with the 607cc genotype . 

there appears little dose effect of the 
 allele a of 607 snp and we could not detect any difference of il-18 production 
 when heterozygote for 137 snp was considered as a whole .  
however , after 
 splitting heterozygote for one snp according to their genotype at other snp , we 
 were able to demonstrate differential haplogenotypic il-18 production .
. increased lps - stimulated production of il-18 was associated with a 
 haplogenotype 607cn/137gn in comparison with 607cn/137cc .   thus alleles 607c and 137 g
are associated 
 with increase of spontaneous production of il-18 , 
 allele 137 g increases spontaneous and stimulated production .
it is possible to assume that 
 only in case of presence of both alleles conducts to increase production of 
 il-18 protein .
the increased frequency of allele 607a with high 
 production il-18 is connected with heterozygotes 607ca ( 16 donors from 19 ) .
.   showed that a 
 polymorphic variant of the il-18 gene influenced il-18 production by monocytes . 

in particular , they showed that spontaneous and lps - stimulated production of 
 il-18 in volunteers with the 137gg genotype 
 was higher than in those with the 137gc genotype , 
 which is in agreement with our results .
il-18 is a pleiotropic 
 proinflammatory cytokine that predominantly influences the differentiation of 
 type 1 t - helper cells , thus participating in the 
 establishment of the cellular immune response and inflammatory reactions .
a study of the relationship between 
 il-18 gene promoter polymorphisms and the likelihood of developing oncological 
 diseases showed that women carrying the c allele in the 137 position of 
 the promoter are at greater risk of developing ovarian carcinoma . on the 
 basis of our results and data from the literature
, one could suppose that the 
 presence of the c allele at the 137 position in 
 the il-18 gene promoter in such patients results in a less efficient antitumor 
 immune response because il-18 production by immunocompetent cells is 
 diminished .
the serum level of the 
 proinflammatory factor il-18 is known to be significantly elevated in patients 
 with atopic asthma during the acute phase .
studies of the association 
 between il-18 polymorphism and atopic asthma have revealed that the g allele at 
 the 137 position 
 leads to an elevated risk of disease development ; moreover , none of the 74 
 patients examined had the cc genotype , which according to our data is 
 associated with reduced il-18 production .
moreover , the linkage disequilibrium 
 was observed between the 137 and 105 polymorphisms of the il-18 gene and the 
 105a allele of the il-18 gene which may be associated with the pathogenesis of asthma .
premature infants with reduced birth weight are at 
 risk of developing necrotizing enterocolitis and il-18 that is also involved in the pathogenesis 
 of this disease .
analysis of the association between disease severity and 
 genotype frequencies at the 607 position of 
 the il-18 gene showed that the aa genotype frequency is significantly higher in 
 patients with the third and most severe stage of necrotizing enterocolitis , 
 when the intestinal wall is perforated . on the basis of present knowledge
, 
 one may assume that allelic variants of the il-18 gene promoter at the 607ca and 137gc positions , which , according to our results , 
 influence the level of protein production , could result in conditions of which 
 the pathogenesis involves a significant role for il-18 . in conclusion
, this study shows 
 that the il-18 genotype frequencies among healthy donors from novosibirsk city 
 ( southwestern siberia ) are as follows : 607cc , 39.2% ; 607ac , 44.7% ; 
 607aa , 16.1% ; 137gg , 45.5% ; 137gc , 42.7% ; and 137cc , 11.8% . 

allelic variants in 
 the il-18 gene promoter at the 607ca and 137gc positions influence the level of production 
 of the mediator by immunocompetent cells .
lps - stimulated production of il-18 in 
 persons with the cc genotype is lower than that in donors with the gg genotype 
 at the 137 position of 
 the promoter .
the c allele frequency is significantly higher in the group with 
 low lps - stimulated il-18 production .
lps - stimulated production of il-18 by pbmc 
 from healthy donors is significantly greater in those carrying ca genotype at 
 the 607 position .
thus alleles 607c and 137 g are associated with the increase 
 of spontaneous production of il-18 and allele 137 g 
 increases spontaneous and stimulated production .
it is possible to assume that only in case of presence of 
 both alleles conducts to increase production of il-18 protein .
 il-18 has proinflammatory effects and participates in both innate and adaptive cellular and humoral immunity . 
 a number of snps that influence il-18 production are found in the gene promoter region . 
 we investigated the association of snps in the il-18 promoter at 607 and 137 with the level of il-18 protein production by pbmc from healthy donors from southwestern siberia . 
 the genetic distribution of these snps in the promoter site was established by pcr . 
 il-18 protein production was determined by elisa . 
 our results showed that pbmc from donors carrying allele 137c have lower levels of both spontaneous and lps - stimulated il-18 production . 
 in contrast , pbmc from donors carrying allele 607a showed significant increases in spontaneous and stimulated il-18 production compared to wild type . 
 our study suggests that the snps 607 and 137 in the promoter region of the il-18 gene influence the level of il-18 protein production by pbmc from healthy donors in southwestern siberia . 