vitamin d modulates hematological parameters and cell...

Research ArticleVitamin D Modulates Hematological Parameters andCell Migration into Peritoneal and Pulmonary Cavities inAlloxan-Diabetic Mice

LeonardoM Bella1 Isis Fieri1 Fernando H G Tessaro1 Eduardo L Nolasco1

Fernanda P B Nunes1 Sabrina S Ferreira1 Carolina B Azevedo2 and Joilson O Martins1

1Laboratory of Immunoendocrinology Department of Clinical and Toxicological Analyses Faculty of Pharmaceutical SciencesUniversity of Sao Paulo (FCFUSP) Sao Paulo SP Brazil2Department of Medicine Rheumatology Division Universidade Federal de Sao Paulo Sao Paulo SP Brazil

Correspondence should be addressed to Joilson O Martins martinsjuspbr

Received 23 January 2017 Accepted 21 March 2017 Published 19 April 2017

Academic Editor Veronika A Myasoedova

Copyright copy 2017 Leonardo M Bella et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

BackgroundAims The effects of cholecalciferol supplementation on the course of diabetes in humans and animals need to bebetter understood Therefore this study investigated the effect of short-term cholecalciferol supplementation on biochemical andhematological parameters inmiceMethodsMale diabetic (alloxan 60mgkg iv 10 days) and nondiabeticmicewere supplementedwith cholecalciferol for seven daysThe following parameters were determined serum levels of 25-hydroxyvitamin D phosphoruscalcium urea creatinine alanine aminotransferase aspartate aminotransferase alkaline phosphatase red blood cell countwhite blood cell count (WBC) hematocrit hemoglobin differential cell counts of peritoneal lavage (PeL) and bronchoalveolarlavage (BAL) fluids and morphological analysis of lung kidney and liver tissues Results Relative to controls cholecalciferolsupplementation increased serum levels of 25-hydroxyvitamin D calcium hemoglobin hematocrit and red blood cell counts anddecreased leukocyte cell counts of PeL and BAL fluids in diabetic mice Diabetic mice that were not treated with cholecalciferol hadlower serum calcium and albumin levels and hemoglobin WBC and mononuclear blood cell counts and higher serum creatinineand urea levels than controls Conclusion Our results suggest that cholecalciferol supplementation improves the hematologicalparameters and reduces leukocyte migration into the PeL and BAL lavage of diabetic mice

1 Introduction

Type 1 diabetes mellitus (T1D) is an autoimmune disorderthat causes destruction of pancreatic 120573 cells and insulitisresulting in loss of insulin secretion [1]

Several studies have demonstrated that diabetic individu-als with uncontrolled hyperglycemia may experience weightloss uncontrolled immune response hematological alter-ations and lung liver and kidney injuries [2 3] In additionrenal impairment in diabetes may affect the metabolism ofurea creatinine [4] hemoglobin (Hb) [5] albumin [6] andvitamin D [7 8]

Anemia is defined as a decrease in the amountof hemoglobin (Hb) or the number of red blood cells

(RBC) in the blood It is a common complication in patientswith diabetic kidney disease and it increases mortality indiabetic individuals but its mechanism of action is still notclear [9]

Albumin is the major plasma protein and plays a criticalrole in maintaining tissue oncotic pressure and in the trans-port of substances such as vitamin D into the bloodstreamStudies have suggested that hepatic albumin synthesis andsecretion are reduced in diabetic rats [10]

Vitamin D is a hormone obtained from the diet andorby endogenous synthesis The vitamin Dmolecule is hydrox-ylated twice once in the liver by the enzyme 25ndashhydroxylaseand once in the kidney by the enzyme 1120572ndashhydroxylase toform calcitriol (125(OH)2D) which is the form of vitamin

HindawiBioMed Research InternationalVolume 2017 Article ID 7651815 10 pageshttpsdoiorg10115520177651815

2 BioMed Research International

D that interacts with vitamin D receptor (VDR) to performits biological activity [7]

The classic activity of calcitriol is to regulate bonemetabolism by maintaining a balance between calcium (Ca)and phosphorus (P) concentrations [8] Studies have reportedthat vitaminDmay affect the course of T1D by immunomod-ulation [11] Although the mechanism of action is stillunknown serum 125(OH)2D levels appear to modulate thelevel of systemic cytokine production and to increase whiteblood cell (WBC) count [12 13]

Although vitamin D supplementation is an importantaid in clinical practice few studies have demonstrated theeffects of vitamin D supplementation on the course of T1Din humans and animals In addition vitamin D supple-mentation studies are often contradictory due to the lackof precise information on regimen and dosage protocols[14]

Thus using a well-established mouse model of T1Dwe investigated the effect of vitamin D supplementationon histological biochemical and hematological parametersand on bronchoalveolar lavage (BAL) and peritoneal lavage(PeL) fluids in male (C57BL6) diabetic and nondiabeticmice The hypothesis is that vitamin D supplementationimproves the hematological parameters and reduces inflam-matory cell migration into the PeL and BAL fluid of diabeticmice

2 Materials and Methods

21 Animals Thirty-one specific pathogen-free maleC57BL6 mice weighing 25 plusmn 2 g at baseline were used Theanimals were maintained at 22∘C under a 12 h light-darkcycle Food and water were provided ad libitum before andduring the experimental period This study was conductedin strict accordance with the principles and guidelines of theNational Council for the Control of Animal Experimentation(CONCEA) and approved by the Ethics Committee onAnimal Use (CEUA) at the School of PharmaceuticalSciences (FCF) University of Sao Paulo Brazil (protocolnumber CEUAFCF389) Surgery was performed underketaminexylazine anesthesia and all efforts were made tominimize animal suffering

22 Induction of Diabetes Mellitus Animals were sepa-rated in four groups control (C) control supplementedwith vitamin D (CV) diabetic (D) and diabetic supple-mented with vitamin D (DV) Briefly diabetes mellituswas induced by intravenous injection of 60mgkg alloxanmonohydrate (ALX) (Sigma Chemical Co St Louis MOUSA) dissolved in physiological saline (09 NaCl) Controlmice were injected with physiological saline only Ten daysafter diabetes induction body weight was measured andperipheral blood (tail vein) glucose level was determinedusing an Accu-Chek Advantage II blood glucose monitor(Roche Diagnostica Sao Paulo SP Brazil) Animals wereconsidered diabetic if glucose level was above 300mgdL[15]

23 Vitamin D Supplementation Animals in the CV andDV groups were supplemented orally with 800 IU of vitaminD (Sanofi Aventis Sao Paulo SP Brazil) for seven days[14] The last dose of vitamin D was administered 24 hbefore the experiment to minimize stress-induced endocrinechanges caused by oral administration of the hormone[16] Body weight and blood glucose were measured onthe first fourth and seventh day of the supplementationperiod

24 Blood Collection Animals were anesthetized by anintraperitoneal injection of 10mgkg xylazine hydrochloride(Ceva Sante Animale Paulınia SP Brazil) and 90mgkgketamine hydrochloride (Ceva Sante Animale) Blood wascollected from anesthetized animals by cardiac punctureSamples of EDTA-anticoagulated blood were used to deter-mine the following hematological parameters red bloodcell (RBC) count white blood cell (WBC) count hemat-ocrit (Htc) and hemoglobin (Hb) All analyses were per-formed using a veterinary hematology analyser ABC Vet(HORIBA UK) Differential cell counts were determinedon stained slides under oil immersion microscopy Based onmorphological criteria 100 cells per sample were countedand classified as either mononuclear or polymorphonuclearA sample aliquot without anticoagulant was centrifuged(10min 3500 rpm) at room temperature Next the serumwasseparated and used for measuring ionic calcium (Ca) phos-phorus (P) albumin alanine transaminase (ALT) aspartateaminotransferase (AST) alkaline phosphatase (ALP) ureaand creatinine by colorimetric assay according to the man-ufacturerrsquos protocol (LabTest Diagnostica Lagoa Santa MGBrazil) Serum 25(OH)D was measured by EUROIMMUN25ndashOHndashVitamin D ELISA kit (Luebeck Germany) Serumsamples were stored at minus80∘C

25 Peritoneal and Bronchoalveolar Lavage The abdomenof all mice was exposed through a ventral midline incisionPeritoneal lavage (PeL)was performedby instillation of 10mL(two 5mL infusions) of phosphate buffered saline (PBS137mM NaCl 27mM KCl 43mM Na2HPO4 and 147mMKH2PO4 pH 74) at room temperature [17] The tracheawas exposed through a midline ventral incision in the neckBronchoalveolar lavage (BAL) was performed by instillationof 5mL (five 1mL infusions) of PBS at room temperaturethrough a 16G times 18810158401015840 BD Angiocath polyethylene tube(BD Franklin Lakes NJ USA) inserted into the tracheaNext PeL and BAL samples were centrifuged (1500 rpm10min 4∘C) supernatants were discarded and the cellswere resuspended in PBS (1mL) The cell suspension wasdiluted 1 2 (v v) with Turkrsquos solution Total cell counts weredetermined using a Neubauer chamber and differential cellcounts were examined on stained slides under oil immersionmicroscopy A total of 100 cells were counted and classifiedas neutrophils lymphocytes or macrophages based on mor-phological criteria

26 Tissue Extraction andHistological Analysis Kidney liverand lung samples were extracted and subsequently fixed

BioMed Research International 3

in formaldehyde solution (10) After fixation tissues weredehydrated in increasing ethanol concentrations (70ndash100)diaphonized in xylol and embedded in paraffin Transversesections (5 120583m) obtained after inclusion were stained withhematoxylin and eosin (HE) After staining the materialwas dehydrated diaphonized and mounted with EntellanSlides containing the tissue were observed under a lightmicroscope (Nikon Eclipse 80i Tokyo Japan) and pho-tographed using the NIS-Elements AR imaging software(Nikon)

27 Data and Statistical Analysis The data were analysedby analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparisons test (when appropriate) orunpaired 119905-test using GraphPad Prism 60 software (LaJolla CA USA) A two-tailed 119901 value with 95 confidenceinterval was computed Data are presented as mean plusmn stan-dard deviation (SD) Data were considered significant when119901 lt 005

3 Results

31 Body Weight Gain and Blood Glucose Levels Ten daysafter administration of ALX diabetic mice exhibited sharplyelevated blood glucose (mean plusmn SD control 1656 plusmn57mgdL 119899 = 18 diabetic 5681 plusmn 100mgdL 119899 = 13 119901 lt0001) levels and a reduction in body weight gain (control08 plusmn 03 g 119899 = 18 diabetic minus13 plusmn 04 g 119899 = 13 119901 lt 005)compared to controls

32 Analysis of Vitamin D Supplementation Animals supple-mented with cholecalciferol (CV and DV groups) exhibitedhigher serum 25(OH)2D (ngmL) levels than their respectivecontrols (C andD groups)These results indicate that vitaminD supplementation was successful (Figure 1(a))

Diabetic mice had lower serum Ca (mgdL) levels thannondiabetic animals However vitamin D supplementationrestored serum Ca levels in the DV group (Figure 1(b))

Serum phosphorus (mgdL) levels were significantlyhigher in vitamin D-treated nondiabetic mice compared tonondiabetic controls (Figure 1(c))

33 Temporal Variation of Blood Glucose Levels and BodyWeight Body weight gain and blood glucose levels weremeasured during the supplementation period Body weightwasmeasured in the first and seventh days of the experimentThere was no significant difference in body weight variationbetween vitamin D3-supplemented animals (CV 01 plusmn 03 g119899 = 9 DV minus13 plusmn 10 g 119899 = 7) and their respective controls(C 03 plusmn 03 g 119899 = 9 D minus11 plusmn 09 g 119899 = 6) Bloodglucose was measured on the first fourth and seventhday of the experimental period (Figure 1(j)) There was nosignificant difference in glycemic evolution between vitaminD3-supplemented (CV and DV) groups and controls (Cand D groups) However blood glucose levels remainedsignificantly higher in diabetic mice (D and DV groups) thanin controls (C and CV)

34 Vitamin D3 Supplementation Improved HematologicalParameters Diabetic mice had lower RBC Hb Htc WBCand peripheral blood mononuclear cell counts than controls(Figure 2) Diabetic mice supplemented with vitamin D3had higher RBC Hb Htc WBC and mononuclear cellcounts than diabetic mice In addition diabetic mice sup-plemented with vitamin D3 had higher Hb and hematocritlevels than nondiabetic mice supplemented with vitamin D3(Figure 2)

35 Vitamin D3 Supplementation in the Kidneys and LiverDiabetic mice had significantly higher serum levels of urea(Figure 1(d)) and creatinine than controls (Figure 1(e)) Inaddition diabetic mice had a significant reduction in serumalbumin concentration compared to controls (Figure 1(f))However there were no significant differences in serum levelsofALPAST andALT (Figures 1(g)ndash1(i)) between vitaminD3-treated mice and their respective controls Compared to con-trols diabetic mice exhibited a thickening of the Bowmanrsquoscapsule However vitaminD supplementation did not restorethis renal parameter (Figure 3) No significant differencesin liver (Figure 4) and lung (Figure 5) morphology wereobserved between vitamin D3-supplemented animals (CVand DV) and their respective controls

36 Cell Composition of Bronchoalveolar Lavage and Peritone-al Lavage Fluids The PeL fluid of vitamin D3-supplementedmice had 49-fold more neutrophils than respective controlsbut no significant difference in total cell counts was observedacross groups Total leukocyte counts in the PeL fluid werereduced by 47 in vitamin D3-treated diabetic mice com-paredwith diabeticmice Similarly a 57-fold decrease in totalleukocyte count was observed in the BAL fluid of vitaminD3-treated diabetic mice (Table 1)

4 Discussion

Alloxan (ALX) is an effective diabetes-inducing agent widelyused in experimental animal models ALX administrationproduces hyperglycemia which leads to polyuria and weightloss [18] In our study blood glucose and body weight weremeasured 10 days after ALX administration During theexperimental period diabetic mice exhibited lower bodyweight gain and hyperglycemia than nondiabetic miceBoth metabolic alterations may occur due to increasedlipolysis and oxidative degradation of amino acids thatincrease tissue energy expenditure and body weight loss[19]

Few studies have demonstrated the effects of vitamin D3supplementation on T1D in animal models Neverthelessresults are controversial due to the use of different animalstrains protocols doses periods and routes of supplemen-tation In our study diabetic and nondiabetic mice sup-plemented with 800 IU (40000 IUkg body weightday) ofvitamin D3 had higher serum 25(OH)2D levels than controlsAccording to Takiishi et al (2014) this dose causes nosignificant alterations in serum P or Ca levels in nonobesediabetic (NOD) mice [14]


C CV D DV

lowast

lowast

0

60

120

18025

(OH

)D (n

gm

L)

(a)C CV D DV

0

4

8

12

Calc

ium

(mg

dL)

(b)

C CV D DV

0

5

10

15

Phos

phor

us (m

gdL

)

(c)C CV D DV

0

50

100

150

Ure

a (m

gdL

)

⦁

(d)

000

025

050

075

C CV D DV

Crea

tinin

e (m

gdL

)

lowast

lowast

(e)C CV D DV

0

1

2

3

Alb

umin

(gd

L)

(f)

C CV D DV0

200

400

600

ALP

(UL

)

(g)C CV D DV

0

45

90

135

ALT

(UL

)

(h)

Figure 1 Continued


C CV D DV0

100

200

300A

ST (U

L)

(i)

Day 1 Day 3 Day 7

lowast

lowastlowast

lowast

lowast

lowast

0

250

500

750

Gly

cem

ia (m

gdL

)

CCV

DDV

(j)

Figure 1 Biochemical and vitamin D levels Data are expressed as mean and standard deviation for 3ndash9 animals per group 119901 lt 005e119901 lt 001 and lowast119901 gt 0001 25(OH)D 25-hydroxyvitamin D ALP alkaline phosphatase ALT alanine transaminase AST aspartateaminotransferase C control CV vitamin D D diabetic DV diabetic + vitamin D

Table 1 Quantification of pulmonary and peritoneal leukocytes

White cells (105 cellsmL)Site Group 119873 Total Macrophage Lymphocyte Neutrophil

Peritoneum

Control 9 112 plusmn 28 81 plusmn 23 295 plusmn 00 01 plusmn 00Vitamin D 7 160 plusmn 32 120 plusmn 22 34 plusmn 15 05 plusmn 01lowast

Diabetic 7 187 plusmn 30 148 plusmn 32 36 plusmn 08 02 plusmn 01Diabetic + vitamin D 7 88 plusmn 21 76 plusmn 17 10 plusmn 05 03 plusmn 02

Lung

Control 3 46 plusmn 20 35 plusmn 19 09 plusmn 06 02 plusmn 01Vitamin D 4 91 plusmn 40 68 plusmn 24 20 plusmn 15 03 plusmn 02Diabetic 3 145 plusmn 40 115 plusmn 21 25 plusmn 14 09 plusmn 06

Diabetic + vitamin D 3 21 plusmn 07 199 plusmn 05 01 plusmn 01 01 plusmn 00Mice were rendered diabetic by the injection of alloxan (60mgkg iv) 10 days before vitamin D supplementation (800 IUday ov) BAL and PeL wereperformed 24 h thereafter Values are expressed as means plusmn SDlowast119901 lt 001 versus corresponding values in control group119901 lt 005 versus corresponding values in diabetic group

The role of vitamin D3 in body weight control is con-troversial The presence of 1120572ndashhydroxylase and VDR inadipose tissue cells suggests the involvement of vitamin D inbody weight regulation [20] Weight loss has been associatedwith Ca metabolism which is regulated by Vitamin D3The increase in Ca availability enhances oxidation promotesapoptosis of adipocytes and reduces the absorption of lipidsdue to the formation of insoluble molecules in the intestine[21] However Takiishi et al found no difference in bodyweight variation between vitamin D3-treated NODmice andcontrols [14] Similarly in our study there was no significantdifference in body weight variation between vitamin D3-treatedmice and controls during the supplementation period

The effects of vitamin D3 supplementation on glycemiccontrol in animal models are controversial Relative tocontrols NOD mice treated with high doses of calcitriol

(5 120583gkgalternate days) showed lower insulitis and a reduc-tion in T cell counts [22] However vitamin D supple-mentation had little effect in reverting overt diabetes instreptozotocin-induced diabetic mice [23] In the currentstudy vitamin D supplementation did not reduce bloodglucose levels in diabetic mice compared to controls

Themajor role of vitaminD3 in the body is to regulate theconcentrations of Ca and P [8] Few studies have focused onhow vitamin D and its analogues may affect Ca homeostasisin T1D [24 25] In addition diabetic individuals can exhibitlow serum Ca and P levels due to renal disorders [26] Inthe current study serum concentrations of these ions weremeasured Diabetic mice had lower serum Ca levels thancontrols and vitamin D3 supplementation increased theselevels in the former High 125(OH)2D levels stimulate boneturnover renal excretion and intestinal Ca absorption [7]


C CV D DV

lowast lowast

0

5

10

15

106

cells

ＧＧ

3)

Red

bloo

d ce

ll co

unt (

(a)C CV D DV

lowast

0

7

14

21

Hem

oglo

bin

(gd

L)

(b)

C CV D DV

lowast

0

25

50

75

Hem

atoc

rit (

)

(c)C CV D DV

00

16

32

48

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(d)

C CV D DV

PolymorphonuclearMononuclear

00

14

28

42

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(e)

Figure 2Hematological parameters Data are expressed asmean and standard deviation for 3ndash9 animals per group 119901 lt 005 and lowast119901 gt 0001C control CV vitamin D D diabetic DV diabetic + vitamin D


C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3

Figure 3 Renal tissue of mice Photomicrograph of the mice kidney tissue C1 C2 and C3 renal tissue of animal control CV1 CV2 andCV3 renal tissue of animal supplemented with vitamin D D1 D2 and D3 renal tissue of diabetic animal DV1 DV2 and DV3 renal tissue ofdiabetic animal supplemented with vitamin D Diabetic animals presented thickening of Bowmanrsquos capsule (C) Presence of glomerulus (G)(bars C1 CV1 D1 and DV1 = 100 120583m C2 CV2 D2 and DV2 = 50 120583m C3 CV3 D3 and DV3 = 10 120583m) Staining HE

However serum P levels in our study were significantlyhigher in vitamin D3-treated nondiabetic mice than controlsThere was no significant difference in P levels betweenvitamin D3-treated and control diabetic mice

Vitamin D is transported by albumin and vitamin D-binding protein in the bloodstream to the liver and kidneyswhere it is activated [7 8] In the current study liver enzymes(ALP ALT and AST) were measured to determine organdysfunction in diabetic and nondiabetic mice The concen-trations of serum ALT AST and ALP were not significantlydifferent between mice supplemented with vitamin D andcontrols Measuring serum urea and creatinine levels is analternative approach to estimate renal function [27] In ourstudy diabetic mice showed low albumin levels and highserum levels of both creatinine and urea and a thickening ofthe Bowmanrsquos capsule compared to nondiabetic mice Theseresults may indicate renal failure [4] However vitamin Dsupplementation did not restore these renal parameters

Alterations in hematological parameters are commonin diabetes patients [28] Hyperglycemia-induced oxidativestress in diabetic conditions leads to functional and mor-phological alterations of the erythrocyte membrane [29]Theincreased nonenzymatic glycosylation of RBC membraneproteins and hyperglycemia could be responsible for thelow Hb levels in these patients [30] Hyperglycemia andoxidation of these proteins increase the production of lipidperoxides that lead to hemolysis of RBC [31] In our studydiabetic mice had lower RBC counts and Hb and Htc levelsthan controls Conversely vitamin D3-treated diabetic miceshowed RBC counts and Htc and Hb levels similar to thoseof nondiabetic mice These findings suggest that vitaminD may affect erythropoiesis Some studies have shown thatVDR is expressed in the bone marrow by specific cell subsetssuch as stromal and accessory cells [32] In addition Aucellaet al reported that patients with chronic kidney diseaseundergoing hemodialysis showed a significant increase in Hb


C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3

Figure 4 Liver tissue of mice Photomicrograph of the mice liver tissue C1 C2 and C3 liver tissue of animal control CV1 CV2 and CV3liver tissue of animal supplemented with vitamin D D1 D2 and D3 liver tissue of diabetic animal DV1 DV2 and DV3 liver tissue of diabeticanimal supplemented with vitamin D Liver sinusoids (S) (Bars C1 CV1 D1 and DV1 = 100120583m C2 CV2 D2 and DV2 = 50 120583m C3 CV3D3 and DV3 = 10120583m) Staining HE

concentration and Htc level after four months of treatmentwith vitamin D [33]

Increased WBC counts are common in acute andchronic diabetes complications [34] However Matough et alobserved lowerWBC counts in diabetic mice as compared tonondiabetic animals [30] In our study diabetic mice showedlower mononuclear cell and WBC counts than control miceConversely vitamin D3 treatment restored mononuclear cellandWBC counts on diabetic mice to levels similar to those ofnondiabetic mice Interestingly vitamin D3-treated diabeticmice showed a reduction in pulmonary and peritonealleukocyte counts compared to diabetic animals Some studieshave shown that VDR expression on leukocyte subsets [35]could increase leukocytemigration from tissues to peripheralblood

Taken together our results suggest that vitamin D sup-plementation may improve hematological parameters and

reduce cell counts of BAL and PeL fluids during the courseof diabetes

Conflicts of Interest

The authors declare that there are no conflicts of interest thatwould prejudice the impartiality of this scientific work

Authorsrsquo Contributions

Leonardo M Bella and Joilson O Martins conceived anddesigned the experiments Leonardo M Bella Isis FieriFernando H G Tessaro Eduardo L Nolasco Fernanda P BNunes and Sabrina S Ferreira performed the experimentsLeonardo M Bella Fernanda P B Nunes Carolina BAzevedo and Joilson OMartins analysed the data Joilson O


C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3

Figure 5 Lung tissue of mice Photomicrograph of the mice lung tissue C1 C2 and C3 lung tissue of animal control CV1 CV2 and CV3lung tissue of animal supplemented with vitamin D D1 D2 and D3 lung tissue of diabetic animal DV1 DV2 and DV3 lung tissue of diabeticanimal supplemented with vitamin D Lung alveolus (A) bronchus (B) bronchiole (Br) (Bars C1 CV1 D1 and DV1 = 100120583m C2 CV2 D2and DV2 = 50 120583m C3 CV3 D3 and DV3 = 10120583m) Staining HE

Martins contributed with reagentsmaterialsanalysis toolsLeonardo M Bella Carolina B Azevedo and Joilson OMartins wrote the paper with the assistance of all the authors

Acknowledgments

The authors would like to sincerely thank Mariana C FSilva for their expert technical help The authors are sup-ported by Sao Paulo Research Foundation (FAPESP) (Grants201002272-0 201223998-4 201320904-1 201405214-1 and201705100-4) National Counsel of Technological and Sci-entific Development (CNPq Projeto Universal 2013 PQ-1D) (Grants 4705232013-1 and 3016172016-3) Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPES)and Pro-reitoria de Pesquisa da Universidade de Sao Paulo(PRPUSP Projeto I and Novos Docentes) Brazil

References

[1] American Diabetes Association ldquoStandards of Medical Care inDiabetes-2015rdquo Diabetes Care vol 38 no 1 pp 1ndash94 2015

[2] S Penckofer J Kouba D E Wallis andM A Emanuele ldquoVita-min D and diabetes let the sunshine inrdquoDiabetes Educator vol34 no 6 pp 939ndash954 2008

[3] S Thomas and M Rampersad ldquoAnaemia in diabetesrdquo ActaDiabetologica vol 41 no 1 pp S13ndashS17 2004

[4] S Gowda P B Desai S S Kulkarni A A K Hull Math andS N Vernekar ldquoMarkers of renal function testsrdquoThe AmericanJournal of the Medical Sciences vol 2 no 4 pp 170ndash173 2010

[5] G P M Strippoli J C Craig C Manno and F P SchenaldquoHemoglobin targets for the anemia of chronic kidney diseasea meta-analysis of randomized controlled trialsrdquo Journal of theAmerican Society of Nephrology vol 15 no 12 pp 3154ndash31652004


[6] UMehdi and R D Toto ldquoAnemia diabetes and chronic kidneydiseaserdquo Diabetes Care vol 32 no 7 pp 1320ndash1326 2009

[7] M F Holick ldquoMedical progress Vitamin D deficiencyrdquo NewEngland Journal of Medicine vol 357 no 3 pp 266ndash281 2007

[8] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 pp 1678ndash1688 2004

[9] T D Johnson-Wimbley and D Y Graham ldquoDiagnosis andmanagement of iron deficiency anemia in the 21st centuryrdquoTherapeutic Advances in Gastroenterology vol 4 no 3 pp 177ndash184 2011

[10] H S Bhonsle A M Korwar S S Kote et al ldquoLow plasmaalbumin levels are associated with increased plasma proteinglycation and HbA1c in diabetesrdquo Journal of Proteome Researchvol 11 no 2 pp 1391ndash1396 2012

[11] K V Q Luong L T Hoang Nguyen and D N Pham NguyenldquoThe role of vitamin D in protecting type 1 diabetes mellitusrdquoDiabetesMetabolism Research and Reviews vol 21 no 4 pp338ndash346 2005

[12] J J Sim P T Lac I L A Liu et al ldquoVitamin D deficiency andanemia a cross-sectional studyrdquo Annals of Hematology vol 89no 5 pp 447ndash452 2010

[13] A Icardi E Paoletti L De Nicola S Mazzaferro R Russo andM Cozzolino ldquoRenal anaemia and EPO hyporesponsivenessassociated with vitamin D deficiency the potential role ofinflammationrdquoNephrology Dialysis Transplantation vol 28 no7 pp 1672ndash1679 2013

[14] T Takiishi L Ding F Baeke et al ldquoDietary supplementationwith high doses of regular vitamin D3 safely reduces diabetesincidence in NOD mice when given early and long termrdquoDiabetes vol 63 no 6 pp 2026ndash2036 2014

[15] F Spiller D Carlos F O Souto et al ldquo1205721-acid glycoproteindecreases neutrophil migration and increases susceptibility tosepsis in diabetic micerdquo Diabetes vol 61 no 6 pp 1584ndash15912012

[16] N George T Peeyush Kumar S Antony S Jayanarayanan andC S Paulose ldquoEffect of vitamin D3 in reducing metabolic andoxidative stress in the liver of streptozotocin-induced diabeticratsrdquo British Journal of Nutrition vol 108 no 8 pp 1410ndash14182012

[17] F P B Nunes S C Sampaio M L Santoro andM C C Sousa-e-Silva ldquoLong-lasting anti-inflammatory properties of Crotalusdurissus terrificus snake venom in micerdquo Toxicon vol 49 no 8pp 1090ndash1098 2007

[18] A Kar B K Choudhary and N G Bandyopadhyay ldquoCom-parative evaluation of hypoglycaemic activity of some Indianmedicinal plants in alloxan diabetic ratsrdquo Journal of Ethnophar-macology vol 84 no 1 pp 105ndash108 2003

[19] G O Oliveira C P Braga and A A H Fernandes ldquoImprove-ment of biochemical parameters in type 1 diabetic rats afterthe roots aqueous extract of yacon [Smallanthus sonchifolius(PoeppampEndl)] treatmentrdquo Food andChemical Toxicology vol59 pp 256ndash260 2013

[20] H M Ochs-Balcom R Chennamaneni A E Millen et alldquoVitamin D receptor gene polymorphisms are associated withadiposity phenotypesrdquo American Journal of Clinical Nutritionvol 93 no 1 pp 5ndash10 2011

[21] E L Melanson W T Donahoo F Dong T Ida and M BZemel ldquoEffect of low- and high-calcium dairy-based diets onmacronutrient oxidation in humansrdquo Obesity Research vol 13no 12 pp 2102ndash2112 2005

[22] K Casteels M Waer J Laureys et al ldquoPrevention of autoim-mune destruction of syngeneic islet grafts in spontaneouslydiabetic nonobese diabetic mice by a combination of a vitaminD3 analog and cyclosporinerdquo Diabetes vol 51 no 9 pp 1367ndash1374 1998

[23] C V Harinarayan ldquoVitamin D and diabetes mellitusrdquo Hor-mones vol 13 no 2 pp 163ndash181 2014

[24] J Del Pino-Montes G E Benito M P Fernandez-Salazar etal ldquoCalcitriol improves streptozotocin-induced diabetes andrecovers bone mineral density in diabetic ratsrdquo Calcified TissueInternational vol 75 no 6 pp 526ndash532 2004

[25] J P Driver D J Lamont C Gysemans C Mathieu and D VSerreze ldquoCalcium insufficiency accelerates type 1 diabetes invitamin D receptor-deficient nonobese diabetic (NOD) micerdquoEndocrinology vol 152 no 12 pp 4620ndash4629 2011

[26] G Liamis E Liberopoulos F Barkas and M Elisaf ldquoDiabetesmellitus and electrolyte disordersrdquo World Journal of ClinicalCases vol 2 no 10 pp 488ndash496 2014

[27] P K Dabla ldquoRenal function in diabetic nephropathyrdquo WorldJournal of Diabetes vol 1 no 2 pp 48ndash56 2010

[28] M C Thomas R J MacIsaac C Tsalamandris D Power andG Jerums ldquoUnrecognized anemia in patients with diabetes across-sectional surveyrdquo Diabetes Care vol 26 no 4 pp 1164ndash1169 2003

[29] S Adak S Chowdhury and M Bhattacharyya ldquoDynamic andelectrokinetic behavior of erythrocyte membrane in diabetesmellitus and diabetic cardiovascular diseaserdquo Biochimica etBiophysica ActamdashGeneral Subjects vol 1780 no 2 pp 108ndash1152008

[30] F Matough S Budin Z Hamid S Louis N Alwahaibi andJ Mohamed ldquoPalm vitamin E reduces oxidative stress andphysical and morphological alterations of erythrocyte mem-branes in streptozotocin-induced diabetic ratsrdquo Oxidants andAntioxidants in Medical Science vol 1 no 1 pp 59ndash68 2012

[31] S O Oyedem M T Yakubu and A J Afolayan ldquoAntidiabeticactivities of aqueous leaves extract of Leonotis leonurus instreptozotocin induced diabetic ratsrdquo Journal of MedicinalPlants Research vol 5 no 1 pp 119ndash125 2011

[32] S Zhou M S LeBoff and J Glowacki ldquoVitamin Dmetabolismand action in human bone marrow stromal cellsrdquo Endocrinol-ogy vol 151 no 6 pp 14ndash22 2010

[33] F Aucella R P Scalzulli G Gatta M Vigilante A MCarella and C Stallone ldquoCalcitriol increases burst-formingunit-erythroid proliferation in chronic renal failure A syner-gistic effect with r-HuEpordquo Nephron Clinical Practice vol 95no 4 pp c121ndashc127 2003

[34] B Menart-Houtermans R Rutter B Nowotny et al ldquoLeuko-cyte profiles differ between type 1 and type 2 diabetes and areassociated withmetabolic phenotypes results from theGermanDiabetes Study (GDS)rdquo Diabetes Care vol 37 no 8 pp 2326ndash2333 2014

[35] A Brennan D R Katz J D Nunn et al ldquoDendritic cells fromhuman tissues express receptors for the immunoregulatoryvitamin D3 metabolite dihydroxycholecalciferolrdquo Immunologyvol 61 no 4 pp 457ndash461 1987

Submit your manuscripts athttpswwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

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Drug DeliveryJournal of



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Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


D that interacts with vitamin D receptor (VDR) to performits biological activity [7]

The classic activity of calcitriol is to regulate bonemetabolism by maintaining a balance between calcium (Ca)and phosphorus (P) concentrations [8] Studies have reportedthat vitaminDmay affect the course of T1D by immunomod-ulation [11] Although the mechanism of action is stillunknown serum 125(OH)2D levels appear to modulate thelevel of systemic cytokine production and to increase whiteblood cell (WBC) count [12 13]

Although vitamin D supplementation is an importantaid in clinical practice few studies have demonstrated theeffects of vitamin D supplementation on the course of T1Din humans and animals In addition vitamin D supple-mentation studies are often contradictory due to the lackof precise information on regimen and dosage protocols[14]

Thus using a well-established mouse model of T1Dwe investigated the effect of vitamin D supplementationon histological biochemical and hematological parametersand on bronchoalveolar lavage (BAL) and peritoneal lavage(PeL) fluids in male (C57BL6) diabetic and nondiabeticmice The hypothesis is that vitamin D supplementationimproves the hematological parameters and reduces inflam-matory cell migration into the PeL and BAL fluid of diabeticmice

2 Materials and Methods

21 Animals Thirty-one specific pathogen-free maleC57BL6 mice weighing 25 plusmn 2 g at baseline were used Theanimals were maintained at 22∘C under a 12 h light-darkcycle Food and water were provided ad libitum before andduring the experimental period This study was conductedin strict accordance with the principles and guidelines of theNational Council for the Control of Animal Experimentation(CONCEA) and approved by the Ethics Committee onAnimal Use (CEUA) at the School of PharmaceuticalSciences (FCF) University of Sao Paulo Brazil (protocolnumber CEUAFCF389) Surgery was performed underketaminexylazine anesthesia and all efforts were made tominimize animal suffering

22 Induction of Diabetes Mellitus Animals were sepa-rated in four groups control (C) control supplementedwith vitamin D (CV) diabetic (D) and diabetic supple-mented with vitamin D (DV) Briefly diabetes mellituswas induced by intravenous injection of 60mgkg alloxanmonohydrate (ALX) (Sigma Chemical Co St Louis MOUSA) dissolved in physiological saline (09 NaCl) Controlmice were injected with physiological saline only Ten daysafter diabetes induction body weight was measured andperipheral blood (tail vein) glucose level was determinedusing an Accu-Chek Advantage II blood glucose monitor(Roche Diagnostica Sao Paulo SP Brazil) Animals wereconsidered diabetic if glucose level was above 300mgdL[15]

23 Vitamin D Supplementation Animals in the CV andDV groups were supplemented orally with 800 IU of vitaminD (Sanofi Aventis Sao Paulo SP Brazil) for seven days[14] The last dose of vitamin D was administered 24 hbefore the experiment to minimize stress-induced endocrinechanges caused by oral administration of the hormone[16] Body weight and blood glucose were measured onthe first fourth and seventh day of the supplementationperiod

24 Blood Collection Animals were anesthetized by anintraperitoneal injection of 10mgkg xylazine hydrochloride(Ceva Sante Animale Paulınia SP Brazil) and 90mgkgketamine hydrochloride (Ceva Sante Animale) Blood wascollected from anesthetized animals by cardiac punctureSamples of EDTA-anticoagulated blood were used to deter-mine the following hematological parameters red bloodcell (RBC) count white blood cell (WBC) count hemat-ocrit (Htc) and hemoglobin (Hb) All analyses were per-formed using a veterinary hematology analyser ABC Vet(HORIBA UK) Differential cell counts were determinedon stained slides under oil immersion microscopy Based onmorphological criteria 100 cells per sample were countedand classified as either mononuclear or polymorphonuclearA sample aliquot without anticoagulant was centrifuged(10min 3500 rpm) at room temperature Next the serumwasseparated and used for measuring ionic calcium (Ca) phos-phorus (P) albumin alanine transaminase (ALT) aspartateaminotransferase (AST) alkaline phosphatase (ALP) ureaand creatinine by colorimetric assay according to the man-ufacturerrsquos protocol (LabTest Diagnostica Lagoa Santa MGBrazil) Serum 25(OH)D was measured by EUROIMMUN25ndashOHndashVitamin D ELISA kit (Luebeck Germany) Serumsamples were stored at minus80∘C

25 Peritoneal and Bronchoalveolar Lavage The abdomenof all mice was exposed through a ventral midline incisionPeritoneal lavage (PeL)was performedby instillation of 10mL(two 5mL infusions) of phosphate buffered saline (PBS137mM NaCl 27mM KCl 43mM Na2HPO4 and 147mMKH2PO4 pH 74) at room temperature [17] The tracheawas exposed through a midline ventral incision in the neckBronchoalveolar lavage (BAL) was performed by instillationof 5mL (five 1mL infusions) of PBS at room temperaturethrough a 16G times 18810158401015840 BD Angiocath polyethylene tube(BD Franklin Lakes NJ USA) inserted into the tracheaNext PeL and BAL samples were centrifuged (1500 rpm10min 4∘C) supernatants were discarded and the cellswere resuspended in PBS (1mL) The cell suspension wasdiluted 1 2 (v v) with Turkrsquos solution Total cell counts weredetermined using a Neubauer chamber and differential cellcounts were examined on stained slides under oil immersionmicroscopy A total of 100 cells were counted and classifiedas neutrophils lymphocytes or macrophages based on mor-phological criteria

26 Tissue Extraction andHistological Analysis Kidney liverand lung samples were extracted and subsequently fixed




3 Results









4 Discussion




C CV D DV

lowast

lowast

0

60

120

18025

(OH

)D (n

gm

L)

(a)C CV D DV

0

4

8

12

Calc

ium

(mg

dL)

(b)

C CV D DV

0

5

10

15

Phos

phor

us (m

gdL

)

(c)C CV D DV

0

50

100

150

Ure

a (m

gdL

)

⦁

(d)

000

025

050

075

C CV D DV

Crea

tinin

e (m

gdL

)

lowast

lowast

(e)C CV D DV

0

1

2

3

Alb

umin

(gd

L)

(f)

C CV D DV0

200

400

600

ALP

(UL

)

(g)C CV D DV

0

45

90

135

ALT

(UL

)

(h)

Figure 1 Continued


C CV D DV0

100

200

300A

ST (U

L)

(i)

Day 1 Day 3 Day 7

lowast

lowastlowast

lowast

lowast

lowast

0

250

500

750

Gly

cem

ia (m

gdL

)

CCV

DDV

(j)




Peritoneum



Lung








C CV D DV

lowast lowast

0

5

10

15

106

cells

ＧＧ

3)

Red

bloo

d ce

ll co

unt (

(a)C CV D DV

lowast

0

7

14

21

Hem

oglo

bin

(gd

L)

(b)

C CV D DV

lowast

0

25

50

75

Hem

atoc

rit (

)

(c)C CV D DV

00

16

32

48

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(d)

C CV D DV


00

14

28

42

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(e)



C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3






C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3











C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3



Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




3 Results









4 Discussion




C CV D DV

lowast

lowast

0

60

120

18025

(OH

)D (n

gm

L)

(a)C CV D DV

0

4

8

12

Calc

ium

(mg

dL)

(b)

C CV D DV

0

5

10

15

Phos

phor

us (m

gdL

)

(c)C CV D DV

0

50

100

150

Ure

a (m

gdL

)

⦁

(d)

000

025

050

075

C CV D DV

Crea

tinin

e (m

gdL

)

lowast

lowast

(e)C CV D DV

0

1

2

3

Alb

umin

(gd

L)

(f)

C CV D DV0

200

400

600

ALP

(UL

)

(g)C CV D DV

0

45

90

135

ALT

(UL

)

(h)

Figure 1 Continued


C CV D DV0

100

200

300A

ST (U

L)

(i)

Day 1 Day 3 Day 7

lowast

lowastlowast

lowast

lowast

lowast

0

250

500

750

Gly

cem

ia (m

gdL

)

CCV

DDV

(j)




Peritoneum



Lung








C CV D DV

lowast lowast

0

5

10

15

106

cells

ＧＧ

3)

Red

bloo

d ce

ll co

unt (

(a)C CV D DV

lowast

0

7

14

21

Hem

oglo

bin

(gd

L)

(b)

C CV D DV

lowast

0

25

50

75

Hem

atoc

rit (

)

(c)C CV D DV

00

16

32

48

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(d)

C CV D DV


00

14

28

42

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(e)



C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3






C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3











C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3



Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


C CV D DV

lowast

lowast

0

60

120

18025

(OH

)D (n

gm

L)

(a)C CV D DV

0

4

8

12

Calc

ium

(mg

dL)

(b)

C CV D DV

0

5

10

15

Phos

phor

us (m

gdL

)

(c)C CV D DV

0

50

100

150

Ure

a (m

gdL

)

⦁

(d)

000

025

050

075

C CV D DV

Crea

tinin

e (m

gdL

)

lowast

lowast

(e)C CV D DV

0

1

2

3

Alb

umin

(gd

L)

(f)

C CV D DV0

200

400

600

ALP

(UL

)

(g)C CV D DV

0

45

90

135

ALT

(UL

)

(h)

Figure 1 Continued


C CV D DV0

100

200

300A

ST (U

L)

(i)

Day 1 Day 3 Day 7

lowast

lowastlowast

lowast

lowast

lowast

0

250

500

750

Gly

cem

ia (m

gdL

)

CCV

DDV

(j)




Peritoneum



Lung








C CV D DV

lowast lowast

0

5

10

15

106

cells

ＧＧ

3)

Red

bloo

d ce

ll co

unt (

(a)C CV D DV

lowast

0

7

14

21

Hem

oglo

bin

(gd

L)

(b)

C CV D DV

lowast

0

25

50

75

Hem

atoc

rit (

)

(c)C CV D DV

00

16

32

48

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(d)

C CV D DV


00

14

28

42

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(e)



C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3






C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3











C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3



Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


C CV D DV0

100

200

300A

ST (U

L)

(i)

Day 1 Day 3 Day 7

lowast

lowastlowast

lowast

lowast

lowast

0

250

500

750

Gly

cem

ia (m

gdL

)

CCV

DDV

(j)




Peritoneum



Lung








C CV D DV

lowast lowast

0

5

10

15

106

cells

ＧＧ

3)

Red

bloo

d ce

ll co

unt (

(a)C CV D DV

lowast

0

7

14

21

Hem

oglo

bin

(gd

L)

(b)

C CV D DV

lowast

0

25

50

75

Hem

atoc

rit (

)

(c)C CV D DV

00

16

32

48

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(d)

C CV D DV


00

14

28

42

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(e)



C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3






C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3











C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3



Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


C CV D DV

lowast lowast

0

5

10

15

106

cells

ＧＧ

3)

Red

bloo

d ce

ll co

unt (

(a)C CV D DV

lowast

0

7

14

21

Hem

oglo

bin

(gd

L)

(b)

C CV D DV

lowast

0

25

50

75

Hem

atoc

rit (

)

(c)C CV D DV

00

16

32

48

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(d)

C CV D DV


00

14

28

42

106

cells

ＧＧ

3)

Whi

te b

lood

cell

coun

t (

(e)



C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3






C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3











C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3



Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3






C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3











C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3



Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3











C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3



Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


C1 C2 C3

CV1 CV2 CV3

D1 D2 D3

DV1 DV2 DV3



Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

vitamin d modulates hematological parameters and cell...

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