Research Article Ascorbic Acid, Ultraviolet C Rays, and ...

Research ArticleAscorbic Acid Ultraviolet C Rays andGlucose but not Hyperthermia Are Elicitors of Human120573-Defensin 1 mRNA in Normal Keratinocytes

Luis Antonio Cruz Diacuteaz1 Mariacutea Guadalupe Flores Miramontes1 Paulina Chaacutevez Hurtado1

Kirk Allen23 Marisela Gonzalez Aacutevila4 and Ernesto Prado Montes de Oca12

1 Molecular Biology Laboratory Biosecurity Area Pharmaceutical and Medical Biotechnology Unit (UBMF)Research Center in Technology and Design Assistance of Jalisco State (CIATEJ AC)National Council of Science and Technology (CONACYT) Guadalajara JAL Mexico

2 In silico Laboratory UBMF CIATEJ AC CONACYT Guadalajara JAL Mexico3 Lancaster Medical School Lancaster University Lancaster UK4Human Digestive Tract Simulator UBMF CIATEJ AC CONACYT Guadalajara JAL Mexico

Correspondence should be addressed to Ernesto Prado Montes de Oca epradociatejmx

Received 31 July 2014 Revised 2 October 2014 Accepted 24 October 2014

Academic Editor Alejandra Ochoa-Zarzosa

Copyright copy 2015 Luis Antonio Cruz Dıaz et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Hostsrsquo innate defense systems are upregulated by antimicrobial peptide elicitors (APEs) Our aim was to investigate the effects ofhyperthermia ultraviolet A rays (UVA) and ultraviolet C rays (UVC) as well as glucose and ascorbic acid (AA) on the regulationof human 120573-defensin 1 (DEFB1) cathelicidin (CAMP) and interferon-120574 (IFNG) genes in normal human keratinocytes (NHK)The indirect in vitro antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes of these potential APEs wastested We found that AA is a more potent APE for DEFB1 than glucose in NHK Glucose but not AA is an APE for CAMP Mildhypo- (35∘C) and hyperthermia (39∘C) are not APEs in NHK AA-dependent DEFB1 upregulation below 20mM predicts in vitroantimicrobial activity as well as glucose- and AA-dependent CAMP and IFNG upregulation UVC upregulates CAMP and DEFB1genes but UVA only upregulates the DEFB1 gene UVC is a previously unrecognized APE in human cells Our results suggest thatglucose upregulates CAMP in an IFN-120574-independent manner AA is an elicitor of innate immunity that will challenge the currentconcept of late activation of adaptive immunity of this vitamin These results could be useful in designing new potential drugs anddevices to combat skin infections

1 Introduction

Keratinocytes control skin microbial colonizationinfectionin part by synthesizing human 120573-defensin 1 (hBD-1) andcathelicidin LL-37 both of which are wide-spectrum antimi-crobial peptides [1ndash4] Antimicrobial peptide elicitors (APEs)are defined as physical (class I) chemical (class II) andbiological (class III) agents that promote upregulation ofendogenous antimicrobial peptides (APs) [5ndash8]

IFNG is a relevant gene in innate and adaptive responsesspecifically its product IFN-120574 is an APE for both CAMP(codes for LL-37) andDEFB1 (codes for hBD-1) inmonocytes

and gingival keratinocytes respectively [6] In this report wewanted to know if keratinocytes upregulate IFNG in responseto APE independently of the IFNGmRNA provided by lym-phocytes in adaptive immunity Probable APEswith potentialapplication in skin therapy against infections in humans arehyperthermia ultraviolet A rays (UVA) ultraviolet C rays(UVC) ascorbic acid (AA or vitamin C) and glucose

In a mice model of influenza infection hyperthermia apotential class I APE is beneficial because it increases leuko-cyte count and diminishes proinflammatory cytokines pre-sumably avoiding damage to infected tissue [9] In humanshyperthermia is beneficial in treating several diseases such as

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 714580 9 pageshttpdxdoiorg1011552015714580

2 BioMed Research International

neurosyphilis some forms of chronic arthritis and cancerFurthermore many infections cause fever during certainphases including fever caused by Rickettsia sp Chlamydiasp viruses or parasites When this fever is associated withendogen pyrogen it leads to the activation of T cells whichenhances the hostrsquos defense system [10]

UVA (320ndash400 nm) are APEs of DEFB1 in sclerodermaskin lesions [11] but they have not been tested in normalkeratinocytes UVC (180ndash280 nm) have been suggested as aprophylactic approach in a mice model of infection againstPseudomonas aeruginosa and Staphylococcus aureus [12] butthe function as an APE of this wavelength has not beendescribed S aureus is a very common human pathogensusceptible to LL-37 and hBD-1 [6] In the case of Listeriasp the pathogen is susceptible to LL-37 These APEs have atherapeutic potential against this pathogen in epithelial cells[13 14] In this study we indirectly assume that microbicidalactivity is due the increment of the level of synthesis of thesepeptides by the APEs as suggested by the mRNA levels andinhibition zones of the antimicrobial assays

AA has been widely used to prevent and treat the com-mon cold malaria and diarrhea infections and pneumoniaAA improves the efficacy of antimicrobial and natural killercell (NK) activities [15] Whether this antimicrobial activityis dependent on induction andor upregulation of APs isunknown Glucose is an APE of DEFB1 in kidney andcolon cells [16] but its effect on both DEFB1 and CAMP inkeratinocytes is unknown

In this study we assessed the capacity of UVA UVCAA glucose and hyperthermia to act as APEs of DEFB1 andCAMP genes and as inductors of IFNG in a cell line of neonatenormal human keratinocytes (NHK)

2 Materials and Methods

21 Cell Culture and Exposure to Potential APEs Normalhuman neonate keratinocytes (ATCC PCS-200-010) weremaintained in K-SFM cell culture media (Gibco) at 37∘C in ahumidified incubator with 5 CO

2 We exposed 1 times 106 cells

toD-(+)-glucose (55 155 255 and 455mM Sigma numberof catalog G8644) or L-ascorbic acid (0 5 10 and 20mMSigma number catalog A4544) for 24 48 and 72 h Theincubation temperature was also assessed as potential APEmimicking human mild hypothermia (35∘C) normothermia(37∘C) and hyperthermia (39∘C) in vitroAdditionally for theascorbic acid at 20mM the interaction between time (24 48and 72 h) and temperature (35 37 and 39∘C) was assessedThe exposition to UV rays (UVA at 365 nm UVC at 254 nm)was performed with a square source of irradiation of 4 Watts(UV lamp UVP-UVGL-25 UVP Cambridge UK number ofcatalog 95-0021-12) at 92 cm from the adhered cells

22 Quantitative Real-Time PCR (qPCR) Nucleic acids wereextracted in a MagNA Pure LC 20 Instrument (Roche) withthe MagNA Pure LC total nucleic acid kit (Roche catalognumber 03038505001) PurifiedRNAwasDNAse-treated andreverse transcribed with a mix containing random primers(Invitrogen) and ArrayScript enzyme (Applied Biosystems)

Quantitative real-time PCR was performed with 33 ng cDNAwith Fast SYBR Green Master Mix (Applied Biosystems)The reactions were run in a Step One Thermocycler(Applied Biosystems) using the following primerssequences designed with Primer Express v 30 software(Applied Biosystems) and tested for specificity in PRIMER-BLAST (httpwwwncbinlmnihgovtoolsprimer-blast)for HPRT (endogenous control suggested for normalhuman keratinocytes according to Allen et al [17]) F51015840-TGTTCAAATTATTACCAGTGAATCTTTGTC-31015840 R51015840 TTTTAAATTTTTGGGAATTTATTGATTTG-31015840 forIFNG F 51015840-GCTGACTAATTATTCGGTAACTGACTTG-31015840 R 51015840-TAGCTGCTGGCGACAGTTCA-31015840 for DEFB1F 51015840-GAGAACTTCCTACCTTCTGCTGTTTAC-31015840R 51015840-AAAGTTACCACCTGAGGCCATCT-31015840 forCAMP F 51015840-ACCCAGACACGCCAAAGC-31015840 R 51015840-TTCACCAGCCCGTCCTTCT-31015840 The PCR program was95∘C for 20 s 40 cycles of denaturing at 95∘C for 3 s andannealingextension at 60∘C for 30 s Melting curves wereperformed at 95∘C for 15 s 60∘C for 1min and 95∘C for 15 sAmplification efficiencies for each gene were calculated andthe relative quantitative expressionwas obtained according tothe Pfaffl method [18] The normalization of gene expressionincluded the input RNA and input cell count The controlswere samples with the vehicle at 24 h or in the case oftemperatures the normothermic sample (37∘C) for each day

23 Antimicrobial Assays Diffusion assays were performedaccording to Kirby et al [19] We tested culture supernatantssamples with a volume of 50120583L poured into 6mm diameterwells The negative control was the supernatant of the culturemedium without the potential APE and the positive controlwas 225120583M (1mgmL) of tetracycline Three biologicalreplicas of each were performed with strains of Listeriamonocytogenes (ATCC 19114 susceptible to LL-37 [13 14])and Staphylococcus aureus (ATCC 25923 susceptible to LL-37 and hBD-1) [6]

24 Cytotoxicity Assays In the experimental conditionswhere gene upregulation was observed experiments wererepeated and cytotoxicity assays were performed in increas-ing concentrations of the APEsThis was assessed by applying005 trypsin by 12min and neutralizing with DMEM (10fetal bovine serum) After centrifugation (400timesg 5min)three 10 120583L aliquots of homogeneous precipitated cells werequantified in a Neubauer chamber The percentage of viablecells adhered to the plate was compared with the controlwithout elicitor considered as being 100 viable Quantifi-cations of each day were compared with the control of thecorresponding days (Cruz Dıaz et al in process)

25 Statistical Analysis Statistical comparisons were done inMicrosoft Excel 2010 To evaluate if variances were differentor equal among treatments we performed119865 tests Dependingof the results of 119865 tests Studentrsquos 119905-tests for equal or nonequalvariances were performed Results are shown as mean +standard error of the mean (SEM) All experiments were

BioMed Research International 3

0

50

100

150

200

250

300

350

400Vi

abili

ty (

)lowastlowastlowast

lowastlowastlowast

24h 48h 72h0mM 455mM 0mM 455mM 0mM 455mM

(a)

01

1

10

100

1000

10000

24 48 72

(h)

55mM155mM

255mM455mM

Fold

chan

geD

EFB1

HPR

T

lowast

(b)

Figure 1 Effect of glucose in normal human keratinocytes (a) Viability percentage of keratinocytes to glucose exposition by 24 h 48 h and72 h in comparison with their controls for each exposure time (b) DEFB1 expression at 24 48 and 72 h of keratinocytes exposed to glucoseReference group was 55mM (normal concentration of culture medium) at each time Error bars represent SEM and significant values ofStudentrsquos 119905 tests are depicted as lowast119875 lt 005 lowastlowastlowast119875 lt 0001

performed at least in triplicate and a 119875 value lt005 wasconsidered statistically significant

3 Results

When we exposed the dermal cells to increasing levels ofglucose simulating increased absorption and distribution ofglucose after feeding the viability of keratinocytes increasedat 24 h and was found to be highly significant at 48 h and 72 h(Figure 1(a)) At 24 h of glucose exposure (455mM) DEFB1expression was significantly upregulated (Figure 1(b))

The viability of keratinocytes is not affected at 10mMof ascorbic acid (AA Figure 2(a)) but begins to be affectedat 20mM and was highly significantly affected at 40mM(Figure 2(b)) When keratinocytes were exposed to 10 and20mM of ascorbic acid DEFB1 was upregulated by almost 5logs after 72 h (Figure 2(c))

At 72 h rather than at 24 h glucose also upregulatedIFNG andCAMP genes (Figure 3) AA also upregulated IFNG(Figure 3) but AA did not affect CAMP expression at anytime or concentration (data not shown) DEFB1 (3 logs)CAMP (1 log) and IFNG (1 log) were upregulated in bothhypothermia and hyperthermia conditions mainly at 72 halthough the results were not significant (data not shown)We then assessed the effect of temperature at 20mMof AA by72 h (highest significant upregulation of DEFB1 Figure 2(c))Compared with normothermia DEFB1 was upregulated at24 h at 35∘C but downregulated at 72 h at both 35 and 39∘C(Figure 2(d)) Interestingly at hypothermia and 20mMofAAboth IFNG andCAMPwere downregulated at 72 h (Figure 3)Furthermore the expressions of DEFB1 and IFNG correlatelinearly with 20mM at 35∘C (r2 = 09)

UVC upregulated CAMP at 5 and 10min of exposure(Figure 4(a)) as well as upregulating DEFB1 (Figure 4(b))

and IFNG genes (Figure 4(c)) Surprisingly UVA slightlyupregulated DEFB1 at 5min of exposure and downregulatedit at 10 and 20min of exposure (Figure 4(d)) UVA do notimpact CAMP or IFNG regulation (data not shown)

The supernatant of NHK in the condition 5mMAA24 h 37∘C that shows upregulation of both CAMP (256-fold) and IFNG (331-fold) showed an increased antimicro-bial activity against both L monocytogenes and S aureus(Figure 5) in spite the diminished expression of DEFB1(05-fold) As expected due to diminished viability bothconditions 37∘C 20mMAA 72 h and 35∘C 20mMAA 24 hdiminish the activity against S aureus significantly even at theformer condition which had the highestDEFB1 expression (4logs)

Both samples from 455mM glucose 24 h and 72 hshowed increased antimicrobial activity against S aureusand L monocytogenes respectively as expected presumablybecause in these samples DEFB1 (90-fold) and CAMP (643-fold) genes were upregulated respectively

4 Discussion

We demonstrate for the first time that AA is an APE forDEFB1 but not for CAMP We therefore propose that theindirect antimicrobial effect of ascorbic acid in NHK couldbe at least partially dependent on DEFB1 induction whichchallenges the traditional view regarding AA only as beingconsidered a player in the later activation of the adaptiveimmunity [20] This also could explain why AA acts as anantitumor agent [21ndash23] since DEFB1 is considered a tumorsuppressor gene [24 25]

Regarding cytotoxicity assay Trypan Blue Exclusion(TBE) Assay overestimates viable cells [26] and assignmentof trypan-stained cells to viable or nonviable categories


O O

HO

HO

OH

OH

H

(a)

0

20

40

60

80

100

120

140

0 10 20 40

Viab

ility

()

(mM)

lowast

lowastlowast

(b)

001

01

1

10

100

1000

10000

100000

1000000

24 48 72

lowast

lowast

(h)

Fold

chan

geD

EFB1

HPR

T

0mM5mM

10mM20mM

(c)

01

1

10

100

lowast

lowast lowast

Fold

chan

geD

EFB1

HPR

T

35 37 39

∘C)24h48h

72h(

(d)

Figure 2 Effect of ascorbic acid alone or in combination with hypothermia or hyperthermia in normal human keratinocytes (a) Structureof L-ascorbic acid (b) Cytotoxicity assays exposed 24 h with ascorbic acid (c) Effect of different concentrations of ascorbic acid on DEFB1expression at 24 48 and 72 h The reference group was 0mM at each time (d) Effect of hypothermia normothermia and hyperthermia allplus interaction with 20mMascorbic acid onDEFB1 expression Reference groupwas normothermia (37∘C) at each time Error bars representSEM and significant values of Studentrsquos 119905 tests are depicted as lowast119875 lt 005 lowastlowast119875 lt 001

was found to be subjective and arbitrary [27] Regarding 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) assay a lineal relationship exists between reducedtetrazolium dye and cell number only up to 2 times 104 cellswellwith no relationship at higher cell numbers andorabsorbance values of greater than 08 [27] Also MTTwas designed to test cytotoxic drugs and the recommendedset of concentrations must include the highest concentrationthat kills most of the cells and the lowest concentrationthat kills none of the cells [28] The main advantages

of the proposed protocol is that in adherent cultures itpermits to obtain a number of viable cells that are alreadyattached because the nonattached cells if any are removedby aspirating the supernatant With our method there isless ambiguity assigning viability as in TBE and chemicalreactions even in the absence of viable cells This does notaffect the results as in MTT (Cruz Dıaz et al in process)

It has been reported that short-term hypothermia (takenas 30ndash34∘C 2ndash4 hours) in cell lines and murine modelsincreases the level of anti-inflammatory cytokines (IL-4


37 37 37 35 37 35 3724 72 72 72 72 72 725 10 20 20 20

255 455

0

5

10

15

20

25

30

35

40Fo

ld ch

ange

HPR

T

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowast

lowast

Temp (∘C)Time (h)AA (mM)Glu (mM)

IFNG CAMP

minus minus minus

minus

minus

minus

minus

Figure 3 Statistically significant results of the effect of ascorbicacid hypothermia andor glucose exposure in IFNG and CAMPexpression in normal human keratinocytes Error bars representSEM and significant values of Studentrsquos 119905 tests are depicted as lowast119875 lt005 lowastlowastlowast119875 lt 0001

and IL-10) and decreases proinflammatory cytokines (IL-1IL-2 IL-6 and TNF-120572) inhibiting lymphocyte proliferationand decreasing HLA-DR expression associated with cellactivation Long term (gt24 hours) hypothermia howeverincreases proinflammatory cytokine levels [29] The hyper-thermic conditioning (41∘C) has been an effective treatmentin mice with sepsis in combination with LL-37 In ratswith hypothermia (32∘C) prior to sepsis the IL-10 levelswere significantly increased compared to normothermic ratsaltering the cytokines profile survival and recruitment ofgranulocytes suggesting immunosuppression [30] Humanhypothermia (32∘C) is associated with elevated frequencyof infectious complications dysfunction of the immuneresponse caused by hypothermia has been demonstratedin both clinical and animal studies [31] Contrary to ourhypothesis hyperthermia does not upregulate the expressionof DEFB1 or CAMP in a significant manner neverthelessthe strong tendency to upregulate mainly DEFB1 (data notshown) deserves further investigation

On the contrary we observed that hypothermia improvedthe potential of AA (20mM) both acting as APE at 24 hHowever in this combination both extreme conditions(hypo- and hyperthermia) diminished DEFB1 expressionat 72 h This could be because in these two scenarios thekeratinocytes are exposed to a prolonged oxidative stressprobably requiring more AA to counteract the adverse envi-ronment Notably the upregulation of DEFB1 with AA doesnot predict antimicrobial activity in vitro probably because inan amount of equal to or higher than 20mM we found thatAA is cytotoxic Also high oral doses of AA in vivo causehyperoxaluria which is the excessive urinary excretion ofoxalate and often the formation of kidney stones [10] Clinical

trials must uncover the optimal doses of AA in fever episodesof diverse infections to reach the expected efficacy of AA asAPEofDEFB1The role ofAAasAPEwas anticipated becauseAA reduces the expression of IL-10 in a dose-dependent way[32] and this cytokine inhibits the expression of hBD-1 [33]

According to our results IFNG mRNA levels probablyprovided the keratinocyte with an innate immunity againstskin pathogens independent of the late lymphocyte-derivedIFN-120574 (adaptive immunity) in an established infection [34]Also we found that IFN-120574 is upregulated earlier thanDEFB1 suggesting that the former could act as an APEin NHK of foreskin as is true in gingival keratinocytes[35 36] macrophages [37] and monocytes [38] The con-dition where IFNG and CAMP were upregulated was theonly one that predicts increment antimicrobial activity inthe two tested Gram-positive pathogens suggesting thatthis IFNG-dependent response is a stronger APE thanIFNG-independent response (eg TNF-120572-mediated) [6]Theantimicrobial in vitro prediction of CAMP upregulation is inconcordance with a recent demonstration that transfectingCAMP mRNA increases resistance to Listeria sp in oralepithelial cells [14]

In diabetic and obese rat models the expression of 120573-defensin 1 (BD-1) was lower than that of slim rat controls[39] and only 120573-defensin 2 was found to be increased indiabetic rats most likely as a result of a proinflammatoryresponse [40] Glucose has been described as an APE ofDEFB1 in human embryonic kidney HEK-293 [16 41] andcolon adenocarcinoma cells HCT-116 [16] but not for ker-atinocytes Our results suggest an impact of intracellularglucose deficiency in susceptibility to skin infections dueto DEFB1 downregulation for example ulcers in diabeticpatients We also demonstrate that glucose induces a fasterand higher expression of DEFB1 compared to CAMP innormal keratinocytes The glucose-dependent expression ofDEFB1 and CAMP has a biological relevance because theyshow constitutive skin expression probably explained bythe constant supply of glucose through blood in a non-starved state andor normal internalization of glucose in anondiabetic host Interestingly Barnea et al did not finddose-dependent response above 10mM of glucose in HEK-293 cells [16] as we found in NHK revealing a possiblehigher tissue-specific response Furthermore some authorsconsider that CAMP is an inducible gene However in oursystem of NHK from neonate foreskin it always showed aconstitutive expression as previously suggested in squamousepithelia of mouth tongue esophagus vagina and cervix[42]

UVA makes up 95 of UV light that reaches the earthrsquossurface [43] UVA inhibits cell proliferation due to the arrestof the S-phase [44] and also acts as an inhibitor of DEFB1in scleroderma lesional skin with no effect in unaffectedskin in these patients [11] On the contrary we found that inNHK UVA is a modest APE of DEFB1 but an antimicrobialpeptide inhibitor (API) at longer exposures These suggestthat in scleroderma lesions the potential of this APE could beaffected by an alteration of DEFB1 overexpression pathways[7 45] probably due to a unbalanced cytokine expressionprofile [6] The lack of upregulation of IFNG in the exposure


0

1

2

3

4

5

0 5 10 20

Fold

chan

geCA

MP

HPR

T

638 Jm2 UVC

lowastlowastlowast

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(a)

0

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2

3

4

5

6

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVC

lowastlowastlowast

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(b)

0

1

2

3

4

5

0 5 10 20

Fold

chan

geIF

NG

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(c)

0

02

04

06

08

1

12

14

16

18

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVA

lowastlowastlowast

lowastlowastlowast

lowastlowast

(d)

Figure 4 Relative gene expression in keratinocytes exposed toUVC (254 nm) andUVA (365 nm) (a)CAMP expression inUVCexposure (b)DEFB1 expression in UVC exposure (c) IFNG expression in UVC exposure (d)DEFB1 expression in UVA exposure Exposure is expressed inminutes In all cases reference group was 0min (not exposed) Error bars represent SEM and significant values of Studentrsquos 119905 tests are depictedas lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001

to UVA suggests that the route of DEFB1 expression withthis APE is IFN-120574-independent but this deserves furtherinvestigation

Our results reinforce and expand the notion that UV light(beyond UVA and UVB) stimulates APs gene expressionFurthermore we were able to induce upregulation of DEFB1and CAMP genes at 425 of energy and 0347 of exposuretime required to upregulate DEFB4 DEFB103 and S100A7

genes [46] In spite of the fact that in nature most of UVCis usually blocked by the stratospheric ozone layer [43] com-pact fluorescent light bulbs expose our skin to UVC as well asUVA [44]UVCcauses degradation of I120581B120572 andnuclear entryof p65RelA thus activating theNF-120581B pathway (independentof I120581B kinase-IKK-activation) [47] This nuclear factor is aprobable regulator of DEFB1 expression as suggested by ourgroup [48] We demonstrate for the first time that UVC in


37 35 37 37 37 3724 24 24 72 24 72

20 5 20455 455

00

05

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30

35

Tet

Inhi

bitio

n di

amet

er (c

m)

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

minus

minus minus minus minus

minus minus


Staphylococcus aureusListeria monocytogenes

Figure 5 Antimicrobial diffusion assays with the supernatantsof APEs-exposed keratinocytes Tet tetracycline was includedas a positive control (225 120583M 1mgmL) The negative controlwas supernatants of cells without elicitor (37∘C 24 h) Error barsrepresent SEM and significant values of Studentrsquos 119905 tests are depictedas lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001

short exposures can be useful as APE of CAMP and DEFB1even though it is well known that longer exposure to UVCcauses DNA damage [44]

5 Conclusion

Mild hypo- (35∘C) and hyperthermia (39∘C) are not APEsin NHK in vitro even when hyperthermia shows a tendencyto act as an APE with probable biological significancein spite of its not being statistically significant Moderateexposure to UVC upregulates CAMP and DEFB1 genes butUVA only upregulates the DEFB1 gene in HNK at 5min ofexposure UVA is an API of DEFB1 at longer exposures Thelater apparently downregulation could be explained becausemRNA of DEFB1 start degrading after 5min of exposure toUVA But this requires further investigation These resultssuggest alternative expression pathways or different induc-tion thresholds for DEFB1 and CAMP UVC is a previouslyunrecognized APE in human cells These findings uncoverthe potential of novel therapeutics in skin infectious diseases

Our results also suggest that glucose upregulates CAMPin an IFN-120574-independent manner and thus probably in aninflammation-independent manner AA is a more potentAPE for DEFB1 than glucose in NHK Glucose but not AAis an APE for CAMP AA-dependent DEFB1 upregulationbelow 20mMpredicts in vitro antimicrobial activity as well asglucose- and AA-dependent CAMP and IFNG upregulationLevels of serum glucose in diabetic patients could be usefulto predict susceptibility to infections due to diminished APsexpression but this requires further clinical studies AA as

APE will challenge the current concept of late activation ofadaptive immunity of this vitamin probably acting at thekeratinocyte level independent of adaptive immunity Thisknowledge could be useful to develop new potential drugsand devices to combat skin infections based on these APEsFuture research in classes I and II APEs holds great promise

Conflict of Interests

The authors declare that they are pursuing patents forthe methods and uses and when applicable the elicitorsinhibitors and derivatives or their combination generated inthe name of CIATEJ AC with E Prado Montes de Oca et alas inventorsTheprimers sequences alsowill be protected andshould be used only for academic purposes

Acknowledgments

This work was supported by Fondos Sectoriales de Cien-cia Basica SEP grant CB-2008-01-105813 from the NationalCouncil of Science and Technology (CONACYT Mexico)Ernesto Prado Montes de Oca and Marisela Gonzalez Avilaare Fellows of the Sistema Nacional de Investigadores (SNICONACYT) Thanks are due to Dr Jim Rheinwald (HarvardMedical School) for provision of his labrsquos protocols to cultivatekeratinocytes

References

[1] J-M Schroder ldquoThe role of keratinocytes in defense againstinfectionrdquo Current Opinion in Infectious Diseases vol 23 no 2pp 106ndash110 2010

[2] M H Braff M Zaiou J Fierer V Nizet and R L GalloldquoKeratinocyte production of cathelicidin provides direct activ-ity against bacterial skin pathogensrdquo Infection and Immunityvol 73 no 10 pp 6771ndash6781 2005

[3] M H Braff A Di Nardo and R L Gallo ldquoKeratinocytesstore the antimicrobial peptide cathelicidin in lamellar bodiesrdquoJournal of InvestigativeDermatology vol 124 no 2 pp 394ndash4002005

[4] J M Schroder and J Harder ldquoAntimicrobial skin peptides andproteinsrdquo Cellular and Molecular Life Sciences vol 63 no 4 pp469ndash486 2006

[5] E PradoMontes deOca ldquoThepotential of antimicrobial peptideinhibitors (APIs) and elicitors (APEs) in leprosy treatmentrdquoin Leprosy Epidemiology Treatment Strategies and CurrentChallenges in Research R S Koop Ed Nova PublishersHauppage NY USA 2014

[6] E Prado Montes de Oca ldquoAntimicrobial peptide elicitors newhope for the post-antibiotic erardquo Innate Immunity vol 19 no 3pp 227ndash241 2013

[7] E Prado Montes de Oca ldquoHuman 120573-defensin 1 a restless war-rior against allergies infections and cancerrdquo The InternationalJournal of Biochemistry amp Cell Biology vol 42 no 6 pp 800ndash804 2010

[8] E Prado-Montes de Oca ldquoAntimicrobial peptide elicitors apotential strategy against infectionsrdquo Gaceta Medica de Mexicovol 145 pp 241ndash243 2009

[9] K A Jhaveri R A Trammell and L A Toth ldquoEffect ofenvironmental temperature on sleep locomotor activity core


body temperature and immune responses of C57BL6J micerdquoBrain Behavior and Immunity vol 21 no 7 pp 975ndash987 2007

[10] R G Petersdorf R D Adams E Braunwald K J Isselbacher JBMartin and J DWilson Eds Principles of InternalMedicineMc Graw Hill New York NY USA 1983

[11] A Kreuter J Hyun M Skrygan et al ldquoUltraviolet A1-induceddownregulation of human 120573-defensins and interleukin-6 andinterleukin-8 correlates with clinical improvement in localizedsclerodermardquoThe British Journal of Dermatology vol 155 no 3pp 600ndash607 2006

[12] T Dai B Garcia C K Murray M S Vrahas and M R Ham-blin ldquoUVC light prophylaxis for cutaneous wound infections inmicerdquoAntimicrobial Agents andChemotherapy vol 56 no 7 pp3841ndash3848 2012

[13] J Turner Y Cho N-N Dinh A J Waring and R I LehrerldquoActivities of LL-37 a cathelin-associated antimicrobial peptideof human neutrophilsrdquo Antimicrobial Agents and Chemother-apy vol 42 no 9 pp 2206ndash2214 1998

[14] X Zou B S Sorenson K F Ross and M C HerzbergldquoAugmentation of epithelial resistance to invading bacteria byusing mRNA transfectionsrdquo Infection and Immunity vol 81 no11 pp 3975ndash3983 2013

[15] E S Wintergerst S Maggini and D H Hornig ldquoImmune-enhancing role of Vitamin C and zinc and effect on clinicalconditionsrdquo Annals of Nutrition and Metabolism vol 50 no 2pp 85ndash94 2006

[16] M Barnea Z Madar and O Froy ldquoGlucose and insulin areneeded for optimal defensin expression in human cell linesrdquoBiochemical and Biophysical Research Communications vol 367no 2 pp 452ndash456 2008

[17] D Allen E Winters P F Kenna P Humphries and G JFarrar ldquoReference gene selection for real-time rtPCR in humanepidermal keratinocytesrdquo Journal of Dermatological Science vol49 no 3 pp 217ndash225 2008

[18] M W Pfaffl ldquoA new mathematical model for relative quantifi-cation in real-time RT-PCRrdquoNucleic Acids Research vol 29 no9 article e45 2001

[19] W M Kirby D M Perry and A W Bauer ldquoTreatment ofstaphylococcal septicemia with vancomycin report of thirty-three casesrdquoThe New England Journal of Medicine vol 262 pp49ndash55 1960

[20] Y B Shaik-Dasthagirisaheb G Varvara G Murmura et alldquoRole of vitamins D e and C in immunity and inflammationrdquoJournal of Biological Regulators and Homeostatic Agents vol 27no 2 pp 291ndash295 2013

[21] H Kawada M Kaneko M Sawanobori et al ldquoHigh concentra-tions of L-ascorbic acid specifically inhibit the growth of humanleukemic cells via downregulation of HIF-1alpha transcriptionrdquoPLoS ONE vol 8 no 4 Article ID e62717 2013

[22] J Kim S-D Lee B Chang et al ldquoEnhanced antitumor activityof vitamin C via p53 in cancer cellsrdquo Free Radical Biology ampMedicine vol 53 no 8 pp 1607ndash1615 2012

[23] RAsada K KageyamaH TanakaMKimura Y Saitoh andNMiwa ldquoCarcinostatic effects of diverse ascorbate derivatives incomparison with aliphatic chain moiety structures promotionby combined hyperthermia and reduced cytotoxicity to normalcellsrdquo Oncology Letters vol 3 no 5 pp 1042ndash1046 2012

[24] C D Donald C Q Sun S D Lim et al ldquoCancer-specific lossof 120573-defensin 1 in renal and prostatic carcinomasrdquo LaboratoryInvestigation vol 83 no 4 pp 501ndash505 2003

[25] C Q Sun R Arnold C Fernandez-Golarz et al ldquoHuman120573-defensin-1 a potential chromosome 8p tumor suppressorcontrol of transcription and induction of apoptosis in renalcell carcinomardquo Cancer Research vol 66 no 17 pp 8542ndash85492006

[26] S A Altman L Renders and G Rao ldquoComparison of trypanblue dye exclusion and fluorometric assays for mammalian cellviability determinationsrdquo Biotechnology Progress vol 9 no 6pp 671ndash674 1993

[27] J A Plumb R Milroy and S B Kaye ldquoEffects of the pH depen-dence of 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide-formazan absorption on chemosensitivity determinedby a novel tetrazolium-based assayrdquoCancer Research vol 49 no16 pp 4435ndash4440 1989

[28] R I Freshney Culture of Animal Cells A Manual of BasicTechnique John Wiley amp Sons Hoboken NJ USA 5th edition2005

[29] V Pongor G Toldi M Szabo and B Vasarhelyi ldquoSystemicand immunomodulatory effects of whole body therapeutichypothermiardquoOrvosi Hetilap vol 152 no 15 pp 575ndash580 2011

[30] A Torossian E Gurschi R Bals T Vassiliou H F Wulf andA Bauhofer ldquoEffects of the antimicrobial peptide LL-37 andhyperthermic preconditioning in septic ratsrdquo Anesthesiologyvol 107 no 3 pp 437ndash441 2007

[31] S Russwurm I Stonans K Schwerter E StonaneWMeissnerand K Reinhart ldquoDirect influence of mild hypothermia oncytokine expression and release in cultures of human peripheralblood mononuclear cellsrdquo Journal of Interferon amp CytokineResearch vol 22 no 2 pp 215ndash221 2002

[32] M Bergman H Salman M Djaldetti L Fish I Punsky andH Bessler ldquoIn vitro immune response of human peripheralblood cells to vitamins C and Erdquo The Journal of NutritionalBiochemistry vol 15 no 1 pp 45ndash50 2004

[33] T Gambichler M Skrygan N S Tomi et al ldquoDifferentialmRNA expression of antimicrobial peptides and proteins inatopic dermatitis as compared to psoriasis vulgaris and healthyskinrdquo International Archives of Allergy and Immunology vol 147no 1 pp 17ndash24 2008

[34] K Schroder P J Hertzog T Ravasi and D A HumeldquoInterferon-120574 an overview of signals mechanisms and func-tionsrdquo Journal of Leukocyte Biology vol 75 no 2 pp 163ndash1892004

[35] S Joly C C Organ G K Johnson P B McCray Jr andJ M Guthmiller ldquoCorrelation between 120573-defensin expressionand induction profiles in gingival keratinocytesrdquo MolecularImmunology vol 42 no 9 pp 1073ndash1084 2005

[36] A A Kalus L P Fredericks B M Hacker et al ldquoAssociation ofa genetic polymorphism (-44 CG SNP) in the human DEFB1gene with expression and inducibility of multiple -defensins ingingival keratinocytesrdquoBMCOral Health vol 9 article 21 2009

[37] M Fabri S Stenger D-M Shin et al ldquoVitamin D is requiredfor IFN-gamma-mediated antimicrobial activity of humanmacrophagesrdquo Science Translational Medicine vol 3 no 104Article ID 104ra102 2011

[38] GMKlug-Micu S Stenger A Sommer and eatl ldquoCD40 ligandand interferon-120574 induce an antimicrobial response againstMycobacterium tuberculosis in human monocytesrdquo Immunol-ogy vol 139 no 1 pp 121ndash128 2013

[39] T Hiratsuka M Nakazato Y Date H Mukae and S Mat-sukura ldquoNucleotide sequence and expression of rat 120573-defensin-1 its significance in diabetic rodent modelsrdquo Nephron vol 88no 1 pp 65ndash70 2001


[40] O Froy A Hananel N Chapnik and Z Madar ldquoDifferentialeffect of insulin treatment on decreased levels of beta-defensinsand Toll-like receptors in diabetic ratsrdquoMolecular Immunologyvol 44 no 5 pp 796ndash802 2007

[41] A N Malik and G Al-Kafaji ldquoGlucose regulation of 120573-defensin-1 mRNA in human renal cellsrdquo Biochemical andBiophysical Research Communications vol 353 no 2 pp 318ndash323 2007

[42] M F Nilsson B Sandstedt O Soslashrensen G Weber NBorregaard and M Stahle-Backdahl ldquoThe human cationicantimicrobial protein (hCAP18) a peptide antibiotic is widelyexpressed in human squamous epithelia and colocalizes withinterleukin-6rdquo Infection and Immunity vol 67 no 5 pp 2561ndash2566 1999

[43] V Muthusamy and T J Piva ldquoThe UV response of the skina review of the MAPK NF120581B and TNF120572 signal transductionpathwaysrdquo Archives of Dermatological Research vol 302 no 1pp 5ndash17 2010

[44] T Mironava M Hadjiargyrou M Simon and M HRafailovich ldquoThe effects of UV emission from compactfluorescent light exposure on human dermal fibroblasts andkeratinocytes in vitrordquo Photochemistry and Photobiology vol88 no 6 pp 1497ndash1506 2012

[45] E Prado Montes de Oca ldquoDEFB1 (defensin beta 1)rdquo in Atlas ofGenetics andCytogenetics inOncology andHaematology 15 2011

[46] R Glaser F Navid W Schuller et al ldquoUV-B radiationinduces the expression of antimicrobial peptides in humankeratinocytes in vitro and in vivordquo The Journal of Allergy andClinical Immunology vol 123 no 5 pp 1117ndash1123 2009

[47] N Li and M Karin ldquoIonizing radiation and short wavelengthUV activate NF-120581B through two distinctmechanismsrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 95 no 22 pp 13012ndash13017 1998

[48] E Prado-Montes de Oca J S Velarde-Felix J J Rios-TostadoV J Picos-Cardenas and L E Figuera ldquoSNP 668C (-44) altersa NF-kappaB1 putative binding site in non-coding strand ofhuman beta-defensin 1 (DEFB1) and is associated with lepro-matous leprosy Infection genetics and evolutionrdquo Journal ofMolecular Epidemiology and Evolutionary Genetics in InfectiousDiseases vol 9 pp 617ndash625 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


neurosyphilis some forms of chronic arthritis and cancerFurthermore many infections cause fever during certainphases including fever caused by Rickettsia sp Chlamydiasp viruses or parasites When this fever is associated withendogen pyrogen it leads to the activation of T cells whichenhances the hostrsquos defense system [10]

UVA (320ndash400 nm) are APEs of DEFB1 in sclerodermaskin lesions [11] but they have not been tested in normalkeratinocytes UVC (180ndash280 nm) have been suggested as aprophylactic approach in a mice model of infection againstPseudomonas aeruginosa and Staphylococcus aureus [12] butthe function as an APE of this wavelength has not beendescribed S aureus is a very common human pathogensusceptible to LL-37 and hBD-1 [6] In the case of Listeriasp the pathogen is susceptible to LL-37 These APEs have atherapeutic potential against this pathogen in epithelial cells[13 14] In this study we indirectly assume that microbicidalactivity is due the increment of the level of synthesis of thesepeptides by the APEs as suggested by the mRNA levels andinhibition zones of the antimicrobial assays

AA has been widely used to prevent and treat the com-mon cold malaria and diarrhea infections and pneumoniaAA improves the efficacy of antimicrobial and natural killercell (NK) activities [15] Whether this antimicrobial activityis dependent on induction andor upregulation of APs isunknown Glucose is an APE of DEFB1 in kidney andcolon cells [16] but its effect on both DEFB1 and CAMP inkeratinocytes is unknown

In this study we assessed the capacity of UVA UVCAA glucose and hyperthermia to act as APEs of DEFB1 andCAMP genes and as inductors of IFNG in a cell line of neonatenormal human keratinocytes (NHK)

2 Materials and Methods

21 Cell Culture and Exposure to Potential APEs Normalhuman neonate keratinocytes (ATCC PCS-200-010) weremaintained in K-SFM cell culture media (Gibco) at 37∘C in ahumidified incubator with 5 CO

2 We exposed 1 times 106 cells

toD-(+)-glucose (55 155 255 and 455mM Sigma numberof catalog G8644) or L-ascorbic acid (0 5 10 and 20mMSigma number catalog A4544) for 24 48 and 72 h Theincubation temperature was also assessed as potential APEmimicking human mild hypothermia (35∘C) normothermia(37∘C) and hyperthermia (39∘C) in vitroAdditionally for theascorbic acid at 20mM the interaction between time (24 48and 72 h) and temperature (35 37 and 39∘C) was assessedThe exposition to UV rays (UVA at 365 nm UVC at 254 nm)was performed with a square source of irradiation of 4 Watts(UV lamp UVP-UVGL-25 UVP Cambridge UK number ofcatalog 95-0021-12) at 92 cm from the adhered cells

22 Quantitative Real-Time PCR (qPCR) Nucleic acids wereextracted in a MagNA Pure LC 20 Instrument (Roche) withthe MagNA Pure LC total nucleic acid kit (Roche catalognumber 03038505001) PurifiedRNAwasDNAse-treated andreverse transcribed with a mix containing random primers(Invitrogen) and ArrayScript enzyme (Applied Biosystems)

Quantitative real-time PCR was performed with 33 ng cDNAwith Fast SYBR Green Master Mix (Applied Biosystems)The reactions were run in a Step One Thermocycler(Applied Biosystems) using the following primerssequences designed with Primer Express v 30 software(Applied Biosystems) and tested for specificity in PRIMER-BLAST (httpwwwncbinlmnihgovtoolsprimer-blast)for HPRT (endogenous control suggested for normalhuman keratinocytes according to Allen et al [17]) F51015840-TGTTCAAATTATTACCAGTGAATCTTTGTC-31015840 R51015840 TTTTAAATTTTTGGGAATTTATTGATTTG-31015840 forIFNG F 51015840-GCTGACTAATTATTCGGTAACTGACTTG-31015840 R 51015840-TAGCTGCTGGCGACAGTTCA-31015840 for DEFB1F 51015840-GAGAACTTCCTACCTTCTGCTGTTTAC-31015840R 51015840-AAAGTTACCACCTGAGGCCATCT-31015840 forCAMP F 51015840-ACCCAGACACGCCAAAGC-31015840 R 51015840-TTCACCAGCCCGTCCTTCT-31015840 The PCR program was95∘C for 20 s 40 cycles of denaturing at 95∘C for 3 s andannealingextension at 60∘C for 30 s Melting curves wereperformed at 95∘C for 15 s 60∘C for 1min and 95∘C for 15 sAmplification efficiencies for each gene were calculated andthe relative quantitative expressionwas obtained according tothe Pfaffl method [18] The normalization of gene expressionincluded the input RNA and input cell count The controlswere samples with the vehicle at 24 h or in the case oftemperatures the normothermic sample (37∘C) for each day

23 Antimicrobial Assays Diffusion assays were performedaccording to Kirby et al [19] We tested culture supernatantssamples with a volume of 50120583L poured into 6mm diameterwells The negative control was the supernatant of the culturemedium without the potential APE and the positive controlwas 225120583M (1mgmL) of tetracycline Three biologicalreplicas of each were performed with strains of Listeriamonocytogenes (ATCC 19114 susceptible to LL-37 [13 14])and Staphylococcus aureus (ATCC 25923 susceptible to LL-37 and hBD-1) [6]

24 Cytotoxicity Assays In the experimental conditionswhere gene upregulation was observed experiments wererepeated and cytotoxicity assays were performed in increas-ing concentrations of the APEsThis was assessed by applying005 trypsin by 12min and neutralizing with DMEM (10fetal bovine serum) After centrifugation (400timesg 5min)three 10 120583L aliquots of homogeneous precipitated cells werequantified in a Neubauer chamber The percentage of viablecells adhered to the plate was compared with the controlwithout elicitor considered as being 100 viable Quantifi-cations of each day were compared with the control of thecorresponding days (Cruz Dıaz et al in process)

25 Statistical Analysis Statistical comparisons were done inMicrosoft Excel 2010 To evaluate if variances were differentor equal among treatments we performed119865 tests Dependingof the results of 119865 tests Studentrsquos 119905-tests for equal or nonequalvariances were performed Results are shown as mean +standard error of the mean (SEM) All experiments were


0

50

100

150

200

250

300

350

400Vi

abili

ty (

)lowastlowastlowast

lowastlowastlowast


(a)

01

1

10

100

1000

10000

24 48 72

(h)

55mM155mM

255mM455mM

Fold

chan

geD

EFB1

HPR

T

lowast

(b)



3 Results








4 Discussion




O O

HO

HO

OH

OH

H

(a)

0

20

40

60

80

100

120

140

0 10 20 40

Viab

ility

()

(mM)

lowast

lowastlowast

(b)

001

01

1

10

100

1000

10000

100000

1000000

24 48 72

lowast

lowast

(h)

Fold

chan

geD

EFB1

HPR

T

0mM5mM

10mM20mM

(c)

01

1

10

100

lowast

lowast lowast

Fold

chan

geD

EFB1

HPR

T

35 37 39

∘C)24h48h

72h(

(d)






37 37 37 35 37 35 3724 72 72 72 72 72 725 10 20 20 20

255 455

0

5

10

15

20

25

30

35

40Fo

ld ch

ange

HPR

T

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowast

lowast


IFNG CAMP

minus minus minus

minus

minus

minus

minus









0

1

2

3

4

5

0 5 10 20

Fold

chan

geCA

MP

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(a)

0

1

2

3

4

5

6

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(b)

0

1

2

3

4

5

0 5 10 20

Fold

chan

geIF

NG

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(c)

0

02

04

06

08

1

12

14

16

18

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVA

lowastlowastlowast

lowastlowastlowast

lowastlowast

(d)






37 35 37 37 37 3724 24 24 72 24 72

20 5 20455 455

00

05

10

15

20

25

30

35

Tet

Inhi

bitio

n di

amet

er (c

m)

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

minus


minus minus





5 Conclusion






Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

50

100

150

200

250

300

350

400Vi

abili

ty (

)lowastlowastlowast

lowastlowastlowast


(a)

01

1

10

100

1000

10000

24 48 72

(h)

55mM155mM

255mM455mM

Fold

chan

geD

EFB1

HPR

T

lowast

(b)



3 Results








4 Discussion




O O

HO

HO

OH

OH

H

(a)

0

20

40

60

80

100

120

140

0 10 20 40

Viab

ility

()

(mM)

lowast

lowastlowast

(b)

001

01

1

10

100

1000

10000

100000

1000000

24 48 72

lowast

lowast

(h)

Fold

chan

geD

EFB1

HPR

T

0mM5mM

10mM20mM

(c)

01

1

10

100

lowast

lowast lowast

Fold

chan

geD

EFB1

HPR

T

35 37 39

∘C)24h48h

72h(

(d)






37 37 37 35 37 35 3724 72 72 72 72 72 725 10 20 20 20

255 455

0

5

10

15

20

25

30

35

40Fo

ld ch

ange

HPR

T

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowast

lowast


IFNG CAMP

minus minus minus

minus

minus

minus

minus









0

1

2

3

4

5

0 5 10 20

Fold

chan

geCA

MP

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(a)

0

1

2

3

4

5

6

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(b)

0

1

2

3

4

5

0 5 10 20

Fold

chan

geIF

NG

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(c)

0

02

04

06

08

1

12

14

16

18

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVA

lowastlowastlowast

lowastlowastlowast

lowastlowast

(d)






37 35 37 37 37 3724 24 24 72 24 72

20 5 20455 455

00

05

10

15

20

25

30

35

Tet

Inhi

bitio

n di

amet

er (c

m)

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

minus


minus minus





5 Conclusion






Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


O O

HO

HO

OH

OH

H

(a)

0

20

40

60

80

100

120

140

0 10 20 40

Viab

ility

()

(mM)

lowast

lowastlowast

(b)

001

01

1

10

100

1000

10000

100000

1000000

24 48 72

lowast

lowast

(h)

Fold

chan

geD

EFB1

HPR

T

0mM5mM

10mM20mM

(c)

01

1

10

100

lowast

lowast lowast

Fold

chan

geD

EFB1

HPR

T

35 37 39

∘C)24h48h

72h(

(d)






37 37 37 35 37 35 3724 72 72 72 72 72 725 10 20 20 20

255 455

0

5

10

15

20

25

30

35

40Fo

ld ch

ange

HPR

T

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowast

lowast


IFNG CAMP

minus minus minus

minus

minus

minus

minus









0

1

2

3

4

5

0 5 10 20

Fold

chan

geCA

MP

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(a)

0

1

2

3

4

5

6

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(b)

0

1

2

3

4

5

0 5 10 20

Fold

chan

geIF

NG

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(c)

0

02

04

06

08

1

12

14

16

18

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVA

lowastlowastlowast

lowastlowastlowast

lowastlowast

(d)






37 35 37 37 37 3724 24 24 72 24 72

20 5 20455 455

00

05

10

15

20

25

30

35

Tet

Inhi

bitio

n di

amet

er (c

m)

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

minus


minus minus





5 Conclusion






Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


37 37 37 35 37 35 3724 72 72 72 72 72 725 10 20 20 20

255 455

0

5

10

15

20

25

30

35

40Fo

ld ch

ange

HPR

T

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowast

lowast


IFNG CAMP

minus minus minus

minus

minus

minus

minus









0

1

2

3

4

5

0 5 10 20

Fold

chan

geCA

MP

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(a)

0

1

2

3

4

5

6

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(b)

0

1

2

3

4

5

0 5 10 20

Fold

chan

geIF

NG

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(c)

0

02

04

06

08

1

12

14

16

18

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVA

lowastlowastlowast

lowastlowastlowast

lowastlowast

(d)






37 35 37 37 37 3724 24 24 72 24 72

20 5 20455 455

00

05

10

15

20

25

30

35

Tet

Inhi

bitio

n di

amet

er (c

m)

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

minus


minus minus





5 Conclusion






Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1

2

3

4

5

0 5 10 20

Fold

chan

geCA

MP

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(a)

0

1

2

3

4

5

6

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(b)

0

1

2

3

4

5

0 5 10 20

Fold

chan

geIF

NG

HPR

T

638 Jm2 UVC

lowastlowastlowast

lowastlowastlowast

(c)

0

02

04

06

08

1

12

14

16

18

0 5 10 20

Fold

chan

geD

EFB1

HPR

T

638 Jm2 UVA

lowastlowastlowast

lowastlowastlowast

lowastlowast

(d)






37 35 37 37 37 3724 24 24 72 24 72

20 5 20455 455

00

05

10

15

20

25

30

35

Tet

Inhi

bitio

n di

amet

er (c

m)

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

minus


minus minus





5 Conclusion






Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


37 35 37 37 37 3724 24 24 72 24 72

20 5 20455 455

00

05

10

15

20

25

30

35

Tet

Inhi

bitio

n di

amet

er (c

m)

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

minus


minus minus





5 Conclusion






Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Ascorbic Acid, Ultraviolet C Rays, and ...

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Transcript of Research Article Ascorbic Acid, Ultraviolet C Rays, and ...