vitamin d can be obtained either from dietary sources or the epidermal layer of the skin via exposure to sunlight .
the endogenously synthesised vitamin d3 and diet - derived d2 and d3 are hydroxylated in the liver to form 25(oh)d .
25(oh)d is the main storage form , which can be stored in muscles and adipose tissue , and is the major circulating metabolite of vitamin d , with a plasma half - life of 2 - 3 weeks .
therefore , the plasma concentration of 25(oh)d is considered to be the primary indicator of vitamin d status .
plasma 25(oh)d values commonly accepted as the reference range   are as follows . in healthy humans ,
25(oh)d > 100  nmol / l are defined as optimal vitamin d status and levels from 50 to 100  nmol / l are defined as adequate .
nmol / l are proposed to define inadequate vitamin d status and values < 30  nmol / l represent vitamin d deficiency .
subsequently , 25(oh)d can be converted in the kidney to the biologically active form , 1 , 25(oh)2d , by 1--hydroxylase , an enzyme which is stimulated by parathyroid hormone when serum calcium and phosphate concentrations fall below the physiological range .
normal concentrations of circulating 1 , 25(oh)2d are approximately 50250  pmol / l , about 1000 times lower than its precursor , 25(oh)d ; the plasma half - life of 1 , 25(oh)2d is 46 hours .
it has only recently been recognised that vitamin d plays an important role in upregulating immunity .
several recent studies have found a negative association between vitamin d status and respiratory illness incidence in young and elderly adults and it has been indicated that individuals with low vitamin d status have a higher risk of respiratory illness incidence and suffer more severe symptoms when a respiratory illness is present [ 57 ] .
moreover , the presence of the vitamin d receptor in almost all immune cells , including t lymphocytes , b lymphocytes , neutrophils , and antigen presenting cells , such as macrophages and dendritic cells has been demonstrated and these immune cells also express the vitamin d - activating enzyme , 1--hydroxylase , and thus possess the ability to convert 25(oh)d to 1 , 25(oh)2d .
this process is regulated by circulating levels of 25(oh)d or induced by activation of specific toll - like receptors which act as detectors of pathogens [ 1 , 8 ] .
therefore , vitamin d could play vital roles in both innate and adaptive immune responses .
furthermore , vitamin d may also influence circulating cytokine levels and cytokine production during periods of infection . within the general population
it has been reported that circulating proinflammatory cytokine concentrations , such as tnf- , ifn- , il-1 , and il-2 , were significantly higher in vitamin d insufficient adults compared with those who were vitamin d sufficient . however , to our knowledge , there has been no study to compare the difference in circulating cytokine levels between vitamin d insufficient and sufficient athletes . moreover
, the anti - inflammatory effects of exercise training   might modify the influence of plasma 25(oh)d on circulating cytokine levels in athletes .
thus , it is of interest to determine the influence of plasma 25(oh)d status on circulating cytokine levels in athletes .
in addition , several recent studies indicate that 1 , 25(oh)2d3 limits the in vitro production of tnf- , ifn- , and il-6 by isolated human pbmc stimulated with lipopolysaccharide ( lps ) and candida albicans and seasonal variations in vitamin d levels correlate with alterations in cytokine production by ex vivo lps stimulated pbmc , with higher tnf- , ifn- , and il-6 production in winter [ 11 , 12 ] . nevertheless , in our previous vitamin d study , we found that vitamin d deficiency was associated with substantially lower production of proinflammatory cytokines , such as tnf- , ifn- , il-1 , and il-6 , in the whole blood culture by both monocytes and lymphocytes in response to a multiantigen challenge containing antigens from a virus and both gram - positive and gram - negative bacteria .
the reason for this discrepancy is unclear and further research is still needed to understand the influence of vitamin d on multi - antigen stimulated cytokine production . in the present study ,
our aims were to determine the influence of plasma 25(oh)d status on the plasma cytokine concentrations of endurance sport athletes and the effects of different doses of 1 , 25(oh)2d3 on the in vitro multi - antigen stimulated cytokine production by whole blood and pbmc cultures .
a total of 225 healthy individuals engaged in regular sports training ( predominantly endurance - based activities such as running , cycling , swimming , triathlon , team games , and racquet sports ) were recruited as subjects from loughborough university , uk ( latitude 53n ) , during november 2011 , in our previous vitamin d study   with the mean age of the study cohort at recruitment being 21  3 years ( mean  sd ) and their self - reported training loads averaged 10  h / week .
subjects were required to complete a comprehensive health - screening questionnaire prior to starting the study and had not taken any regular medication or antibiotics in the 3 months prior to the study .
all subjects were fully informed about the rationale for the study and of all experimental procedures to be undertaken .
subjects provided written consent to participate in the study , which had earlier received the approval of loughborough university ethical advisory committee .
for the visit to the laboratory , subjects arrived in the morning at 08:3010:30 following an overnight fast of approximately 12  h and their body mass and height were recorded .
information about the study was given to them and they then signed an informed consent form .
subsequently , a resting venous blood sample ( 5  ml ) was obtained by venepuncture from an antecubital forearm vein into a vacutainer tube ( becton dickinson , oxford , uk ) containing k3edta .
haematological analysis was immediately carried out on this sample ( including haemoglobin , haematocrit , and total and differential leukocyte counts ) using an automated cell - counter ( ac.t 5diff haematology analyser , beckman coulter , high wycombe , uk ) .
the remaining edta blood was centrifuged for 10  min at 1500  g and 4c and the plasma was stored at 80c prior to analysis .
edta plasma samples from 43 athletes with high and low vitamin d status ( high level : 25(oh)d > 80 
nmol / l , n = 18 ; low level : 25(oh)d < 40  nmol / l , n = 25 ) with sufficient volume were selected for assay of the circulating cytokine concentrations .
there was no significant difference in the physical characteristics of participants between high - level and low - level vitamin d status groups ( table 1 ) .
for the study of 1 , 25(oh)2d3  in vitro incubation , 6 healthy male individuals ( age : 28.8  4.1 ; height : 176.3  10.0 ; weight : 83.9  7.8 ; bmi : 27.1  2.9 ) who had not taken any regular medication or antibiotics in the 3 months prior to the study were recruited as subjects from loughborough university . for the visit to the laboratory
, subjects arrived in the morning at 08:3010:30 following an overnight fast of approximately 12  h and subsequently , a resting venous blood sample ( 20  ml ) was obtained by venepuncture from an antecubital forearm vein into vacutainer tubes containing k3edta and lithium heparin .
haematological analysis was immediately carried out on the edta sample using an automated cell counter .
the whole blood samples and the pbmc isolated from the heparin tubes were used immediately for in vitro incubation with a multi - antigen vaccine and different doses of 1 , 25(oh)2d3 .
pbmc were isolated by density centrifugation on ficoll - hypaque , histopaque 1077 ( sigma chemicals , poole , uk ) .
the heparinized whole blood was diluted 1  :  1 with room temperature 10  mm phosphate buffered saline ( pbs , sigma chemicals , poole , uk ) and 20  ml of diluted blood was layered onto 10  ml of room temperature ficoll - hypaque .
cells were collected from interphase and washed twice with rpmi 1640 medium ( sigma chemicals , poole , uk ) supplemented with 10% fetal bovine serum ( sigma chemicals , poole , uk ) and then spun at 250  g for 10 minutes at 20c .
subsequently , cells were resuspended in rpmi 1640 medium supplemented with 10% fetal bovine serum .
cells were counted in an automated cell counter and the number of cells was adjusted to 2  10 cells / ml in rpmi 1640 medium supplemented with 10% fetal bovine serum . in our previous vitamin d study , 225 edta plasma samples were analysed for 25(oh)d3 and 25(oh)d2 with a high pressure liquid chromatography tandem mass spectrometer ( waters acuity , manchester , uk ) after a maximum of 10 months in storage with no previous freeze - thaw cycles as described previously .
briefly , 25(oh)d2 , 25(oh)d3 , and deuterated internal standard were extracted from plasma samples , following protein precipitation , using isolute c18 solid phase extraction cartridges .
potential interfering compounds were removed by initial elution with 50% methanol followed by elution of the vitamins using 10% tetrahydrofuran in acetonitrile .
dried extracts were reconstituted prior to injection into a high performance liquid chromatography tandem mass spectrometer in the multiple reaction mode ( mrm ) . the mrm transitions ( m / z ) used were 413.2 > 395.3 , 401.1
> 383.3 , and 407.5 > 107.2 for 25(oh)d2 , 25(oh)d3 , and hexa - deuterated(oh)d3 ( internal standard ) , respectively .
intra - assay cvs were < 10% across a working range of 2.5624  nmol / l for both 25(oh)d3 and 25(oh)d2 .
measurements were performed in a laboratory ( norwich university hospital , norwich , uk ) meeting the performance target set by the vitamin d external quality assessment scheme ( deqas ) advisory panel for 25(oh)d assays .
the whole blood samples and pbmc were incubated in vitro with a multi - antigen vaccine and different doses of 1 , 25(oh)2d3 .
the stimulant was a commercially available multi - antigen vaccine ( pediacel vaccine , sanofi pasteur , uk ) containing diphtheria , tetanus , acellular pertussis , poliomyelitis , and haemophilus influenzae type b antigens . 1 ,
25(oh)2d3 was purchased from sigma - aldrich ( missouri , usa ) and dissolved in absolute ethanol to give a stock solution of 10  m .
aliquots were subsequently diluted with rpmi 1640 medium for use in the in vitro incubations . stimulated whole
blood and pbmc culture production of cytokines ( ifn- , tnf- , il-1 , il-2 , il-4 , il-6 , and il-10 ) were determined as described previously .
briefly , for the determination of baseline unstimulated cytokine production , 0.25  ml of heparinized whole blood or pbmc were added to 0.75  ml of rpmi 1640 medium and incubated at 37c and 5% co2 for 24  h. additionally , 0.25  ml of heparinized whole blood or pbmc were added to 0.70  ml of rpmi 1640 medium containing different doses of 1 , 25(oh)2d3 ( 0 , 100 , 200 , 1000 , and 10000  pmol / l ) with an added 50  l of pediacel vaccine cocktail ( sanofi pasteur msd limited , maidenhead , uk ) at a dilution of 1  :  100 , before being incubated at 37c and 5% co2 for 24  h. the stimulant dilution of 1  :  100 used in this study was based on a separate experiment ( unpublished data ) , which established the dose - response curve for the measured cytokines over the dilution range of 1  :  1001  :  20 000 .
samples were then centrifuged at 13000  g for 4  min at 4c , following which the supernatant fluid was harvested and stored at 80c prior to analysis of cytokine concentrations .
cytokine concentrations were determined using an evidence investigator system and the high sensitivity cytokine biochip array ev3513 ( randox , county antrim , uk ) .
the measured cytokine concentrations for the monocyte - derived cytokines ( tnf- , il-1 , and il-6 ) and lymphocyte - derived cytokines ( il-2 , il-4 , and ifn- ) were divided by the monocyte and lymphocyte counts , respectively , to give cytokine production per 10 cells . the independent t - test was used to test for the differences in the physical characteristics of participants between high - level and low - level vitamin d groups .
mann - whitney u - test was used to test for the differences in the circulating cytokine concentrations between high - level and low - level vitamin d groups . the correlation between circulating cytokine and plasma 25(oh)d concentrations
wilcoxon signed rank test performed in the previous studies by khoo and colleagues [ 11 , 12 ] was also used in the present study to examine for the differences in the stimulated whole blood and pbmc culture production of cytokines between different doses of 1 , 25(oh)2d3  in vitro incubation .
differences in the multi - antigen stimulated cytokine production without the addition of 1 , 25(oh)2d3 by whole blood and pbmc cultures were compared with the mann - whitney u test .
data are presented as mean ( sd ) or median and interquartile range ( iqr ) and the accepted level of significance was p < 0.05 .
the plasma 25(oh)d concentrations were 101.3  13.9 and 31.8  3.5  nmol / l in the high - level and low - level vitamin d status groups , respectively .
the anti - inflammatory cytokine il-10 concentration in the high - level vitamin d status group was marginally but significantly higher than in the low - level group ( p = 0.020 ) , and the proinflammatory cytokine ifn- concentration in the high - level vitamin d status group was substantially and significantly higher than in the low - level group ( p = 0.004 ) ( table 2 ) .
however , there was no significant difference in the circulating tnf- , il-1 , il-2 , il-4 , and il-6 concentrations between high - level and low - level vitamin d status groups ( table 2 ) .
in addition , there was a positive correlation between the plasma 25(oh)d and ifn- concentrations ( r = 0.374 , p = 0.014 ) , but plasma 25(oh)d concentrations did not correlate with tnf- , il-1 , il-2 , il-4 , il-6 , or il-10 levels ( table 3 ) .
the antigen - stimulated production of the monocyte - derived cytokines , tnf- and il-6 , was both attenuated by 1 , 25(oh)2d3 , but production of il-1 was not significantly influenced by 1 , 25(oh)2d3 .
pmol / l and 10000  pmol / l of 1 , 25(oh)2d3 compared with its production without the addition of 1 , 25(oh)2d3 and the production of il-6 was significantly lower in the presence of 10000 
pmol / l of 1 , 25(oh)2d3 compared with its production without the addition of 1 , 25(oh)2d3 ( figure 1 ) .
in addition , the antigen - stimulated production of the lymphocyte - derived cytokines , ifn- and il-2 , was both attenuated by 1 , 25(oh)2d3 , but production of il-4 was not significantly influenced by 1 , 25(oh)2d3 .
pmol / l and 10000  pmol / l of 1 , 25(oh)2d3 compared with its production without the addition of 1 , 25(oh)2d3 and the production of ifn- was significantly lower in the presence of 10000  pmol / l of 1 , 25(oh)2d3 compared with its production without the addition of 1 , 25(oh)2d3 ( figure 1 ) .
the anti - inflammatory cytokine il-10 is produced by both monocytes and lymphocytes , so it is not appropriate to normalise its production by cell counts .
pmol / l of 1 , 25(oh)2d3 compared with its production without the addition of 1 , 25(oh)2d3 ( 0 
pmol / l : 4.5  2.1  pg / ml ; p = 0.028 ) , but no differences were observed at the other concentration of added 1 , 25(oh)2d3 .
the antigen - stimulated production of the monocyte - derived cytokine , il-6 , was attenuated by 1 , 25(oh)2d3 , but production of tnf- and il-1 was not significantly influenced by 1 , 25(oh)2d3 .
pmol / l of 1 , 25(oh)2d3 compared with its production without the addition of 1 , 25(oh)2d3 ( figure 2 ) .
however , the antigen - stimulated production of the lymphocyte - derived cytokines , ifn- , il-2 , and il-4 , was not significantly influenced by the incubation of different doses of 1 , 25(oh)2d3 ( figure 2 ) .
in addition , the production of il-10 was also not significantly influenced by 1 , 25(oh)2d3 .
the production of the lymphocyte - derived cytokines , ifn- , il-2 , and il-4 , and the monocyte - derived cytokine , il-1 , by multi - antigen stimulated whole blood culture without the addition of 1 , 25(oh)2d3 was significantly higher than their respective production by the pbmc culture ( table 4 ) .
however , there was no significant difference in the antigen - stimulated production of tnf- , il-6 , and il-10 between the whole blood and pbmc cultures ( table 4 ) .
the aims of this research were to determine the influence of plasma 25(oh)d status on the circulating cytokine levels in endurance sport athletes and the effects of different doses of 1 , 25(oh)2d3 on the multi - antigen stimulated cytokine production by whole blood and pbmc cultures .
the main findings of the present study were that the circulating il-10 and ifn- concentrations were significantly higher in the vitamin d sufficient athletes compared with the vitamin d insufficient athletes and there was a positive correlation between the plasma 25(oh)d and ifn- concentrations in endurance athletes .
furthermore , the production of tnf- , il-6 , ifn- , il-2 , and il-10 by multi - antigen stimulated whole blood culture was significantly influenced by 1 , 25(oh)2d3 of concentrations of 1000  pmol / l and/or 10000  pmol / l . nevertheless , a similar effect was only observed for the production of il-6 by multi - antigen stimulated pbmc culture .
in addition , the production of lymphocyte - derived and monocyte - derived cytokines , such as ifn- , il-2 , il-4 , and il-1 , was significantly different in the multi - antigen stimulated whole blood culture without the addition of 1 , 25(oh)2d3 compared with its production in the pbmc culture .
on the basis of the present data , we found that the circulating il-10 and ifn- concentrations in the vitamin d sufficient athletes were significantly higher than in the vitamin d insufficient athletes and there was a positive correlation between the plasma 25(oh)d and ifn- concentrations in endurance athletes .
in contrast , a previous study has reported an elevation of circulating proinflammatory cytokines in vitamin d insufficient adults .
barker et al .   indicated that circulating proinflammatory cytokine concentrations , such as tnf- , ifn- , il-1 , and il-2 , were significantly higher in vitamin d insufficient compared to vitamin d sufficient adults but the anti - inflammatory cytokine , il-10 , was not significantly different between vitamin d insufficient and sufficient adults .
moreover , barker and colleagues   found an inverse correlation between the plasma 25(oh)d and ifn- concentrations .
the reason for this discrepancy is still unclear but may be due to the difference of the cut - off point used for vitamin d insufficiency in the two studies . in healthy humans ,
plasma 25(oh)d serum levels > 100  nmol / l are defined as optimal vitamin d status and levels from 50 to 100  nmol / l are defined as adequate .
nmol / l are proposed to define inadequate vitamin d status and values < 30  nmol / l represent vitamin d deficiency .
therefore , the commonly used cut - off point for vitamin d insufficiency in clinical practice and research reports is the threshold concentration of 25(oh)d of < 50  nmol / l . however , the cut - off point for vitamin d insufficiency used in the barker et al .  
study was < 80  nmol / l and the average serum 25(oh)d concentration of vitamin d of insufficient adults was around 60 
thus , the vitamin d insufficient adults in their study were not even close to being vitamin d deficient .
in addition , another confounding factor may be due to the anti - inflammatory effects of exercise training .
the participants recruited in our previous vitamin d study   were the endurance sport athletes engaged in regular sports training ( predominantly endurance - based activities such as running , cycling , swimming , triathlon , team games , and racquet sports ) and their self - reported training loads averaged 10  h / week .
the anti - inflammatory effects of exercise training on cytokine responses , such as the release of il-6 from contracting muscle and the increased levels of circulating il-10 , cortisol , and adrenaline , are discussed in the review article by gleeson et al . .
it has been demonstrated that the active skeletal muscle significantly increases both cellular and circulating levels of il-6 during and following exercise of sufficient load and the transient rise in circulating il-6 during exercise appears to be responsible for a subsequent rise in circulating levels of anti - inflammatory cytokines , including il-10 , which is a potent promoter of an anti - inflammatory state .
increases in circulating cortisol and adrenaline during exercise are due to activation of the hypothalamic - pituitary - adrenal axis and the sympathetic nervous system , respectively .
cortisol is known to have potent anti - inflammatory effects and also augmented by the rise in circulating il-6 from contracting skeletal muscle .
therefore , the influence of vitamin d on circulating cytokines might be different between athletes and nonathletes .
further research is still needed to understand the influence of vitamin d on circulating cytokines between athletes and nonathletes .
the finding of higher circulating ifn- concentrations in the vitamin d sufficient athletes and a positive correlation between the plasma 25(oh)d and ifn- concentrations in the present study might be helpful to support the arguments that vitamin d could play a role in reducing both the severity and duration of upper respiratory tract illness symptoms in our previous vitamin d study .
it has been reported that increasing ifn- , in the circulation contributes to the conversion of 25(oh)d to its active hormonal form , 1 , 25(oh)2d , in the circulation during inflammatory stress in humans . 1
, 25(oh)2d has a vital role in upregulating the production of antimicrobial proteins and peptides , such as cathelicidin and -defensin , which are produced by both epithelial cells and macrophages and have a broad range of activities against microorganisms including the direct inactivation of viruses   and the hormone also acts to maintain a balance between inflammatory th1/th17 cells and immunosuppressive th2/regulatory t cells to temper inflammation and tissue damage .
therefore , the vitamin d sufficient athletes with higher circulating ifn- concentrations may have less severe symptoms and shorter duration when the upper respiratory tract illness is present . according to the findings from the present study
, it seems likely that the multi - antigen stimulated cytokine production by whole blood culture was influenced by 1 , 25(oh)2d3 only in concentrations that were 1000  pmol / l or more .
although the production of proinflammatory cytokines , tnf- , il-6 , ifn- and il-2 by multi - antigen stimulated whole blood culture was significantly lower in the presence of 1000  pmol / l or 10000  pmol / l of 1 , 25(oh)2d3 and the production of anti - inflammatory cytokine , il-10 , was significantly higher in the presence of 10000 
pmol / l of 1 , 25(oh)2d3 , we found that both proinflammatory and anti - inflammatory cytokine production by multi - antigen stimulated whole blood culture were not influenced by 1 , 25(oh)2d3 in added concentrations of 100 
pmol / l and 200  pmol / l . in contrast , the results from our previous study   showed that vitamin d status ( determined by plasma levels of 25(oh)d ) did significantly influence lymphocyte and monocyte proinflammatory cytokine production by antigen - stimulated whole blood culture .
individuals with high levels of plasma 25(oh)d would be expected to have higher levels of 1 , 25(oh)2d , so it is perhaps surprising that in the in vitro situation variations in 1 , 25(oh)2d within the normal physiological range appear to have no significant effect on leukocyte cytokine production . in healthy humans , normal concentrations of circulating 1 ,
25(oh)2d are approximately 50250  pmol / l , about 1000 times lower than its precursor , 25(oh)d .
inhibition of proinflammatory cytokine production and elevation of anti - inflammatory cytokine production in the present study were only observed when the whole blood was incubated with 1 , 25(oh)2d in concentrations that were 10100-fold above the normal healthy range for plasma 1 , 25(oh)2d .
a limitation of the present study is that the participants ' plasma concentration of 1 , 25(oh)2d was not determined .
further research is still needed to understand the influence of the normal healthy range of plasma 1 , 25(oh)2d on the multi - antigen stimulated cytokine production by whole blood culture . the data from our study show that there was a significant difference in the production of multi - antigen stimulated lymphocyte - derived and monocyte - derived cytokines , such as ifn- , il-2 , il-4 , and il-1 , between the whole blood and pbmc cultures and it seems likely that there was a better dose - dependent response in the cytokine production of multi - antigen stimulated whole blood culture with different doses of 1 , 25(oh)2d3 than in the pbmc culture .
the whole blood culture retains the normal cellular , hormonal , and cytokine milieu that the leukocytes are normally exposed to in the circulation .
this model probably comes closest to the natural environment avoiding artefacts from cell isolation and preparation and allowing natural interactions between immune components and antigens within the normal hormonal milieu .
moreover , the stimulant we used was a vaccine containing antigens from a virus and both gram - positive and gram - negative bacteria and the multiple antigen challenge used in the present study provides valuable information on cytokine production since not all cytokines respond to the same antigen .
the capacity of leukocytes to produce cytokines upon adequate challenge ( e.g. , with mitogen , antigen , endotoxin , or pathogen exposure ) has potentially far reaching consequences for the entire functional capacity of the immune system .
therefore , the whole blood culture stimulated with multi - antigen is highly likely to reflect the capacity of an individual to defend itself against intruding microorganisms . in conclusion
, the present study demonstrated that the influence of vitamin d on circulating cytokines might be different in athletes compared with previous studies on nonathletes and the higher circulating ifn- concentrations in vitamin d sufficient athletes might be helpful to support the arguments that vitamin d could play a role in reducing both the severity and duration of upper respiratory tract illness symptoms .
in addition , we found that both proinflammatory and anti - inflammatory cytokines by multi - antigen stimulated whole blood culture were not influenced by 1 , 25(oh)2d3 in concentrations within the normal healthy range .
furthermore , the whole blood culture stimulated with multi - antigen might be a better in vitro method of simulating responses to an infection than cultures using pbmcs .
 
 aim . 
 our aims were to determine the influence of plasma total 25-hydroxy vitamin d ( 25(oh)d ) status on the plasma cytokine concentrations in athletes and the in vitro effects of different doses of 1 , 25 dihydroxyvitamin d3 ( 1 , 25(oh)2d3 ) on multiantigen stimulated cytokine production by whole blood and peripheral blood mononuclear cell ( pbmc ) cultures . 
 methods . 
 plasma samples from 43 athletes with high and low levels of 25(oh)d were assayed for the concentrations of cytokines . 
 the whole blood samples and pbmcs from healthy subjects were incubated in vitro with a multi - antigen vaccine and different doses of added 1 , 25(oh)2d3 . 
 the circulating cytokines and stimulated whole blood and pbmc culture production of cytokines were determined using a biochip assay . 
 results . 
 the circulating interleukin-(il-)10 and interferon-(ifn- )  concentrations were significantly higher in the vitamin d sufficient athletes . 
 furthermore , the production of tumour necrosis factor-(tnf- )  , il-6 , ifn- , il-2 , and il-10 by whole blood culture was significantly inhibited by 1 , 25(oh)2d3 concentrations of 1000  pmol / l or 10000  pmol / l . conclusions . 
 we found that the influence of vitamin d on circulating cytokines might be different in athletes compared with nonathletes and cytokines production by whole blood culture was not influenced by 1 , 25(oh)2d3 in concentrations within the normal healthy range . 
1. Introduction
2. Methods
3. Results
4. Discussion