research article mild systemic oxidative stress in the...
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Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 609019 8 pageshttpdxdoiorg1011552013609019
Research ArticleMild Systemic Oxidative Stress in the Subclinical Stage ofAlzheimerrsquos Disease
Leandro Giavarotti1 Karin A Simon2 Ligia A Azzalis2 Fernando L A Fonseca23
Alessandra F Lima2 Maria C V Freitas2 Milena K C Brunialti4
Reinaldo Salomatildeo4 Alcione A V S Moscardi5 Maria B M M Montantildeo5
Luiz R Ramos5 and Virgiacutenia B C Junqueira2
1 Instituto de Quımica USP Cidade Universitaria Avenida Lineu Prestes 748 05508-900 Sao Paulo SP Brazil2 Instituto de Ciencias Ambientais Quımica e Farmaceuticas UNIFESP Rua Arthur Riedel 275 09972-270 Diadema SP Brazil3 Departamento de Hematologia e Oncologia FMABC Avenida Prıncipe de Gales 821 09060-650 Santo Andre SP Brazil4Departamento de Medicina UNIFESP Rua Sena Madureira 1500 04021-001 Sao Paulo SP Brazil5 Departamento de Medicina Preventiva UNIFESP Rua Sena Madureira 1500 04021-001 Sao Paulo SP Brazil
Correspondence should be addressed to Virgınia B C Junqueira virginiajunqueiragmailcom
Received 14 July 2013 Accepted 8 November 2013
Academic Editor Sara Mostafalou
Copyright copy 2013 Leandro Giavarotti 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
Alzheimerrsquos disease (AD) is a late-onset progressive degenerative disorder that affects mainly the judgment emotional stabilityand memory domains AD is the outcome of a complex interaction among several factors which are not fully understood yetnevertheless it is clear that oxidative stress and inflammatory pathways are among these factors 65 elderly subjects (42 cognitivelyintact and 23 with probable Alzheimerrsquos disease) were selected for this study We evaluated erythrocyte activities of superoxidedismutase catalase and glutathione peroxidase as well as plasma levels of total glutathione 120572-tocopherol 120573-carotene lycopeneand coenzyme Q10 These antioxidant parameters were confronted with plasmatic levels of protein and lipid oxidation productsAdditionally we measured basal expression of monocyte HLA-DR and CD-11b as well as monocyte production of cytokines IL1-120572IL-6 and TNF-120572 AD patients presented lower plasmatic levels of 120572-tocopherol when compared to control ones and also higherbasal monocyte HLA-DR expression associated with higher IL-1120572 production when stimulated by LPS These findings support theinflammatory theory of AD and point out that this disease is associated with a higher basal activation of circulating monocytes thatmay be a result of 120572-tocopherol stock depletion
1 Introduction
Alzheimerrsquos disease (AD) is a neurodegenerative disorderresulting in progressive neuronal death and memory loss[1] Histopathologically the disease is characterized by intra-neuronal neurofibrillary tangles (NFT) (aggregations of ahyperphosphorylated form of the microtubule-associatedprotein tau) and extracellular deposits of neuritic plaquescomposed of amyloid-120573 (A120573) protein a 40ndash43 amino acidproteolytic fragment derived from the amyloid precursorprotein [2]
Although AD is considered a neurodegenerative diseaseboth inflammation and oxidative stress are present early
and throughout its pathogenesis One of the early clues asto AD as an inflammatory process was the finding thatrheumatoid arthritis patients taking high levels of anti-inflammatory drugs had a low incidence of AD [3] Inflam-matory mechanisms may significantly contribute to diseaseprogression and chronicity with proinflammatory cytokines(notably IL-1120572 and IL-1120573) being found throughout the brainof individuals with AD when analyzed at autopsy [1] Manyneuroinflammatorymediators including complement activa-tors and inhibitors chemokines cytokines reactive speciesand inflammatory enzymes are generated and released bymicroglia astrocytes and neurons [4]
2 Oxidative Medicine and Cellular Longevity
However the precise implications of the inflammatoryresponse for neurodegeneration have not been elucidatedA current hypothesis considers that an extracellular insultto neurons could trigger the production of inflammatorycytokines by astrocytes andmicrogliaThe cytokines namelyIL-1120573 TNF-120572 and IL-6 could affect the normal behavior ofneuronal cells Therefore dysfunction at this core level maylead to abnormalities such as neurofibrillary degeneration inAD [5]
It is well characterized that there is oxidative stress in theAD brain Lower plasma antioxidant levels and alterations inantioxidant enzyme activities were reported inmild cognitiveimpairment (MCI) patients and patients at early AD stagessuggesting an imbalance between reactive species productionand antioxidant defense systems in the plasma ofADpatientsThis was substantiated by an increase in DNA lipid andprotein oxidation products found in blood and cerebrospinalfluid (CSF) obtained from AD patients in comparison withcontrols [6]
At the same time microglial cells activation is prominentduring AD Microglia are bone marrow-derived cells of themonocyte lineages that like peripheral tissue macrophagesbecome phagocytic and produce reactive oxygen species [3]However the evaluation of microglia activity during thedevelopment of AD in human patients is impossible due toits localization thus justifying demand for models that useparameters of easy access and reflecting the state of activationof microglia However the studies with monocytes couldcontribute to the understanding of inflammatory compo-nent in AD establishing at the same time the possibilityof using a minimally invasive technique such as bloodcollection
As exposed AD has an important component thatinvolves inflammatory reactions associated with reactivespecies release and lower antioxidant levels Thus this paperaims at verifying blood oxidative markers and monocyteinflammatory parameters in elderly subjects who presentwith very mild cognitive impairment
2 Methods
21 Subjects Patients participating in this study were partof the last wave of Epidoso Project receiving monitoring onaging by a multidisciplinary team [7]
65 elderly subjects (mean age 82 years) were selected forthis study All patients had their functional capacity and labo-ratory profile evaluatedThese seniors were initially subjectedto a clinical survey conducted by a geriatrician consistingof history taking physical examination and evaluation ofmental autonomy through the degree of cognitive functionPatients with a history of cardiovascular disease cancer orchronic inflammatory diseases as well as individuals withcurrent inflammatory alterations assessed by erythrocytesedimentation rate or plasma levels of C-reactive proteinwere not included in this study
The study protocol was submitted to the Ethics Commit-tee of UNIFESP and was approved under number 085903All patients or their guardians signed informed consent
22 Assessment of Patientsrsquo Cognitive Ability Minimentalstate examination was used for screening cognitive deficitsamong patients [8] The maximum score is 30 and scoreslower than 24 were considered indicative of some kind ofcognitive dysfunction
Patients who scored less than 24 points in MMSE werethen tested with CDR (Clinical Dementia Rating) [9 10]
Patients were considered cognitively intact (INT group119899 = 42) when CDR was lt1 while others were included inthe group of patients with probable Alzheimerrsquos disease (ADgroup 119899 = 23) if CDR was ge1
23 Measurements of Erythrocytes Parameters Blood sam-ples from participants were obtained after 12 hours offasting Part of whole blood was used for flow cytometryAfter plasma separation erythrocytes were used to measuretotal glutathione content (TGSH) [11] and the remaininghemolysate to assess the activities of antioxidant enzymes[7] Cu-Zn superoxide dismutase (SOD) catalase (CAT) andglutathione peroxidase (GPx) all adjusted by hemoglobinconcentration
24 Measurements of Plasma Parameters Plasma sampleswere stored at minus80∘C for later determination of 120572-tocopherol120573-carotene lycopene coenzyme Q10 and vitamin C [7] Forextraction 20120583L plasma aliquots were mixed with 100120583Lextraction solvent (50 ethanol proanalysis (pa) 50 1-butanol pa containing 5mg butylhydroxytoluenemL and8 120583mol TocolL as an internal standard) to extract thevitamins and to denature plasma proteins The sample wasthen mixed by vortex for 30 s and centrifuged at 21 000timesgfor 10min at 4∘C The supernatant was transferred to apolypropylene vial This method uses precolumn and reversephase C18 column and UV-VIS detector with deuteriumlamp with a mobile phase consisting of 80 acetonitrile 3methanol (100mM ammonium acetate 01 triethylamine)and 15 dioxan The mobile phase is pumped through thesystem by a flow of 15mLmin with maximum pressureof 350Kgf and the minimum of 0 The wavelength wasestablished by the method for each parameter and run timefor it was 4 to 6 minutes
Plasma was also used to determine the concentration ofthiobarbituric acid reactive substances (TBARS) [7] and levelof oxidized proteins expressed as plasma carbonyls [12]
25 Monocyte Activation Parameters In order to evaluatebasal activation of circulating monocytes cell surface HLA-DR and CD-11b levels were assessed by flow cytometry asdescribed elsewhere [13] Monocytes were identified bymon-oclonal antibodies anti-CD14-pernidin chlorophyll protein(PerCP) neutrophils were excluded with CD66b-fluoresceinisothiocyanate (FITC) labeling Cell surface molecules weremarked with anti-HLA-DR-phycoerythrin (PE) and anti-CD11b-allophycocyanin (APC) controls were incubated withanti-HLA-DR-PE and CD11b-APC isotypes
Intracellular production of IL-1120572 IL-6 and TNF-120572was quantified by flow cytometry in monocytes stimulatedwith LPS and treated with monensin in order to prevent
Oxidative Medicine and Cellular Longevity 3
Table 1 Oxidative stress parameters in erythrocyte (RBC) Antiox-idant enzymes superoxide dismutase (SOD) catalase (CAT) andglutathione peroxidase (GPx) and total glutathione level (TGSH)INT (cognitively intact patients 119899 = 42) and AD (patients withprobable Alzheimerrsquos disease 119899 = 23) Values shown correspondto the means plusmn SEM Comparison between average results ofthe groups was performed using Studentrsquos 119905-test for independentsamples
Parameter INT ADRBC SOD (Umg Hb) 782 plusmn 024 82 plusmn 031
RBC CAT (Umg Hb) 187 plusmn 7 184 plusmn 8
RBC GPx (Umg Hb) 128 plusmn 04 126 plusmn 07
RBC TGSH (mmolHb) 767 plusmn 021 745 plusmn 036
119875 lt 005 was considered significant
cytokine release [13] Monocytes were identified by mon-oclonal antibodies for surface staining (anti-CD66b-FITCand anti-CD14-PerCP) and cytokine productionwas detectedusing monoclonal antibodies anti-IL-1120572-PE anti-IL-6-PEand anti-TNF120572-APC or their respective isotype controls
Data were acquired using a FACSCalibur flow cytometerand CellQuest software (both from BD ImmunocytometrySystems)
26 Reactive Oxygen Species Assay Whole blood sampleswere first stained with anti-CD14 PerCP for monocyte iden-tification samples were subsequently incubated with 2101584071015840-dichlorofluorescein diacetate (DCFH-DA) a stable com-pound nonfluorescent which diffuses into the cells andis hydrolyzed to 2101584071015840-dichlorofluorescein (DCFH) Uponcell stimulation with LPS DCFH was oxidized to DCF byreactive species mostly H
2O2 emitting high levels of green
fluorescence which was detected by flow cytometry [14]using a FACSCalibur flow cytometer and CellQuest software(both from BD Immunocytometry Systems)
27 Statistical Analysis Normality of data distribution wasassessed by Shapiro-Wilk test Comparison between averageresults of the groups was performed using Student t test forindependent samples Correlation analysis between data wasperformed using Spearman correlation test All data wereprocessed and analyzed by appropriate statistical tests usingSPSS 17 software
3 Results
No changes were observed on plasma activity of the majorerythrocyte antioxidant enzymes SOD catalase and glu-tathione peroxidase (Table 1) Table 1 also shows that totalglutathione levels were similar in both cognitively intact(INT) and in group of patients with probable AD
Lipid peroxidation (measured as TBARS) and plasmaticprotein oxidation measured as the amount of circulatingprotein carbonyls did not significantly differ between thegroups INT and AD (Table 2)
Plasma vitamin C levels were equivalent in both groupsas illustrated in Table 2 Values of circulating 120572-tocopherol
Table 2 Oxidative stress parameters in plasma INT (cognitivelyintact patients 119899 = 42) and AD (patients with probable Alzheimerrsquosdisease 119899 = 23) Values shown correspond to the means plusmn SEMComparison between average results of the groups was performedusing Studentrsquos 119905-test for independent samples
Parameter INT ADVitamin C (120583M) 48 plusmn 3 52 plusmn 5
120572-Tocopherol (120583M) 21 plusmn 1 17 plusmn 1lowast
120573-Carotene (120583M) 079 plusmn 09 076 plusmn 1
Lycopene (120583M) 073 plusmn 009 067 plusmn 013
Coenzyme Q10 (120583M) 015 plusmn 002 013 plusmn 003
TBARS (thiobarbituric acid reactivesubstances) (nmolmL) 582 plusmn 051 583 plusmn 062
Oxidized proteins (nmolmg prot) 014 plusmn 001 016 plusmn 002
lowast
119875 lt 005 was considered significant
were about 20 lower in patients with probable Alzheimerrsquosdisease compared with cognitively intact patients This dif-ference was statistically significant It was observed that thetwo groups studied had similar plasma levels of 120573-carotenelycopene and coenzyme Q10 (Table 2)
Table 3 shows that Cu Zn-SOD activity had a positivecorrelation with catalase and glutathione peroxidase activi-ties as well as total glutathione content Catalase activity inturn also had positive correlation with glutathione peroxi-dase activity and total glutathione content in erythrocytesGlutathione peroxidase activity and total glutathione contentin red blood cells also had a significant positive correlationThe same two parameters negatively correlated with lipidperoxidation products in plasma
Monocytes of patients with probable Alzheimerrsquos dis-ease expressed approximately 70 more HLA-DR than thecognitively intact patients On the other hand there wereno differences on the expression of CD11b by monocytesobtained from the two groups (Figure 1 and Table 4)
The proportion of cells producing IL-1120572 was approxi-mately 25 higher in patients with probable diagnosis ofthe disease as compared to those cognitively intact (Table 4)In the case of IL-6 and TNF-120572 there were no statisticallysignificant differences in the proportion of cells that producedthose interleukins (Figure 2 and Table 4)
The oxidativemetabolism ofmonocytes studiedwith flowcytometry techniques showed no differences between groups(Table 4)
4 Discussion
The aim of this study was to investigate the interrelationshipbetween AD blood parameters of oxidative stress andindicators of inflammatory activity in monocytes It followsthe reasoning by which monocytes are circulating cells thatresemble the brain microglia andmay even be recruited fromblood to brain tissue [15] in AD Microglia are consideredthe main element for the localized inflammatory events thataccompany this disease [16] This would lead to localizedinflammation and increased production of reactive oxygen
4 Oxidative Medicine and Cellular Longevity
Forward scatter
Side
scat
ter
1000
800
600
400
200
010008006004002000
R1
100
101
102
103
104
80
0
Cou
nts
CD11b-APC
INT
100
101
102
103
104
80
0
Cou
nts
AD
CD11b-APC
100
101
102
103
104
80
0
Cou
nts
M1
INT isotype
mlgG2b-APC
100
101
102
103
104
80
0
Cou
nts
M1
AD isotype
mlgG2b-APC
100
101
102
103
104
80
0
Cou
nts
AD
HLA-DR-PE
M1
100
101
102
103
104
80
0
Cou
nts
AD isotype
mlgG2a-PE
M1
HLA-DR-PE
INT
100
101
102
103
104
80
0
Cou
nts
CD14-PerCP
CD
66b-
FITC
R3
R2
100
101
102
103
104
100
101
102
103
104
M1
M1
INT isotype
100
101
102
103
104
80
0C
ount
s
mlgG2a-PE
Figure 1 Representative flow cytometric analysis of whole blood samples stainedwith anti-CD11b-APC (right column) and anti-HLA-DR-PE(left column) monoclonal antibodies From all events acquired in gate R1 (upper left graph) CD14-positive monocytes (gate R2 separatedfrom CD66b-positive neutrophils in gate R3 upper right graph) were used for histogram calculation of the geometric mean fluorescenceintensity (GMFI) A similar analysis was performed for the detection of fluorescent DCF in the ROS assay (histogram not shown) INT(sample from cognitively intact patient) and AD (sample from patient with probable Alzheimerrsquos disease)
Oxidative Medicine and Cellular Longevity 5
INT
100
101
102
103
104
100
101
102
103
104
INT + LPS
IL-11205721000
0
1000
0
AD
Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0
Forw
ard
scat
ter AD + LPS
IL-1120572-PE IL-1120572-PE
IL-1120572-PE IL-1120572-PE
(a)
IL-6
IL-6-PE IL-6-PE
IL-6-PE IL-6-PE
INT
100
101
102
103
104
100
101
102
103
104
INT + LPS1000
0
1000
0
AD
Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter AD + LPS
(b)
TNF-120572
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter
INT INT + LPS
AD AD + LPS
TNF120572-APC TNF120572-APC
TNF120572-APC TNF120572-APC
(c)
Figure 2 Representative flow cytometric detection ofmonocyte intracellular production of IL-1120572 IL-6 and TNF-120572 Monocytes were selectedin a similar manner as described in Figure 1 LPS-induced production of each cytokine was determined by the percentage of events in theupper right quadrant (URQ) Only upper quadrants are shown INT (sample from cognitively intact patient) and AD (sample from patientwith probable Alzheimerrsquos disease)
species causing oxidative stress which could be reflected bycirculating parameters [17]
In the conditions of this work AD is not associatedwith any evidence of systemic oxidative stress Althoughthe activities of erythrocyte antioxidant enzymes do notshow changes in patients with AD Spearman rank correla-tion test reveals a positive correlation between the mutualactivities of erythrocyte superoxide dismutase catalase andglutathione peroxidase and erythrocyte total glutathionelevels These data confirm the validity of the postulate thatsays that the bodyrsquos antioxidant systems act together inan integrated manner Spearman test has also identified anegative correlation between the levels of lipid peroxidationproducts plasma glutathione peroxidase activity and totalglutathione levels in erythrocytes This fact indicates that
lipid peroxidation is influenced by the activity of antioxidantglutathione peroxidase-glutathione system in this experi-mental model Indeed glutathione-glutathione peroxidasesystem is an important component of antioxidant protectionnetwork to cells [18] As a counterpoint to the measurementof erythrocyte antioxidant activities circulating levels ofoxidation products of macromolecules in the body have beenevaluated To this end we analyzed levels of plasma proteincarbonyls and plasma levels of lipid peroxidation productsPlasma level of protein oxidation products is not altered inAD compared with cognitively intact group This lack ofchange is found in some studies [18] On the other handsome authors have found an increase of oxidized proteinsin plasma from AD patients [19 20] This difference maybe due to difference of sensitivity of the methods employed
6 Oxidative Medicine and Cellular Longevity
Table 3 Correlation between antioxidant erythrocytes parameters and plasmatic oxidation products Superoxide dismutase (SOD) catalase(CAT) glutathione peroxidase (GPx) total glutathione level (TGSH) and thiobarbituric acid reactive substances (TBARS) Analysis wasperformed using the Spearman correlation test
Spearman test SOD CAT GPX GSH TBARSSOD
Coefficient of correlation 1000 0415lowast 0316lowast 0406lowastlowast 0097Significance 0001 0011 0001 0244
CATCoefficient of correlation 0415lowast 1000 0368lowastlowast 0317lowast 0158Significance 0001 0003 0010 0129
GPXCoefficient of correlation 0316lowast 0368lowastlowast 1000 0416lowastlowast 0323lowastlowast
Significance 0011 0003 0001 0009TGSH
Coefficient of correlation 0406lowastlowast 0317lowast 0416lowastlowast 1000 0253lowast
Significance 0001 0010 0001 0034TBARS
Coefficient of correlation 0097 0158 0323lowastlowast 0253lowast 1000Significance 0244 0129 0009 0034
lowastlowastP lt 001 lowastP lt 005
Table 4 Monocyte activation parameters INT (cognitively intactpatients 119899 = 42) and AD (patients with probable Alzheimerrsquosdisease 119899 = 23) GMFI geometric mean fluorescence intensity URQ percentage of events in the upper right quadrant Valuesshown correspond to the means plusmn SEM Comparison betweenaverage results of the groups was performed using Student 119905 test forindependent samples
Parameter INT ADHLADR (GMFI) 10993 plusmn 1059 17774 plusmn 3064
lowast
CD11B (GMFI) 40674 plusmn 3924 41292 plusmn 4897
IL-1120572 (URQ) 6578 plusmn 246 8282 plusmn 652lowast
IL-6 (URQ) 5289 plusmn 431 4971 plusmn 657
TNF-120572 (URQ) 685 plusmn 128 826 plusmn 247
Oxidative metabolism (GMFI) 26632 plusmn 3899 25777 plusmn 6307lowast
119875 lt 005 was considered significant
in the mentioned studies (two-dimensional electrophoresismass spectrometry) in relation to enzyme-immunoassaymethod chosen for this work and the possible differences inquality of care and feeding among the target populations ofeach study Concentration of circulating lipid peroxidationproducts is equivalent in both groups results that agree withthose commonly found in the literature [18] In generalthe population studied seems to enjoy a privileged statusin relation to nutritional populations examined in similarstudies in other locations In addition to the antioxidantprofile outlined above measurements of plasma levels ofnonenzymatic antioxidants of lowmolecularweightwere alsocarried out
Values of vitamin C 120573-carotene and 120572-tocopherolobtained in this study are within the range previouslydescribed for a healthy population group in that age [721] A review of several published papers performed by
Stocker and Frei [22] shows populational values for plasmaconcentrations of vitamin C 120572-tocopherol and 120573-caroteneranging from 30 to 150 120583M 15 to 40 120583M and 03 to 06 120583Mrespectively Other authors report that for cardiovasculardisease and cancer prevention concentrations above 30120583M120572-tocopherol in combination with concentrations greaterthan 50 120583M of vitamin C 04 120583M 120573-carotene and 05120583Mlycopene are desired [23] Thus the two groups of elderlypatients studied have positioned themselves within the limitsfound in several other studies to molecular plasma levels ofantioxidantsThese parameters reflect part of the antioxidantdefense system of the body of patients evaluating compo-nents that can be absorbed directly from the diet so furtherpromoted by the antioxidant defense mentioned above Ofall lowmolecular weight antioxidants studied only vitamin Esignificantly differs between groupsThe INT group has levelsof plasma vitamin E higher than those in AD patients
This apparent homogeneity in antioxidant levels betweenthe various groups opposed to other studies in the literature[24ndash27] This finding as previously mentioned reinforcesthe notion that the oxidative processes that are triggeredduring the course of AD are not always reflected in circu-lating blood and even when that occurs it may be onlyan indication of other underlying mechanisms Of specialinterest is the fact that the establishment of AD expressedby the comparison between the INT and AD groups inour study is accompanied by the decrease of circulatingvitamin E which possesses antioxidant capabilities as wellas recognized anti-inflammatory activities due to its actionon signal transduction mechanisms in phagocytes [28 29]Not surprisingly studies that have obtained the best results inpreventing or treating AD in recent years used 120572-tocopherolas an agent [30]
To complete the picture outlined up to this point circulat-ingmonocytes were analyzed by flow cytometry as indicators
Oxidative Medicine and Cellular Longevity 7
of inflammatory activity involved in AD To this end wequantified the level of expression of surface molecules HLA-DR as an indicator of activation of circulating monocytesand CD-11b an adhesion molecule involved in the processof marginalization and migration of monocytes The resultsshow that AD patients have circulating monocytes thatexpress higher amounts of HLA-DR on the surface evenwithout external stimulus indicating a higher basal activationstate of these cells confirming the data available in theliterature [31] Markers of increased activation of monocytesin the bloodstream are found in patients from a number ofneurodegenerative diseases [32] As previously mentionedthese circulatingmonocytes can cross the blood-brain barrierandmigrate to the brain tissue differentiating into microgliaSince circulating monocytes of AD patients are more activeat baseline than those of INT it can be postulated thatmicroglia derived from these cells react more intensely toinflammatory stimuli producing greater injury to brainLevel of expression of surface protein CD-11b is the same inboth groups indicating that the potential for adhesion andmigration of these cells is not altered in this pathology
Also by flow cytometry monocytes production of IL-1120572 IL-6 and TNF-120572 was measured This measurement wasintended to evaluate the capacity of circulating monocytesto stimulate the inflammatory process by attracting newmonocytes to the inflammatory site The production of IL-1120572is higher in monocytes from AD patients than in INT groupSince IL-1120572 is an important proinflammatory cytokine it isplausible to assume thatmigration anddifferentiation of thesemonocytes produce more active microglia This increasedproduction of IL-1120572 was shown in vitro in human monocyteslineage when stimulated by P120573A by means of mechanismsof signal transduction mediated by tyrosine kinases whichreinforces the above hypothesis [33]
Levels of IL-6 produced by stimulated peripheral mono-cytes are similar in both groups as well as of TNF-120572 agreeingwith Beloosesky et al [34] Other authors detected anincrease in IL-6 production bymonocytes of ADpatients [35]using however a different stimulus (phytohemagglutinin)than that used in this present study (LPS)
From the data shown here it is reasonable to concludethat AD is accompanied by the activation of circulatingmonocytes and a decrease in circulating levels of vitamin ESeveral studies show 120572-tocopherol modulating inflammationactivity [28 36] In our study the group of patients whereplasma 120572-tocopherol is decreased also showed more acti-vated monocytes that respond to stimuli with an increasedproduction of proinflammatory cytokine IL-1120572 This studypoints out that AD is associated with a higher basal activationof circulating monocytes that may be a result of 120572-tocopherolstock depletion
Abbreviations
AD Alzheimerrsquos diseaseAPC AllophycocyaninCDR Clinical dementia ratingFITC Fluorescein isothiocyanateINT Cognitively intact group
GMFI Geometric mean fluorescenceintensity
LPS LipopolysaccharideMMSE Minimental state examinationPE PhycoerythrinPerCP Periidinin chlorophyll proteinROS Reactive oxygen speciesURQ Upper right quadrant
References
[1] B C Dickerson A Bakkour D H Salat et al ldquoThe corticalsignature of Alzheimerrsquos disease regionally specific corticalthinning relates to symptom severity in very mild to mild ADdementia and is detectable in asymptomatic amyloid-positiveindividualsrdquo Cerebral Cortex vol 19 no 3 pp 497ndash510 2009
[2] A Jana and K Pahan ldquoFibrillar amyloid-120573 peptides kill humanprimary neurons via NADPH oxidase-mediated activation ofneutral sphingomyelinase Implications for Alzheimerrsquos dis-easerdquo Journal of Biological Chemistry vol 279 no 49 pp 51451ndash51459 2004
[3] C E Finch and T E Morgan ldquoInflammatory processes ofAlzheimer disease and agingrdquoProceedings of the IndianNationalScience Academy B vol 69 pp 165ndash178 2003
[4] E Zotova J A Nicoll R Kalaria C Holmes and D BocheldquoInflammation in Alzheimerrsquos disease relevance to pathogene-sis and therapyrdquo Alzheimerrsquos Research and Therapy vol 2 no 1article 1 2010
[5] L E Rojo J A Fernandez A A Maccioni J M Jimenezand R B Maccioni ldquoNeuroinflammation implications for thepathogenesis and molecular diagnosis of Alzheimerrsquos diseaserdquoArchives of Medical Research vol 39 no 1 pp 1ndash16 2008
[6] K Jomova D Vondrakova M Lawson and M Valko ldquoMetalsoxidative stress and neurodegenerative disordersrdquo Molecularand Cellular Biochemistry vol 345 no 1-2 pp 91ndash104 2010
[7] V B C Junqueira S B M Barros S S Chan et al ldquoAging andoxidative stressrdquoMolecular Aspects of Medicine vol 25 no 1-2pp 5ndash16 2004
[8] M F Folstein S E Folstein and P R McHugh ldquoMini-mentalstate A practical method for grading the cognitive state ofpatients for the clinicianrdquo Journal of Psychiatric Research vol12 no 3 pp 189ndash198 1975
[9] J C Morris ldquoThe Clinical Dementia Rating (CDR) currentversion and scoring rulesrdquo Neurology vol 43 no 11 pp 2412ndash2414 1993
[10] M B MMontano and L R Ramos ldquoValidity of the Portugueseversion of Clinical Dementia Ratingrdquo Revista de Saude Publicavol 39 no 6 pp 912ndash917 2005
[11] F Tietze ldquoEnzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione appli-cations to mammalian blood and other tissuesrdquo AnalyticalBiochemistry vol 27 no 3 pp 502ndash522 1969
[12] H Buss T P Chan K B Sluis N B Domigan and C CWinterbourn ldquoProtein carbonyl measurement by a sensitiveELISA methodrdquo Free Radical Biology and Medicine vol 23 no3 pp 361ndash366 1998
[13] M K C Brunialti E G Kallas M Freudenberg C Galanosand R Salomao ldquoInfluence of EDTA and heparin onlipopolysaccharide binding and cell activation evaluatedat single-cell level in whole bloodrdquo Clinical Cytometry vol 50no 1 pp 14ndash18 2002
8 Oxidative Medicine and Cellular Longevity
[14] M Hasui Y Hirabayashi and Y Kobayashi ldquoSimultaneousmeasurement by flow cytometry of phagocytosis and hydrogenperoxide production of neutrophils in whole bloodrdquo Journal ofImmunological Methods vol 117 no 1 pp 53ndash58 1989
[15] J Wegiel H Imaki K-C Wang et al ldquoOrigin and turnover ofmicroglial cells in fibrillar plaques of APPsw transgenic micerdquoActa Neuropathologica vol 105 no 4 pp 393ndash402 2003
[16] M R DrsquoAndrea G M Cole and M D Ard ldquoThe microglialphagocytic role with specific plaque types in the Alzheimerdisease brainrdquoNeurobiology of Aging vol 25 no 5 pp 675ndash6832004
[17] J Vina A Lloret ROrtı andDAlonso ldquoMolecular bases of thetreatment of Alzheimerrsquos disease with antioxidants preventionof oxidative stressrdquo Molecular Aspects of Medicine vol 25 no1-2 pp 117ndash123 2004
[18] M C Polidori P Mattioli S Aldred et al ldquoPlasma antioxidantstatus immunoglobulin G oxidation and lipid peroxidation indemented patients relevance to Alzheimer disease and vasculardementiardquo Dementia and Geriatric Cognitive Disorders vol 18no 3-4 pp 265ndash270 2004
[19] C C Conrad P L Marshall J M Talent C A MalakowskyJ Choi and R W Gracy ldquoOxidized proteins in Alzheimerrsquosplasmardquo Biochemical and Biophysical Research Communica-tions vol 275 no 2 pp 678ndash681 2000
[20] J Choi C A Malakowsky J M Talent C C Conradand R W Gracy ldquoIdentification of oxidized plasma proteinsin Alzheimerrsquos diseaserdquo Biochemical and Biophysical ResearchCommunications vol 293 no 5 pp 1566ndash1570 2002
[21] H Heseker and R Schneider ldquoRequirement and supply ofvitamin C E and 120573-carotene for elderly men and womenrdquoEuropean Journal of Clinical Nutrition vol 48 no 2 pp 118ndash1271994
[22] R Stocker and B Frei ldquoEndogenous antioxidant defencesin human blood plasmardquo in Oxidative Stress Oxidants andAntioxidants H Sies Ed Academic Press London UK 1991
[23] K F Gey ldquoPrevention of early stages of cardiovascular diseaseand cancer may require concurrent optimization of all majorantioxidants and other nutrients an update and reevaluationof observational data and intervention trialsrdquo in Free RadicalsLipoprotein Oxidation and Atherosclerosis C Rice-Evans EdRichelieu Press London UK 1995
[24] C Behl and B Moosmann ldquoAntioxidant neuroprotection inAlzheimerrsquos disease as preventive and therapeutic approachrdquoFree Radical Biology and Medicine vol 33 no 2 pp 182ndash1912002
[25] E Esposito D Rotilio V di Matteo C di Giulio M Cacchioand S Algeri ldquoA review of specific dietary antioxidants and theeffects on biochemical mechanisms related to neurodegenera-tive processesrdquoNeurobiology of Aging vol 23 no 5 pp 719ndash7352002
[26] P Mecocci M Cristina Polidori A Cherubini et al ldquoLym-phocyte oxidative DNA damage and plasma antioxidants inAlzheimer diseaserdquoArchives ofNeurology vol 59 no 5 pp 794ndash798 2002
[27] M Sano ldquoDo dietary antioxidants prevent Alzheimerrsquos dis-easerdquoThe Lancet Neurology vol 1 no 6 p 342 2002
[28] S S Chan H P Monteiro F Schindler A Stern and V B CJunqueira ldquo120572-tocopherol modulates tyrosine phosphorylationin human neutrophils by inhibition of protein kinas C activityand activation of tyrosine phosphatasesrdquo Free Radical Researchvol 35 no 6 pp 843ndash856 2001
[29] E M Bulger and R V Maier ldquoAn argument for vitamin Esupplementation in the management of systemic inflammatoryresponse syndromerdquo Shock vol 19 no 2 pp 99ndash103 2003
[30] P P Zandi J C Anthony A S Khachaturian et al ldquoReducedrisk of Alzheimer disease in users of antioxidant vitaminsupplements the Cache County studyrdquo Archives of Neurologyvol 61 no 1 pp 82ndash88 2004
[31] L Kusdra H Rempel K Yaffe and L Pulliam ldquoElevation ofCD69+ monocytemacrophages in patients with Alzheimerrsquosdiseaserdquo Immunobiology vol 202 no 1 pp 26ndash33 2000
[32] J A Casal A Robles and J C Tutor ldquoSerum markers ofmonocytemacrophage activation in patients with Alzheimerrsquosdisease and other types of dementiardquo Clinical Biochemistry vol36 no 7 pp 553ndash556 2003
[33] C K Combs J Colleen Karlo S-C Kao and G E Landrethldquo120573-amyloid stimulation of microglia anti monocytes results inTNF120572-dependent expression of inducible nitric oxide synthaseand neuronal apoptosisrdquo Journal of Neuroscience vol 21 no 4pp 1179ndash1188 2001
[34] Y Beloosesky H Salman M Bergman H Bessler and MDjaldetti ldquoCytokine levels and phagocytic activity in patientswith Alzheimerrsquos diseaserdquo Gerontology vol 48 no 3 pp 128ndash132 2002
[35] F Shalit B Sredni L Stern E Kott and M HubermanldquoElevated interleukin-6 secretion levels by mononuclear cells ofAlzheimerrsquos patientsrdquo Neuroscience Letters vol 174 no 2 pp130ndash132 1994
[36] A Azzi R Gysin P Kempna et al ldquoThe role of 120572-tocopherolin preventing disease from epidemiology to molecular eventsrdquoMolecular Aspects of Medicine vol 24 no 6 pp 325ndash336 2003
Submit your manuscripts athttpwwwhindawicom
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Oxidative Medicine and Cellular Longevity
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2 Oxidative Medicine and Cellular Longevity
However the precise implications of the inflammatoryresponse for neurodegeneration have not been elucidatedA current hypothesis considers that an extracellular insultto neurons could trigger the production of inflammatorycytokines by astrocytes andmicrogliaThe cytokines namelyIL-1120573 TNF-120572 and IL-6 could affect the normal behavior ofneuronal cells Therefore dysfunction at this core level maylead to abnormalities such as neurofibrillary degeneration inAD [5]
It is well characterized that there is oxidative stress in theAD brain Lower plasma antioxidant levels and alterations inantioxidant enzyme activities were reported inmild cognitiveimpairment (MCI) patients and patients at early AD stagessuggesting an imbalance between reactive species productionand antioxidant defense systems in the plasma ofADpatientsThis was substantiated by an increase in DNA lipid andprotein oxidation products found in blood and cerebrospinalfluid (CSF) obtained from AD patients in comparison withcontrols [6]
At the same time microglial cells activation is prominentduring AD Microglia are bone marrow-derived cells of themonocyte lineages that like peripheral tissue macrophagesbecome phagocytic and produce reactive oxygen species [3]However the evaluation of microglia activity during thedevelopment of AD in human patients is impossible due toits localization thus justifying demand for models that useparameters of easy access and reflecting the state of activationof microglia However the studies with monocytes couldcontribute to the understanding of inflammatory compo-nent in AD establishing at the same time the possibilityof using a minimally invasive technique such as bloodcollection
As exposed AD has an important component thatinvolves inflammatory reactions associated with reactivespecies release and lower antioxidant levels Thus this paperaims at verifying blood oxidative markers and monocyteinflammatory parameters in elderly subjects who presentwith very mild cognitive impairment
2 Methods
21 Subjects Patients participating in this study were partof the last wave of Epidoso Project receiving monitoring onaging by a multidisciplinary team [7]
65 elderly subjects (mean age 82 years) were selected forthis study All patients had their functional capacity and labo-ratory profile evaluatedThese seniors were initially subjectedto a clinical survey conducted by a geriatrician consistingof history taking physical examination and evaluation ofmental autonomy through the degree of cognitive functionPatients with a history of cardiovascular disease cancer orchronic inflammatory diseases as well as individuals withcurrent inflammatory alterations assessed by erythrocytesedimentation rate or plasma levels of C-reactive proteinwere not included in this study
The study protocol was submitted to the Ethics Commit-tee of UNIFESP and was approved under number 085903All patients or their guardians signed informed consent
22 Assessment of Patientsrsquo Cognitive Ability Minimentalstate examination was used for screening cognitive deficitsamong patients [8] The maximum score is 30 and scoreslower than 24 were considered indicative of some kind ofcognitive dysfunction
Patients who scored less than 24 points in MMSE werethen tested with CDR (Clinical Dementia Rating) [9 10]
Patients were considered cognitively intact (INT group119899 = 42) when CDR was lt1 while others were included inthe group of patients with probable Alzheimerrsquos disease (ADgroup 119899 = 23) if CDR was ge1
23 Measurements of Erythrocytes Parameters Blood sam-ples from participants were obtained after 12 hours offasting Part of whole blood was used for flow cytometryAfter plasma separation erythrocytes were used to measuretotal glutathione content (TGSH) [11] and the remaininghemolysate to assess the activities of antioxidant enzymes[7] Cu-Zn superoxide dismutase (SOD) catalase (CAT) andglutathione peroxidase (GPx) all adjusted by hemoglobinconcentration
24 Measurements of Plasma Parameters Plasma sampleswere stored at minus80∘C for later determination of 120572-tocopherol120573-carotene lycopene coenzyme Q10 and vitamin C [7] Forextraction 20120583L plasma aliquots were mixed with 100120583Lextraction solvent (50 ethanol proanalysis (pa) 50 1-butanol pa containing 5mg butylhydroxytoluenemL and8 120583mol TocolL as an internal standard) to extract thevitamins and to denature plasma proteins The sample wasthen mixed by vortex for 30 s and centrifuged at 21 000timesgfor 10min at 4∘C The supernatant was transferred to apolypropylene vial This method uses precolumn and reversephase C18 column and UV-VIS detector with deuteriumlamp with a mobile phase consisting of 80 acetonitrile 3methanol (100mM ammonium acetate 01 triethylamine)and 15 dioxan The mobile phase is pumped through thesystem by a flow of 15mLmin with maximum pressureof 350Kgf and the minimum of 0 The wavelength wasestablished by the method for each parameter and run timefor it was 4 to 6 minutes
Plasma was also used to determine the concentration ofthiobarbituric acid reactive substances (TBARS) [7] and levelof oxidized proteins expressed as plasma carbonyls [12]
25 Monocyte Activation Parameters In order to evaluatebasal activation of circulating monocytes cell surface HLA-DR and CD-11b levels were assessed by flow cytometry asdescribed elsewhere [13] Monocytes were identified bymon-oclonal antibodies anti-CD14-pernidin chlorophyll protein(PerCP) neutrophils were excluded with CD66b-fluoresceinisothiocyanate (FITC) labeling Cell surface molecules weremarked with anti-HLA-DR-phycoerythrin (PE) and anti-CD11b-allophycocyanin (APC) controls were incubated withanti-HLA-DR-PE and CD11b-APC isotypes
Intracellular production of IL-1120572 IL-6 and TNF-120572was quantified by flow cytometry in monocytes stimulatedwith LPS and treated with monensin in order to prevent
Oxidative Medicine and Cellular Longevity 3
Table 1 Oxidative stress parameters in erythrocyte (RBC) Antiox-idant enzymes superoxide dismutase (SOD) catalase (CAT) andglutathione peroxidase (GPx) and total glutathione level (TGSH)INT (cognitively intact patients 119899 = 42) and AD (patients withprobable Alzheimerrsquos disease 119899 = 23) Values shown correspondto the means plusmn SEM Comparison between average results ofthe groups was performed using Studentrsquos 119905-test for independentsamples
Parameter INT ADRBC SOD (Umg Hb) 782 plusmn 024 82 plusmn 031
RBC CAT (Umg Hb) 187 plusmn 7 184 plusmn 8
RBC GPx (Umg Hb) 128 plusmn 04 126 plusmn 07
RBC TGSH (mmolHb) 767 plusmn 021 745 plusmn 036
119875 lt 005 was considered significant
cytokine release [13] Monocytes were identified by mon-oclonal antibodies for surface staining (anti-CD66b-FITCand anti-CD14-PerCP) and cytokine productionwas detectedusing monoclonal antibodies anti-IL-1120572-PE anti-IL-6-PEand anti-TNF120572-APC or their respective isotype controls
Data were acquired using a FACSCalibur flow cytometerand CellQuest software (both from BD ImmunocytometrySystems)
26 Reactive Oxygen Species Assay Whole blood sampleswere first stained with anti-CD14 PerCP for monocyte iden-tification samples were subsequently incubated with 2101584071015840-dichlorofluorescein diacetate (DCFH-DA) a stable com-pound nonfluorescent which diffuses into the cells andis hydrolyzed to 2101584071015840-dichlorofluorescein (DCFH) Uponcell stimulation with LPS DCFH was oxidized to DCF byreactive species mostly H
2O2 emitting high levels of green
fluorescence which was detected by flow cytometry [14]using a FACSCalibur flow cytometer and CellQuest software(both from BD Immunocytometry Systems)
27 Statistical Analysis Normality of data distribution wasassessed by Shapiro-Wilk test Comparison between averageresults of the groups was performed using Student t test forindependent samples Correlation analysis between data wasperformed using Spearman correlation test All data wereprocessed and analyzed by appropriate statistical tests usingSPSS 17 software
3 Results
No changes were observed on plasma activity of the majorerythrocyte antioxidant enzymes SOD catalase and glu-tathione peroxidase (Table 1) Table 1 also shows that totalglutathione levels were similar in both cognitively intact(INT) and in group of patients with probable AD
Lipid peroxidation (measured as TBARS) and plasmaticprotein oxidation measured as the amount of circulatingprotein carbonyls did not significantly differ between thegroups INT and AD (Table 2)
Plasma vitamin C levels were equivalent in both groupsas illustrated in Table 2 Values of circulating 120572-tocopherol
Table 2 Oxidative stress parameters in plasma INT (cognitivelyintact patients 119899 = 42) and AD (patients with probable Alzheimerrsquosdisease 119899 = 23) Values shown correspond to the means plusmn SEMComparison between average results of the groups was performedusing Studentrsquos 119905-test for independent samples
Parameter INT ADVitamin C (120583M) 48 plusmn 3 52 plusmn 5
120572-Tocopherol (120583M) 21 plusmn 1 17 plusmn 1lowast
120573-Carotene (120583M) 079 plusmn 09 076 plusmn 1
Lycopene (120583M) 073 plusmn 009 067 plusmn 013
Coenzyme Q10 (120583M) 015 plusmn 002 013 plusmn 003
TBARS (thiobarbituric acid reactivesubstances) (nmolmL) 582 plusmn 051 583 plusmn 062
Oxidized proteins (nmolmg prot) 014 plusmn 001 016 plusmn 002
lowast
119875 lt 005 was considered significant
were about 20 lower in patients with probable Alzheimerrsquosdisease compared with cognitively intact patients This dif-ference was statistically significant It was observed that thetwo groups studied had similar plasma levels of 120573-carotenelycopene and coenzyme Q10 (Table 2)
Table 3 shows that Cu Zn-SOD activity had a positivecorrelation with catalase and glutathione peroxidase activi-ties as well as total glutathione content Catalase activity inturn also had positive correlation with glutathione peroxi-dase activity and total glutathione content in erythrocytesGlutathione peroxidase activity and total glutathione contentin red blood cells also had a significant positive correlationThe same two parameters negatively correlated with lipidperoxidation products in plasma
Monocytes of patients with probable Alzheimerrsquos dis-ease expressed approximately 70 more HLA-DR than thecognitively intact patients On the other hand there wereno differences on the expression of CD11b by monocytesobtained from the two groups (Figure 1 and Table 4)
The proportion of cells producing IL-1120572 was approxi-mately 25 higher in patients with probable diagnosis ofthe disease as compared to those cognitively intact (Table 4)In the case of IL-6 and TNF-120572 there were no statisticallysignificant differences in the proportion of cells that producedthose interleukins (Figure 2 and Table 4)
The oxidativemetabolism ofmonocytes studiedwith flowcytometry techniques showed no differences between groups(Table 4)
4 Discussion
The aim of this study was to investigate the interrelationshipbetween AD blood parameters of oxidative stress andindicators of inflammatory activity in monocytes It followsthe reasoning by which monocytes are circulating cells thatresemble the brain microglia andmay even be recruited fromblood to brain tissue [15] in AD Microglia are consideredthe main element for the localized inflammatory events thataccompany this disease [16] This would lead to localizedinflammation and increased production of reactive oxygen
4 Oxidative Medicine and Cellular Longevity
Forward scatter
Side
scat
ter
1000
800
600
400
200
010008006004002000
R1
100
101
102
103
104
80
0
Cou
nts
CD11b-APC
INT
100
101
102
103
104
80
0
Cou
nts
AD
CD11b-APC
100
101
102
103
104
80
0
Cou
nts
M1
INT isotype
mlgG2b-APC
100
101
102
103
104
80
0
Cou
nts
M1
AD isotype
mlgG2b-APC
100
101
102
103
104
80
0
Cou
nts
AD
HLA-DR-PE
M1
100
101
102
103
104
80
0
Cou
nts
AD isotype
mlgG2a-PE
M1
HLA-DR-PE
INT
100
101
102
103
104
80
0
Cou
nts
CD14-PerCP
CD
66b-
FITC
R3
R2
100
101
102
103
104
100
101
102
103
104
M1
M1
INT isotype
100
101
102
103
104
80
0C
ount
s
mlgG2a-PE
Figure 1 Representative flow cytometric analysis of whole blood samples stainedwith anti-CD11b-APC (right column) and anti-HLA-DR-PE(left column) monoclonal antibodies From all events acquired in gate R1 (upper left graph) CD14-positive monocytes (gate R2 separatedfrom CD66b-positive neutrophils in gate R3 upper right graph) were used for histogram calculation of the geometric mean fluorescenceintensity (GMFI) A similar analysis was performed for the detection of fluorescent DCF in the ROS assay (histogram not shown) INT(sample from cognitively intact patient) and AD (sample from patient with probable Alzheimerrsquos disease)
Oxidative Medicine and Cellular Longevity 5
INT
100
101
102
103
104
100
101
102
103
104
INT + LPS
IL-11205721000
0
1000
0
AD
Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0
Forw
ard
scat
ter AD + LPS
IL-1120572-PE IL-1120572-PE
IL-1120572-PE IL-1120572-PE
(a)
IL-6
IL-6-PE IL-6-PE
IL-6-PE IL-6-PE
INT
100
101
102
103
104
100
101
102
103
104
INT + LPS1000
0
1000
0
AD
Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter AD + LPS
(b)
TNF-120572
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter
INT INT + LPS
AD AD + LPS
TNF120572-APC TNF120572-APC
TNF120572-APC TNF120572-APC
(c)
Figure 2 Representative flow cytometric detection ofmonocyte intracellular production of IL-1120572 IL-6 and TNF-120572 Monocytes were selectedin a similar manner as described in Figure 1 LPS-induced production of each cytokine was determined by the percentage of events in theupper right quadrant (URQ) Only upper quadrants are shown INT (sample from cognitively intact patient) and AD (sample from patientwith probable Alzheimerrsquos disease)
species causing oxidative stress which could be reflected bycirculating parameters [17]
In the conditions of this work AD is not associatedwith any evidence of systemic oxidative stress Althoughthe activities of erythrocyte antioxidant enzymes do notshow changes in patients with AD Spearman rank correla-tion test reveals a positive correlation between the mutualactivities of erythrocyte superoxide dismutase catalase andglutathione peroxidase and erythrocyte total glutathionelevels These data confirm the validity of the postulate thatsays that the bodyrsquos antioxidant systems act together inan integrated manner Spearman test has also identified anegative correlation between the levels of lipid peroxidationproducts plasma glutathione peroxidase activity and totalglutathione levels in erythrocytes This fact indicates that
lipid peroxidation is influenced by the activity of antioxidantglutathione peroxidase-glutathione system in this experi-mental model Indeed glutathione-glutathione peroxidasesystem is an important component of antioxidant protectionnetwork to cells [18] As a counterpoint to the measurementof erythrocyte antioxidant activities circulating levels ofoxidation products of macromolecules in the body have beenevaluated To this end we analyzed levels of plasma proteincarbonyls and plasma levels of lipid peroxidation productsPlasma level of protein oxidation products is not altered inAD compared with cognitively intact group This lack ofchange is found in some studies [18] On the other handsome authors have found an increase of oxidized proteinsin plasma from AD patients [19 20] This difference maybe due to difference of sensitivity of the methods employed
6 Oxidative Medicine and Cellular Longevity
Table 3 Correlation between antioxidant erythrocytes parameters and plasmatic oxidation products Superoxide dismutase (SOD) catalase(CAT) glutathione peroxidase (GPx) total glutathione level (TGSH) and thiobarbituric acid reactive substances (TBARS) Analysis wasperformed using the Spearman correlation test
Spearman test SOD CAT GPX GSH TBARSSOD
Coefficient of correlation 1000 0415lowast 0316lowast 0406lowastlowast 0097Significance 0001 0011 0001 0244
CATCoefficient of correlation 0415lowast 1000 0368lowastlowast 0317lowast 0158Significance 0001 0003 0010 0129
GPXCoefficient of correlation 0316lowast 0368lowastlowast 1000 0416lowastlowast 0323lowastlowast
Significance 0011 0003 0001 0009TGSH
Coefficient of correlation 0406lowastlowast 0317lowast 0416lowastlowast 1000 0253lowast
Significance 0001 0010 0001 0034TBARS
Coefficient of correlation 0097 0158 0323lowastlowast 0253lowast 1000Significance 0244 0129 0009 0034
lowastlowastP lt 001 lowastP lt 005
Table 4 Monocyte activation parameters INT (cognitively intactpatients 119899 = 42) and AD (patients with probable Alzheimerrsquosdisease 119899 = 23) GMFI geometric mean fluorescence intensity URQ percentage of events in the upper right quadrant Valuesshown correspond to the means plusmn SEM Comparison betweenaverage results of the groups was performed using Student 119905 test forindependent samples
Parameter INT ADHLADR (GMFI) 10993 plusmn 1059 17774 plusmn 3064
lowast
CD11B (GMFI) 40674 plusmn 3924 41292 plusmn 4897
IL-1120572 (URQ) 6578 plusmn 246 8282 plusmn 652lowast
IL-6 (URQ) 5289 plusmn 431 4971 plusmn 657
TNF-120572 (URQ) 685 plusmn 128 826 plusmn 247
Oxidative metabolism (GMFI) 26632 plusmn 3899 25777 plusmn 6307lowast
119875 lt 005 was considered significant
in the mentioned studies (two-dimensional electrophoresismass spectrometry) in relation to enzyme-immunoassaymethod chosen for this work and the possible differences inquality of care and feeding among the target populations ofeach study Concentration of circulating lipid peroxidationproducts is equivalent in both groups results that agree withthose commonly found in the literature [18] In generalthe population studied seems to enjoy a privileged statusin relation to nutritional populations examined in similarstudies in other locations In addition to the antioxidantprofile outlined above measurements of plasma levels ofnonenzymatic antioxidants of lowmolecularweightwere alsocarried out
Values of vitamin C 120573-carotene and 120572-tocopherolobtained in this study are within the range previouslydescribed for a healthy population group in that age [721] A review of several published papers performed by
Stocker and Frei [22] shows populational values for plasmaconcentrations of vitamin C 120572-tocopherol and 120573-caroteneranging from 30 to 150 120583M 15 to 40 120583M and 03 to 06 120583Mrespectively Other authors report that for cardiovasculardisease and cancer prevention concentrations above 30120583M120572-tocopherol in combination with concentrations greaterthan 50 120583M of vitamin C 04 120583M 120573-carotene and 05120583Mlycopene are desired [23] Thus the two groups of elderlypatients studied have positioned themselves within the limitsfound in several other studies to molecular plasma levels ofantioxidantsThese parameters reflect part of the antioxidantdefense system of the body of patients evaluating compo-nents that can be absorbed directly from the diet so furtherpromoted by the antioxidant defense mentioned above Ofall lowmolecular weight antioxidants studied only vitamin Esignificantly differs between groupsThe INT group has levelsof plasma vitamin E higher than those in AD patients
This apparent homogeneity in antioxidant levels betweenthe various groups opposed to other studies in the literature[24ndash27] This finding as previously mentioned reinforcesthe notion that the oxidative processes that are triggeredduring the course of AD are not always reflected in circu-lating blood and even when that occurs it may be onlyan indication of other underlying mechanisms Of specialinterest is the fact that the establishment of AD expressedby the comparison between the INT and AD groups inour study is accompanied by the decrease of circulatingvitamin E which possesses antioxidant capabilities as wellas recognized anti-inflammatory activities due to its actionon signal transduction mechanisms in phagocytes [28 29]Not surprisingly studies that have obtained the best results inpreventing or treating AD in recent years used 120572-tocopherolas an agent [30]
To complete the picture outlined up to this point circulat-ingmonocytes were analyzed by flow cytometry as indicators
Oxidative Medicine and Cellular Longevity 7
of inflammatory activity involved in AD To this end wequantified the level of expression of surface molecules HLA-DR as an indicator of activation of circulating monocytesand CD-11b an adhesion molecule involved in the processof marginalization and migration of monocytes The resultsshow that AD patients have circulating monocytes thatexpress higher amounts of HLA-DR on the surface evenwithout external stimulus indicating a higher basal activationstate of these cells confirming the data available in theliterature [31] Markers of increased activation of monocytesin the bloodstream are found in patients from a number ofneurodegenerative diseases [32] As previously mentionedthese circulatingmonocytes can cross the blood-brain barrierandmigrate to the brain tissue differentiating into microgliaSince circulating monocytes of AD patients are more activeat baseline than those of INT it can be postulated thatmicroglia derived from these cells react more intensely toinflammatory stimuli producing greater injury to brainLevel of expression of surface protein CD-11b is the same inboth groups indicating that the potential for adhesion andmigration of these cells is not altered in this pathology
Also by flow cytometry monocytes production of IL-1120572 IL-6 and TNF-120572 was measured This measurement wasintended to evaluate the capacity of circulating monocytesto stimulate the inflammatory process by attracting newmonocytes to the inflammatory site The production of IL-1120572is higher in monocytes from AD patients than in INT groupSince IL-1120572 is an important proinflammatory cytokine it isplausible to assume thatmigration anddifferentiation of thesemonocytes produce more active microglia This increasedproduction of IL-1120572 was shown in vitro in human monocyteslineage when stimulated by P120573A by means of mechanismsof signal transduction mediated by tyrosine kinases whichreinforces the above hypothesis [33]
Levels of IL-6 produced by stimulated peripheral mono-cytes are similar in both groups as well as of TNF-120572 agreeingwith Beloosesky et al [34] Other authors detected anincrease in IL-6 production bymonocytes of ADpatients [35]using however a different stimulus (phytohemagglutinin)than that used in this present study (LPS)
From the data shown here it is reasonable to concludethat AD is accompanied by the activation of circulatingmonocytes and a decrease in circulating levels of vitamin ESeveral studies show 120572-tocopherol modulating inflammationactivity [28 36] In our study the group of patients whereplasma 120572-tocopherol is decreased also showed more acti-vated monocytes that respond to stimuli with an increasedproduction of proinflammatory cytokine IL-1120572 This studypoints out that AD is associated with a higher basal activationof circulating monocytes that may be a result of 120572-tocopherolstock depletion
Abbreviations
AD Alzheimerrsquos diseaseAPC AllophycocyaninCDR Clinical dementia ratingFITC Fluorescein isothiocyanateINT Cognitively intact group
GMFI Geometric mean fluorescenceintensity
LPS LipopolysaccharideMMSE Minimental state examinationPE PhycoerythrinPerCP Periidinin chlorophyll proteinROS Reactive oxygen speciesURQ Upper right quadrant
References
[1] B C Dickerson A Bakkour D H Salat et al ldquoThe corticalsignature of Alzheimerrsquos disease regionally specific corticalthinning relates to symptom severity in very mild to mild ADdementia and is detectable in asymptomatic amyloid-positiveindividualsrdquo Cerebral Cortex vol 19 no 3 pp 497ndash510 2009
[2] A Jana and K Pahan ldquoFibrillar amyloid-120573 peptides kill humanprimary neurons via NADPH oxidase-mediated activation ofneutral sphingomyelinase Implications for Alzheimerrsquos dis-easerdquo Journal of Biological Chemistry vol 279 no 49 pp 51451ndash51459 2004
[3] C E Finch and T E Morgan ldquoInflammatory processes ofAlzheimer disease and agingrdquoProceedings of the IndianNationalScience Academy B vol 69 pp 165ndash178 2003
[4] E Zotova J A Nicoll R Kalaria C Holmes and D BocheldquoInflammation in Alzheimerrsquos disease relevance to pathogene-sis and therapyrdquo Alzheimerrsquos Research and Therapy vol 2 no 1article 1 2010
[5] L E Rojo J A Fernandez A A Maccioni J M Jimenezand R B Maccioni ldquoNeuroinflammation implications for thepathogenesis and molecular diagnosis of Alzheimerrsquos diseaserdquoArchives of Medical Research vol 39 no 1 pp 1ndash16 2008
[6] K Jomova D Vondrakova M Lawson and M Valko ldquoMetalsoxidative stress and neurodegenerative disordersrdquo Molecularand Cellular Biochemistry vol 345 no 1-2 pp 91ndash104 2010
[7] V B C Junqueira S B M Barros S S Chan et al ldquoAging andoxidative stressrdquoMolecular Aspects of Medicine vol 25 no 1-2pp 5ndash16 2004
[8] M F Folstein S E Folstein and P R McHugh ldquoMini-mentalstate A practical method for grading the cognitive state ofpatients for the clinicianrdquo Journal of Psychiatric Research vol12 no 3 pp 189ndash198 1975
[9] J C Morris ldquoThe Clinical Dementia Rating (CDR) currentversion and scoring rulesrdquo Neurology vol 43 no 11 pp 2412ndash2414 1993
[10] M B MMontano and L R Ramos ldquoValidity of the Portugueseversion of Clinical Dementia Ratingrdquo Revista de Saude Publicavol 39 no 6 pp 912ndash917 2005
[11] F Tietze ldquoEnzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione appli-cations to mammalian blood and other tissuesrdquo AnalyticalBiochemistry vol 27 no 3 pp 502ndash522 1969
[12] H Buss T P Chan K B Sluis N B Domigan and C CWinterbourn ldquoProtein carbonyl measurement by a sensitiveELISA methodrdquo Free Radical Biology and Medicine vol 23 no3 pp 361ndash366 1998
[13] M K C Brunialti E G Kallas M Freudenberg C Galanosand R Salomao ldquoInfluence of EDTA and heparin onlipopolysaccharide binding and cell activation evaluatedat single-cell level in whole bloodrdquo Clinical Cytometry vol 50no 1 pp 14ndash18 2002
8 Oxidative Medicine and Cellular Longevity
[14] M Hasui Y Hirabayashi and Y Kobayashi ldquoSimultaneousmeasurement by flow cytometry of phagocytosis and hydrogenperoxide production of neutrophils in whole bloodrdquo Journal ofImmunological Methods vol 117 no 1 pp 53ndash58 1989
[15] J Wegiel H Imaki K-C Wang et al ldquoOrigin and turnover ofmicroglial cells in fibrillar plaques of APPsw transgenic micerdquoActa Neuropathologica vol 105 no 4 pp 393ndash402 2003
[16] M R DrsquoAndrea G M Cole and M D Ard ldquoThe microglialphagocytic role with specific plaque types in the Alzheimerdisease brainrdquoNeurobiology of Aging vol 25 no 5 pp 675ndash6832004
[17] J Vina A Lloret ROrtı andDAlonso ldquoMolecular bases of thetreatment of Alzheimerrsquos disease with antioxidants preventionof oxidative stressrdquo Molecular Aspects of Medicine vol 25 no1-2 pp 117ndash123 2004
[18] M C Polidori P Mattioli S Aldred et al ldquoPlasma antioxidantstatus immunoglobulin G oxidation and lipid peroxidation indemented patients relevance to Alzheimer disease and vasculardementiardquo Dementia and Geriatric Cognitive Disorders vol 18no 3-4 pp 265ndash270 2004
[19] C C Conrad P L Marshall J M Talent C A MalakowskyJ Choi and R W Gracy ldquoOxidized proteins in Alzheimerrsquosplasmardquo Biochemical and Biophysical Research Communica-tions vol 275 no 2 pp 678ndash681 2000
[20] J Choi C A Malakowsky J M Talent C C Conradand R W Gracy ldquoIdentification of oxidized plasma proteinsin Alzheimerrsquos diseaserdquo Biochemical and Biophysical ResearchCommunications vol 293 no 5 pp 1566ndash1570 2002
[21] H Heseker and R Schneider ldquoRequirement and supply ofvitamin C E and 120573-carotene for elderly men and womenrdquoEuropean Journal of Clinical Nutrition vol 48 no 2 pp 118ndash1271994
[22] R Stocker and B Frei ldquoEndogenous antioxidant defencesin human blood plasmardquo in Oxidative Stress Oxidants andAntioxidants H Sies Ed Academic Press London UK 1991
[23] K F Gey ldquoPrevention of early stages of cardiovascular diseaseand cancer may require concurrent optimization of all majorantioxidants and other nutrients an update and reevaluationof observational data and intervention trialsrdquo in Free RadicalsLipoprotein Oxidation and Atherosclerosis C Rice-Evans EdRichelieu Press London UK 1995
[24] C Behl and B Moosmann ldquoAntioxidant neuroprotection inAlzheimerrsquos disease as preventive and therapeutic approachrdquoFree Radical Biology and Medicine vol 33 no 2 pp 182ndash1912002
[25] E Esposito D Rotilio V di Matteo C di Giulio M Cacchioand S Algeri ldquoA review of specific dietary antioxidants and theeffects on biochemical mechanisms related to neurodegenera-tive processesrdquoNeurobiology of Aging vol 23 no 5 pp 719ndash7352002
[26] P Mecocci M Cristina Polidori A Cherubini et al ldquoLym-phocyte oxidative DNA damage and plasma antioxidants inAlzheimer diseaserdquoArchives ofNeurology vol 59 no 5 pp 794ndash798 2002
[27] M Sano ldquoDo dietary antioxidants prevent Alzheimerrsquos dis-easerdquoThe Lancet Neurology vol 1 no 6 p 342 2002
[28] S S Chan H P Monteiro F Schindler A Stern and V B CJunqueira ldquo120572-tocopherol modulates tyrosine phosphorylationin human neutrophils by inhibition of protein kinas C activityand activation of tyrosine phosphatasesrdquo Free Radical Researchvol 35 no 6 pp 843ndash856 2001
[29] E M Bulger and R V Maier ldquoAn argument for vitamin Esupplementation in the management of systemic inflammatoryresponse syndromerdquo Shock vol 19 no 2 pp 99ndash103 2003
[30] P P Zandi J C Anthony A S Khachaturian et al ldquoReducedrisk of Alzheimer disease in users of antioxidant vitaminsupplements the Cache County studyrdquo Archives of Neurologyvol 61 no 1 pp 82ndash88 2004
[31] L Kusdra H Rempel K Yaffe and L Pulliam ldquoElevation ofCD69+ monocytemacrophages in patients with Alzheimerrsquosdiseaserdquo Immunobiology vol 202 no 1 pp 26ndash33 2000
[32] J A Casal A Robles and J C Tutor ldquoSerum markers ofmonocytemacrophage activation in patients with Alzheimerrsquosdisease and other types of dementiardquo Clinical Biochemistry vol36 no 7 pp 553ndash556 2003
[33] C K Combs J Colleen Karlo S-C Kao and G E Landrethldquo120573-amyloid stimulation of microglia anti monocytes results inTNF120572-dependent expression of inducible nitric oxide synthaseand neuronal apoptosisrdquo Journal of Neuroscience vol 21 no 4pp 1179ndash1188 2001
[34] Y Beloosesky H Salman M Bergman H Bessler and MDjaldetti ldquoCytokine levels and phagocytic activity in patientswith Alzheimerrsquos diseaserdquo Gerontology vol 48 no 3 pp 128ndash132 2002
[35] F Shalit B Sredni L Stern E Kott and M HubermanldquoElevated interleukin-6 secretion levels by mononuclear cells ofAlzheimerrsquos patientsrdquo Neuroscience Letters vol 174 no 2 pp130ndash132 1994
[36] A Azzi R Gysin P Kempna et al ldquoThe role of 120572-tocopherolin preventing disease from epidemiology to molecular eventsrdquoMolecular Aspects of Medicine vol 24 no 6 pp 325ndash336 2003
Submit your manuscripts athttpwwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
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Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 3
Table 1 Oxidative stress parameters in erythrocyte (RBC) Antiox-idant enzymes superoxide dismutase (SOD) catalase (CAT) andglutathione peroxidase (GPx) and total glutathione level (TGSH)INT (cognitively intact patients 119899 = 42) and AD (patients withprobable Alzheimerrsquos disease 119899 = 23) Values shown correspondto the means plusmn SEM Comparison between average results ofthe groups was performed using Studentrsquos 119905-test for independentsamples
Parameter INT ADRBC SOD (Umg Hb) 782 plusmn 024 82 plusmn 031
RBC CAT (Umg Hb) 187 plusmn 7 184 plusmn 8
RBC GPx (Umg Hb) 128 plusmn 04 126 plusmn 07
RBC TGSH (mmolHb) 767 plusmn 021 745 plusmn 036
119875 lt 005 was considered significant
cytokine release [13] Monocytes were identified by mon-oclonal antibodies for surface staining (anti-CD66b-FITCand anti-CD14-PerCP) and cytokine productionwas detectedusing monoclonal antibodies anti-IL-1120572-PE anti-IL-6-PEand anti-TNF120572-APC or their respective isotype controls
Data were acquired using a FACSCalibur flow cytometerand CellQuest software (both from BD ImmunocytometrySystems)
26 Reactive Oxygen Species Assay Whole blood sampleswere first stained with anti-CD14 PerCP for monocyte iden-tification samples were subsequently incubated with 2101584071015840-dichlorofluorescein diacetate (DCFH-DA) a stable com-pound nonfluorescent which diffuses into the cells andis hydrolyzed to 2101584071015840-dichlorofluorescein (DCFH) Uponcell stimulation with LPS DCFH was oxidized to DCF byreactive species mostly H
2O2 emitting high levels of green
fluorescence which was detected by flow cytometry [14]using a FACSCalibur flow cytometer and CellQuest software(both from BD Immunocytometry Systems)
27 Statistical Analysis Normality of data distribution wasassessed by Shapiro-Wilk test Comparison between averageresults of the groups was performed using Student t test forindependent samples Correlation analysis between data wasperformed using Spearman correlation test All data wereprocessed and analyzed by appropriate statistical tests usingSPSS 17 software
3 Results
No changes were observed on plasma activity of the majorerythrocyte antioxidant enzymes SOD catalase and glu-tathione peroxidase (Table 1) Table 1 also shows that totalglutathione levels were similar in both cognitively intact(INT) and in group of patients with probable AD
Lipid peroxidation (measured as TBARS) and plasmaticprotein oxidation measured as the amount of circulatingprotein carbonyls did not significantly differ between thegroups INT and AD (Table 2)
Plasma vitamin C levels were equivalent in both groupsas illustrated in Table 2 Values of circulating 120572-tocopherol
Table 2 Oxidative stress parameters in plasma INT (cognitivelyintact patients 119899 = 42) and AD (patients with probable Alzheimerrsquosdisease 119899 = 23) Values shown correspond to the means plusmn SEMComparison between average results of the groups was performedusing Studentrsquos 119905-test for independent samples
Parameter INT ADVitamin C (120583M) 48 plusmn 3 52 plusmn 5
120572-Tocopherol (120583M) 21 plusmn 1 17 plusmn 1lowast
120573-Carotene (120583M) 079 plusmn 09 076 plusmn 1
Lycopene (120583M) 073 plusmn 009 067 plusmn 013
Coenzyme Q10 (120583M) 015 plusmn 002 013 plusmn 003
TBARS (thiobarbituric acid reactivesubstances) (nmolmL) 582 plusmn 051 583 plusmn 062
Oxidized proteins (nmolmg prot) 014 plusmn 001 016 plusmn 002
lowast
119875 lt 005 was considered significant
were about 20 lower in patients with probable Alzheimerrsquosdisease compared with cognitively intact patients This dif-ference was statistically significant It was observed that thetwo groups studied had similar plasma levels of 120573-carotenelycopene and coenzyme Q10 (Table 2)
Table 3 shows that Cu Zn-SOD activity had a positivecorrelation with catalase and glutathione peroxidase activi-ties as well as total glutathione content Catalase activity inturn also had positive correlation with glutathione peroxi-dase activity and total glutathione content in erythrocytesGlutathione peroxidase activity and total glutathione contentin red blood cells also had a significant positive correlationThe same two parameters negatively correlated with lipidperoxidation products in plasma
Monocytes of patients with probable Alzheimerrsquos dis-ease expressed approximately 70 more HLA-DR than thecognitively intact patients On the other hand there wereno differences on the expression of CD11b by monocytesobtained from the two groups (Figure 1 and Table 4)
The proportion of cells producing IL-1120572 was approxi-mately 25 higher in patients with probable diagnosis ofthe disease as compared to those cognitively intact (Table 4)In the case of IL-6 and TNF-120572 there were no statisticallysignificant differences in the proportion of cells that producedthose interleukins (Figure 2 and Table 4)
The oxidativemetabolism ofmonocytes studiedwith flowcytometry techniques showed no differences between groups(Table 4)
4 Discussion
The aim of this study was to investigate the interrelationshipbetween AD blood parameters of oxidative stress andindicators of inflammatory activity in monocytes It followsthe reasoning by which monocytes are circulating cells thatresemble the brain microglia andmay even be recruited fromblood to brain tissue [15] in AD Microglia are consideredthe main element for the localized inflammatory events thataccompany this disease [16] This would lead to localizedinflammation and increased production of reactive oxygen
4 Oxidative Medicine and Cellular Longevity
Forward scatter
Side
scat
ter
1000
800
600
400
200
010008006004002000
R1
100
101
102
103
104
80
0
Cou
nts
CD11b-APC
INT
100
101
102
103
104
80
0
Cou
nts
AD
CD11b-APC
100
101
102
103
104
80
0
Cou
nts
M1
INT isotype
mlgG2b-APC
100
101
102
103
104
80
0
Cou
nts
M1
AD isotype
mlgG2b-APC
100
101
102
103
104
80
0
Cou
nts
AD
HLA-DR-PE
M1
100
101
102
103
104
80
0
Cou
nts
AD isotype
mlgG2a-PE
M1
HLA-DR-PE
INT
100
101
102
103
104
80
0
Cou
nts
CD14-PerCP
CD
66b-
FITC
R3
R2
100
101
102
103
104
100
101
102
103
104
M1
M1
INT isotype
100
101
102
103
104
80
0C
ount
s
mlgG2a-PE
Figure 1 Representative flow cytometric analysis of whole blood samples stainedwith anti-CD11b-APC (right column) and anti-HLA-DR-PE(left column) monoclonal antibodies From all events acquired in gate R1 (upper left graph) CD14-positive monocytes (gate R2 separatedfrom CD66b-positive neutrophils in gate R3 upper right graph) were used for histogram calculation of the geometric mean fluorescenceintensity (GMFI) A similar analysis was performed for the detection of fluorescent DCF in the ROS assay (histogram not shown) INT(sample from cognitively intact patient) and AD (sample from patient with probable Alzheimerrsquos disease)
Oxidative Medicine and Cellular Longevity 5
INT
100
101
102
103
104
100
101
102
103
104
INT + LPS
IL-11205721000
0
1000
0
AD
Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0
Forw
ard
scat
ter AD + LPS
IL-1120572-PE IL-1120572-PE
IL-1120572-PE IL-1120572-PE
(a)
IL-6
IL-6-PE IL-6-PE
IL-6-PE IL-6-PE
INT
100
101
102
103
104
100
101
102
103
104
INT + LPS1000
0
1000
0
AD
Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter AD + LPS
(b)
TNF-120572
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter
INT INT + LPS
AD AD + LPS
TNF120572-APC TNF120572-APC
TNF120572-APC TNF120572-APC
(c)
Figure 2 Representative flow cytometric detection ofmonocyte intracellular production of IL-1120572 IL-6 and TNF-120572 Monocytes were selectedin a similar manner as described in Figure 1 LPS-induced production of each cytokine was determined by the percentage of events in theupper right quadrant (URQ) Only upper quadrants are shown INT (sample from cognitively intact patient) and AD (sample from patientwith probable Alzheimerrsquos disease)
species causing oxidative stress which could be reflected bycirculating parameters [17]
In the conditions of this work AD is not associatedwith any evidence of systemic oxidative stress Althoughthe activities of erythrocyte antioxidant enzymes do notshow changes in patients with AD Spearman rank correla-tion test reveals a positive correlation between the mutualactivities of erythrocyte superoxide dismutase catalase andglutathione peroxidase and erythrocyte total glutathionelevels These data confirm the validity of the postulate thatsays that the bodyrsquos antioxidant systems act together inan integrated manner Spearman test has also identified anegative correlation between the levels of lipid peroxidationproducts plasma glutathione peroxidase activity and totalglutathione levels in erythrocytes This fact indicates that
lipid peroxidation is influenced by the activity of antioxidantglutathione peroxidase-glutathione system in this experi-mental model Indeed glutathione-glutathione peroxidasesystem is an important component of antioxidant protectionnetwork to cells [18] As a counterpoint to the measurementof erythrocyte antioxidant activities circulating levels ofoxidation products of macromolecules in the body have beenevaluated To this end we analyzed levels of plasma proteincarbonyls and plasma levels of lipid peroxidation productsPlasma level of protein oxidation products is not altered inAD compared with cognitively intact group This lack ofchange is found in some studies [18] On the other handsome authors have found an increase of oxidized proteinsin plasma from AD patients [19 20] This difference maybe due to difference of sensitivity of the methods employed
6 Oxidative Medicine and Cellular Longevity
Table 3 Correlation between antioxidant erythrocytes parameters and plasmatic oxidation products Superoxide dismutase (SOD) catalase(CAT) glutathione peroxidase (GPx) total glutathione level (TGSH) and thiobarbituric acid reactive substances (TBARS) Analysis wasperformed using the Spearman correlation test
Spearman test SOD CAT GPX GSH TBARSSOD
Coefficient of correlation 1000 0415lowast 0316lowast 0406lowastlowast 0097Significance 0001 0011 0001 0244
CATCoefficient of correlation 0415lowast 1000 0368lowastlowast 0317lowast 0158Significance 0001 0003 0010 0129
GPXCoefficient of correlation 0316lowast 0368lowastlowast 1000 0416lowastlowast 0323lowastlowast
Significance 0011 0003 0001 0009TGSH
Coefficient of correlation 0406lowastlowast 0317lowast 0416lowastlowast 1000 0253lowast
Significance 0001 0010 0001 0034TBARS
Coefficient of correlation 0097 0158 0323lowastlowast 0253lowast 1000Significance 0244 0129 0009 0034
lowastlowastP lt 001 lowastP lt 005
Table 4 Monocyte activation parameters INT (cognitively intactpatients 119899 = 42) and AD (patients with probable Alzheimerrsquosdisease 119899 = 23) GMFI geometric mean fluorescence intensity URQ percentage of events in the upper right quadrant Valuesshown correspond to the means plusmn SEM Comparison betweenaverage results of the groups was performed using Student 119905 test forindependent samples
Parameter INT ADHLADR (GMFI) 10993 plusmn 1059 17774 plusmn 3064
lowast
CD11B (GMFI) 40674 plusmn 3924 41292 plusmn 4897
IL-1120572 (URQ) 6578 plusmn 246 8282 plusmn 652lowast
IL-6 (URQ) 5289 plusmn 431 4971 plusmn 657
TNF-120572 (URQ) 685 plusmn 128 826 plusmn 247
Oxidative metabolism (GMFI) 26632 plusmn 3899 25777 plusmn 6307lowast
119875 lt 005 was considered significant
in the mentioned studies (two-dimensional electrophoresismass spectrometry) in relation to enzyme-immunoassaymethod chosen for this work and the possible differences inquality of care and feeding among the target populations ofeach study Concentration of circulating lipid peroxidationproducts is equivalent in both groups results that agree withthose commonly found in the literature [18] In generalthe population studied seems to enjoy a privileged statusin relation to nutritional populations examined in similarstudies in other locations In addition to the antioxidantprofile outlined above measurements of plasma levels ofnonenzymatic antioxidants of lowmolecularweightwere alsocarried out
Values of vitamin C 120573-carotene and 120572-tocopherolobtained in this study are within the range previouslydescribed for a healthy population group in that age [721] A review of several published papers performed by
Stocker and Frei [22] shows populational values for plasmaconcentrations of vitamin C 120572-tocopherol and 120573-caroteneranging from 30 to 150 120583M 15 to 40 120583M and 03 to 06 120583Mrespectively Other authors report that for cardiovasculardisease and cancer prevention concentrations above 30120583M120572-tocopherol in combination with concentrations greaterthan 50 120583M of vitamin C 04 120583M 120573-carotene and 05120583Mlycopene are desired [23] Thus the two groups of elderlypatients studied have positioned themselves within the limitsfound in several other studies to molecular plasma levels ofantioxidantsThese parameters reflect part of the antioxidantdefense system of the body of patients evaluating compo-nents that can be absorbed directly from the diet so furtherpromoted by the antioxidant defense mentioned above Ofall lowmolecular weight antioxidants studied only vitamin Esignificantly differs between groupsThe INT group has levelsof plasma vitamin E higher than those in AD patients
This apparent homogeneity in antioxidant levels betweenthe various groups opposed to other studies in the literature[24ndash27] This finding as previously mentioned reinforcesthe notion that the oxidative processes that are triggeredduring the course of AD are not always reflected in circu-lating blood and even when that occurs it may be onlyan indication of other underlying mechanisms Of specialinterest is the fact that the establishment of AD expressedby the comparison between the INT and AD groups inour study is accompanied by the decrease of circulatingvitamin E which possesses antioxidant capabilities as wellas recognized anti-inflammatory activities due to its actionon signal transduction mechanisms in phagocytes [28 29]Not surprisingly studies that have obtained the best results inpreventing or treating AD in recent years used 120572-tocopherolas an agent [30]
To complete the picture outlined up to this point circulat-ingmonocytes were analyzed by flow cytometry as indicators
Oxidative Medicine and Cellular Longevity 7
of inflammatory activity involved in AD To this end wequantified the level of expression of surface molecules HLA-DR as an indicator of activation of circulating monocytesand CD-11b an adhesion molecule involved in the processof marginalization and migration of monocytes The resultsshow that AD patients have circulating monocytes thatexpress higher amounts of HLA-DR on the surface evenwithout external stimulus indicating a higher basal activationstate of these cells confirming the data available in theliterature [31] Markers of increased activation of monocytesin the bloodstream are found in patients from a number ofneurodegenerative diseases [32] As previously mentionedthese circulatingmonocytes can cross the blood-brain barrierandmigrate to the brain tissue differentiating into microgliaSince circulating monocytes of AD patients are more activeat baseline than those of INT it can be postulated thatmicroglia derived from these cells react more intensely toinflammatory stimuli producing greater injury to brainLevel of expression of surface protein CD-11b is the same inboth groups indicating that the potential for adhesion andmigration of these cells is not altered in this pathology
Also by flow cytometry monocytes production of IL-1120572 IL-6 and TNF-120572 was measured This measurement wasintended to evaluate the capacity of circulating monocytesto stimulate the inflammatory process by attracting newmonocytes to the inflammatory site The production of IL-1120572is higher in monocytes from AD patients than in INT groupSince IL-1120572 is an important proinflammatory cytokine it isplausible to assume thatmigration anddifferentiation of thesemonocytes produce more active microglia This increasedproduction of IL-1120572 was shown in vitro in human monocyteslineage when stimulated by P120573A by means of mechanismsof signal transduction mediated by tyrosine kinases whichreinforces the above hypothesis [33]
Levels of IL-6 produced by stimulated peripheral mono-cytes are similar in both groups as well as of TNF-120572 agreeingwith Beloosesky et al [34] Other authors detected anincrease in IL-6 production bymonocytes of ADpatients [35]using however a different stimulus (phytohemagglutinin)than that used in this present study (LPS)
From the data shown here it is reasonable to concludethat AD is accompanied by the activation of circulatingmonocytes and a decrease in circulating levels of vitamin ESeveral studies show 120572-tocopherol modulating inflammationactivity [28 36] In our study the group of patients whereplasma 120572-tocopherol is decreased also showed more acti-vated monocytes that respond to stimuli with an increasedproduction of proinflammatory cytokine IL-1120572 This studypoints out that AD is associated with a higher basal activationof circulating monocytes that may be a result of 120572-tocopherolstock depletion
Abbreviations
AD Alzheimerrsquos diseaseAPC AllophycocyaninCDR Clinical dementia ratingFITC Fluorescein isothiocyanateINT Cognitively intact group
GMFI Geometric mean fluorescenceintensity
LPS LipopolysaccharideMMSE Minimental state examinationPE PhycoerythrinPerCP Periidinin chlorophyll proteinROS Reactive oxygen speciesURQ Upper right quadrant
References
[1] B C Dickerson A Bakkour D H Salat et al ldquoThe corticalsignature of Alzheimerrsquos disease regionally specific corticalthinning relates to symptom severity in very mild to mild ADdementia and is detectable in asymptomatic amyloid-positiveindividualsrdquo Cerebral Cortex vol 19 no 3 pp 497ndash510 2009
[2] A Jana and K Pahan ldquoFibrillar amyloid-120573 peptides kill humanprimary neurons via NADPH oxidase-mediated activation ofneutral sphingomyelinase Implications for Alzheimerrsquos dis-easerdquo Journal of Biological Chemistry vol 279 no 49 pp 51451ndash51459 2004
[3] C E Finch and T E Morgan ldquoInflammatory processes ofAlzheimer disease and agingrdquoProceedings of the IndianNationalScience Academy B vol 69 pp 165ndash178 2003
[4] E Zotova J A Nicoll R Kalaria C Holmes and D BocheldquoInflammation in Alzheimerrsquos disease relevance to pathogene-sis and therapyrdquo Alzheimerrsquos Research and Therapy vol 2 no 1article 1 2010
[5] L E Rojo J A Fernandez A A Maccioni J M Jimenezand R B Maccioni ldquoNeuroinflammation implications for thepathogenesis and molecular diagnosis of Alzheimerrsquos diseaserdquoArchives of Medical Research vol 39 no 1 pp 1ndash16 2008
[6] K Jomova D Vondrakova M Lawson and M Valko ldquoMetalsoxidative stress and neurodegenerative disordersrdquo Molecularand Cellular Biochemistry vol 345 no 1-2 pp 91ndash104 2010
[7] V B C Junqueira S B M Barros S S Chan et al ldquoAging andoxidative stressrdquoMolecular Aspects of Medicine vol 25 no 1-2pp 5ndash16 2004
[8] M F Folstein S E Folstein and P R McHugh ldquoMini-mentalstate A practical method for grading the cognitive state ofpatients for the clinicianrdquo Journal of Psychiatric Research vol12 no 3 pp 189ndash198 1975
[9] J C Morris ldquoThe Clinical Dementia Rating (CDR) currentversion and scoring rulesrdquo Neurology vol 43 no 11 pp 2412ndash2414 1993
[10] M B MMontano and L R Ramos ldquoValidity of the Portugueseversion of Clinical Dementia Ratingrdquo Revista de Saude Publicavol 39 no 6 pp 912ndash917 2005
[11] F Tietze ldquoEnzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione appli-cations to mammalian blood and other tissuesrdquo AnalyticalBiochemistry vol 27 no 3 pp 502ndash522 1969
[12] H Buss T P Chan K B Sluis N B Domigan and C CWinterbourn ldquoProtein carbonyl measurement by a sensitiveELISA methodrdquo Free Radical Biology and Medicine vol 23 no3 pp 361ndash366 1998
[13] M K C Brunialti E G Kallas M Freudenberg C Galanosand R Salomao ldquoInfluence of EDTA and heparin onlipopolysaccharide binding and cell activation evaluatedat single-cell level in whole bloodrdquo Clinical Cytometry vol 50no 1 pp 14ndash18 2002
8 Oxidative Medicine and Cellular Longevity
[14] M Hasui Y Hirabayashi and Y Kobayashi ldquoSimultaneousmeasurement by flow cytometry of phagocytosis and hydrogenperoxide production of neutrophils in whole bloodrdquo Journal ofImmunological Methods vol 117 no 1 pp 53ndash58 1989
[15] J Wegiel H Imaki K-C Wang et al ldquoOrigin and turnover ofmicroglial cells in fibrillar plaques of APPsw transgenic micerdquoActa Neuropathologica vol 105 no 4 pp 393ndash402 2003
[16] M R DrsquoAndrea G M Cole and M D Ard ldquoThe microglialphagocytic role with specific plaque types in the Alzheimerdisease brainrdquoNeurobiology of Aging vol 25 no 5 pp 675ndash6832004
[17] J Vina A Lloret ROrtı andDAlonso ldquoMolecular bases of thetreatment of Alzheimerrsquos disease with antioxidants preventionof oxidative stressrdquo Molecular Aspects of Medicine vol 25 no1-2 pp 117ndash123 2004
[18] M C Polidori P Mattioli S Aldred et al ldquoPlasma antioxidantstatus immunoglobulin G oxidation and lipid peroxidation indemented patients relevance to Alzheimer disease and vasculardementiardquo Dementia and Geriatric Cognitive Disorders vol 18no 3-4 pp 265ndash270 2004
[19] C C Conrad P L Marshall J M Talent C A MalakowskyJ Choi and R W Gracy ldquoOxidized proteins in Alzheimerrsquosplasmardquo Biochemical and Biophysical Research Communica-tions vol 275 no 2 pp 678ndash681 2000
[20] J Choi C A Malakowsky J M Talent C C Conradand R W Gracy ldquoIdentification of oxidized plasma proteinsin Alzheimerrsquos diseaserdquo Biochemical and Biophysical ResearchCommunications vol 293 no 5 pp 1566ndash1570 2002
[21] H Heseker and R Schneider ldquoRequirement and supply ofvitamin C E and 120573-carotene for elderly men and womenrdquoEuropean Journal of Clinical Nutrition vol 48 no 2 pp 118ndash1271994
[22] R Stocker and B Frei ldquoEndogenous antioxidant defencesin human blood plasmardquo in Oxidative Stress Oxidants andAntioxidants H Sies Ed Academic Press London UK 1991
[23] K F Gey ldquoPrevention of early stages of cardiovascular diseaseand cancer may require concurrent optimization of all majorantioxidants and other nutrients an update and reevaluationof observational data and intervention trialsrdquo in Free RadicalsLipoprotein Oxidation and Atherosclerosis C Rice-Evans EdRichelieu Press London UK 1995
[24] C Behl and B Moosmann ldquoAntioxidant neuroprotection inAlzheimerrsquos disease as preventive and therapeutic approachrdquoFree Radical Biology and Medicine vol 33 no 2 pp 182ndash1912002
[25] E Esposito D Rotilio V di Matteo C di Giulio M Cacchioand S Algeri ldquoA review of specific dietary antioxidants and theeffects on biochemical mechanisms related to neurodegenera-tive processesrdquoNeurobiology of Aging vol 23 no 5 pp 719ndash7352002
[26] P Mecocci M Cristina Polidori A Cherubini et al ldquoLym-phocyte oxidative DNA damage and plasma antioxidants inAlzheimer diseaserdquoArchives ofNeurology vol 59 no 5 pp 794ndash798 2002
[27] M Sano ldquoDo dietary antioxidants prevent Alzheimerrsquos dis-easerdquoThe Lancet Neurology vol 1 no 6 p 342 2002
[28] S S Chan H P Monteiro F Schindler A Stern and V B CJunqueira ldquo120572-tocopherol modulates tyrosine phosphorylationin human neutrophils by inhibition of protein kinas C activityand activation of tyrosine phosphatasesrdquo Free Radical Researchvol 35 no 6 pp 843ndash856 2001
[29] E M Bulger and R V Maier ldquoAn argument for vitamin Esupplementation in the management of systemic inflammatoryresponse syndromerdquo Shock vol 19 no 2 pp 99ndash103 2003
[30] P P Zandi J C Anthony A S Khachaturian et al ldquoReducedrisk of Alzheimer disease in users of antioxidant vitaminsupplements the Cache County studyrdquo Archives of Neurologyvol 61 no 1 pp 82ndash88 2004
[31] L Kusdra H Rempel K Yaffe and L Pulliam ldquoElevation ofCD69+ monocytemacrophages in patients with Alzheimerrsquosdiseaserdquo Immunobiology vol 202 no 1 pp 26ndash33 2000
[32] J A Casal A Robles and J C Tutor ldquoSerum markers ofmonocytemacrophage activation in patients with Alzheimerrsquosdisease and other types of dementiardquo Clinical Biochemistry vol36 no 7 pp 553ndash556 2003
[33] C K Combs J Colleen Karlo S-C Kao and G E Landrethldquo120573-amyloid stimulation of microglia anti monocytes results inTNF120572-dependent expression of inducible nitric oxide synthaseand neuronal apoptosisrdquo Journal of Neuroscience vol 21 no 4pp 1179ndash1188 2001
[34] Y Beloosesky H Salman M Bergman H Bessler and MDjaldetti ldquoCytokine levels and phagocytic activity in patientswith Alzheimerrsquos diseaserdquo Gerontology vol 48 no 3 pp 128ndash132 2002
[35] F Shalit B Sredni L Stern E Kott and M HubermanldquoElevated interleukin-6 secretion levels by mononuclear cells ofAlzheimerrsquos patientsrdquo Neuroscience Letters vol 174 no 2 pp130ndash132 1994
[36] A Azzi R Gysin P Kempna et al ldquoThe role of 120572-tocopherolin preventing disease from epidemiology to molecular eventsrdquoMolecular Aspects of Medicine vol 24 no 6 pp 325ndash336 2003
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Oxidative Medicine and Cellular Longevity
Forward scatter
Side
scat
ter
1000
800
600
400
200
010008006004002000
R1
100
101
102
103
104
80
0
Cou
nts
CD11b-APC
INT
100
101
102
103
104
80
0
Cou
nts
AD
CD11b-APC
100
101
102
103
104
80
0
Cou
nts
M1
INT isotype
mlgG2b-APC
100
101
102
103
104
80
0
Cou
nts
M1
AD isotype
mlgG2b-APC
100
101
102
103
104
80
0
Cou
nts
AD
HLA-DR-PE
M1
100
101
102
103
104
80
0
Cou
nts
AD isotype
mlgG2a-PE
M1
HLA-DR-PE
INT
100
101
102
103
104
80
0
Cou
nts
CD14-PerCP
CD
66b-
FITC
R3
R2
100
101
102
103
104
100
101
102
103
104
M1
M1
INT isotype
100
101
102
103
104
80
0C
ount
s
mlgG2a-PE
Figure 1 Representative flow cytometric analysis of whole blood samples stainedwith anti-CD11b-APC (right column) and anti-HLA-DR-PE(left column) monoclonal antibodies From all events acquired in gate R1 (upper left graph) CD14-positive monocytes (gate R2 separatedfrom CD66b-positive neutrophils in gate R3 upper right graph) were used for histogram calculation of the geometric mean fluorescenceintensity (GMFI) A similar analysis was performed for the detection of fluorescent DCF in the ROS assay (histogram not shown) INT(sample from cognitively intact patient) and AD (sample from patient with probable Alzheimerrsquos disease)
Oxidative Medicine and Cellular Longevity 5
INT
100
101
102
103
104
100
101
102
103
104
INT + LPS
IL-11205721000
0
1000
0
AD
Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0
Forw
ard
scat
ter AD + LPS
IL-1120572-PE IL-1120572-PE
IL-1120572-PE IL-1120572-PE
(a)
IL-6
IL-6-PE IL-6-PE
IL-6-PE IL-6-PE
INT
100
101
102
103
104
100
101
102
103
104
INT + LPS1000
0
1000
0
AD
Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter AD + LPS
(b)
TNF-120572
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter
INT INT + LPS
AD AD + LPS
TNF120572-APC TNF120572-APC
TNF120572-APC TNF120572-APC
(c)
Figure 2 Representative flow cytometric detection ofmonocyte intracellular production of IL-1120572 IL-6 and TNF-120572 Monocytes were selectedin a similar manner as described in Figure 1 LPS-induced production of each cytokine was determined by the percentage of events in theupper right quadrant (URQ) Only upper quadrants are shown INT (sample from cognitively intact patient) and AD (sample from patientwith probable Alzheimerrsquos disease)
species causing oxidative stress which could be reflected bycirculating parameters [17]
In the conditions of this work AD is not associatedwith any evidence of systemic oxidative stress Althoughthe activities of erythrocyte antioxidant enzymes do notshow changes in patients with AD Spearman rank correla-tion test reveals a positive correlation between the mutualactivities of erythrocyte superoxide dismutase catalase andglutathione peroxidase and erythrocyte total glutathionelevels These data confirm the validity of the postulate thatsays that the bodyrsquos antioxidant systems act together inan integrated manner Spearman test has also identified anegative correlation between the levels of lipid peroxidationproducts plasma glutathione peroxidase activity and totalglutathione levels in erythrocytes This fact indicates that
lipid peroxidation is influenced by the activity of antioxidantglutathione peroxidase-glutathione system in this experi-mental model Indeed glutathione-glutathione peroxidasesystem is an important component of antioxidant protectionnetwork to cells [18] As a counterpoint to the measurementof erythrocyte antioxidant activities circulating levels ofoxidation products of macromolecules in the body have beenevaluated To this end we analyzed levels of plasma proteincarbonyls and plasma levels of lipid peroxidation productsPlasma level of protein oxidation products is not altered inAD compared with cognitively intact group This lack ofchange is found in some studies [18] On the other handsome authors have found an increase of oxidized proteinsin plasma from AD patients [19 20] This difference maybe due to difference of sensitivity of the methods employed
6 Oxidative Medicine and Cellular Longevity
Table 3 Correlation between antioxidant erythrocytes parameters and plasmatic oxidation products Superoxide dismutase (SOD) catalase(CAT) glutathione peroxidase (GPx) total glutathione level (TGSH) and thiobarbituric acid reactive substances (TBARS) Analysis wasperformed using the Spearman correlation test
Spearman test SOD CAT GPX GSH TBARSSOD
Coefficient of correlation 1000 0415lowast 0316lowast 0406lowastlowast 0097Significance 0001 0011 0001 0244
CATCoefficient of correlation 0415lowast 1000 0368lowastlowast 0317lowast 0158Significance 0001 0003 0010 0129
GPXCoefficient of correlation 0316lowast 0368lowastlowast 1000 0416lowastlowast 0323lowastlowast
Significance 0011 0003 0001 0009TGSH
Coefficient of correlation 0406lowastlowast 0317lowast 0416lowastlowast 1000 0253lowast
Significance 0001 0010 0001 0034TBARS
Coefficient of correlation 0097 0158 0323lowastlowast 0253lowast 1000Significance 0244 0129 0009 0034
lowastlowastP lt 001 lowastP lt 005
Table 4 Monocyte activation parameters INT (cognitively intactpatients 119899 = 42) and AD (patients with probable Alzheimerrsquosdisease 119899 = 23) GMFI geometric mean fluorescence intensity URQ percentage of events in the upper right quadrant Valuesshown correspond to the means plusmn SEM Comparison betweenaverage results of the groups was performed using Student 119905 test forindependent samples
Parameter INT ADHLADR (GMFI) 10993 plusmn 1059 17774 plusmn 3064
lowast
CD11B (GMFI) 40674 plusmn 3924 41292 plusmn 4897
IL-1120572 (URQ) 6578 plusmn 246 8282 plusmn 652lowast
IL-6 (URQ) 5289 plusmn 431 4971 plusmn 657
TNF-120572 (URQ) 685 plusmn 128 826 plusmn 247
Oxidative metabolism (GMFI) 26632 plusmn 3899 25777 plusmn 6307lowast
119875 lt 005 was considered significant
in the mentioned studies (two-dimensional electrophoresismass spectrometry) in relation to enzyme-immunoassaymethod chosen for this work and the possible differences inquality of care and feeding among the target populations ofeach study Concentration of circulating lipid peroxidationproducts is equivalent in both groups results that agree withthose commonly found in the literature [18] In generalthe population studied seems to enjoy a privileged statusin relation to nutritional populations examined in similarstudies in other locations In addition to the antioxidantprofile outlined above measurements of plasma levels ofnonenzymatic antioxidants of lowmolecularweightwere alsocarried out
Values of vitamin C 120573-carotene and 120572-tocopherolobtained in this study are within the range previouslydescribed for a healthy population group in that age [721] A review of several published papers performed by
Stocker and Frei [22] shows populational values for plasmaconcentrations of vitamin C 120572-tocopherol and 120573-caroteneranging from 30 to 150 120583M 15 to 40 120583M and 03 to 06 120583Mrespectively Other authors report that for cardiovasculardisease and cancer prevention concentrations above 30120583M120572-tocopherol in combination with concentrations greaterthan 50 120583M of vitamin C 04 120583M 120573-carotene and 05120583Mlycopene are desired [23] Thus the two groups of elderlypatients studied have positioned themselves within the limitsfound in several other studies to molecular plasma levels ofantioxidantsThese parameters reflect part of the antioxidantdefense system of the body of patients evaluating compo-nents that can be absorbed directly from the diet so furtherpromoted by the antioxidant defense mentioned above Ofall lowmolecular weight antioxidants studied only vitamin Esignificantly differs between groupsThe INT group has levelsof plasma vitamin E higher than those in AD patients
This apparent homogeneity in antioxidant levels betweenthe various groups opposed to other studies in the literature[24ndash27] This finding as previously mentioned reinforcesthe notion that the oxidative processes that are triggeredduring the course of AD are not always reflected in circu-lating blood and even when that occurs it may be onlyan indication of other underlying mechanisms Of specialinterest is the fact that the establishment of AD expressedby the comparison between the INT and AD groups inour study is accompanied by the decrease of circulatingvitamin E which possesses antioxidant capabilities as wellas recognized anti-inflammatory activities due to its actionon signal transduction mechanisms in phagocytes [28 29]Not surprisingly studies that have obtained the best results inpreventing or treating AD in recent years used 120572-tocopherolas an agent [30]
To complete the picture outlined up to this point circulat-ingmonocytes were analyzed by flow cytometry as indicators
Oxidative Medicine and Cellular Longevity 7
of inflammatory activity involved in AD To this end wequantified the level of expression of surface molecules HLA-DR as an indicator of activation of circulating monocytesand CD-11b an adhesion molecule involved in the processof marginalization and migration of monocytes The resultsshow that AD patients have circulating monocytes thatexpress higher amounts of HLA-DR on the surface evenwithout external stimulus indicating a higher basal activationstate of these cells confirming the data available in theliterature [31] Markers of increased activation of monocytesin the bloodstream are found in patients from a number ofneurodegenerative diseases [32] As previously mentionedthese circulatingmonocytes can cross the blood-brain barrierandmigrate to the brain tissue differentiating into microgliaSince circulating monocytes of AD patients are more activeat baseline than those of INT it can be postulated thatmicroglia derived from these cells react more intensely toinflammatory stimuli producing greater injury to brainLevel of expression of surface protein CD-11b is the same inboth groups indicating that the potential for adhesion andmigration of these cells is not altered in this pathology
Also by flow cytometry monocytes production of IL-1120572 IL-6 and TNF-120572 was measured This measurement wasintended to evaluate the capacity of circulating monocytesto stimulate the inflammatory process by attracting newmonocytes to the inflammatory site The production of IL-1120572is higher in monocytes from AD patients than in INT groupSince IL-1120572 is an important proinflammatory cytokine it isplausible to assume thatmigration anddifferentiation of thesemonocytes produce more active microglia This increasedproduction of IL-1120572 was shown in vitro in human monocyteslineage when stimulated by P120573A by means of mechanismsof signal transduction mediated by tyrosine kinases whichreinforces the above hypothesis [33]
Levels of IL-6 produced by stimulated peripheral mono-cytes are similar in both groups as well as of TNF-120572 agreeingwith Beloosesky et al [34] Other authors detected anincrease in IL-6 production bymonocytes of ADpatients [35]using however a different stimulus (phytohemagglutinin)than that used in this present study (LPS)
From the data shown here it is reasonable to concludethat AD is accompanied by the activation of circulatingmonocytes and a decrease in circulating levels of vitamin ESeveral studies show 120572-tocopherol modulating inflammationactivity [28 36] In our study the group of patients whereplasma 120572-tocopherol is decreased also showed more acti-vated monocytes that respond to stimuli with an increasedproduction of proinflammatory cytokine IL-1120572 This studypoints out that AD is associated with a higher basal activationof circulating monocytes that may be a result of 120572-tocopherolstock depletion
Abbreviations
AD Alzheimerrsquos diseaseAPC AllophycocyaninCDR Clinical dementia ratingFITC Fluorescein isothiocyanateINT Cognitively intact group
GMFI Geometric mean fluorescenceintensity
LPS LipopolysaccharideMMSE Minimental state examinationPE PhycoerythrinPerCP Periidinin chlorophyll proteinROS Reactive oxygen speciesURQ Upper right quadrant
References
[1] B C Dickerson A Bakkour D H Salat et al ldquoThe corticalsignature of Alzheimerrsquos disease regionally specific corticalthinning relates to symptom severity in very mild to mild ADdementia and is detectable in asymptomatic amyloid-positiveindividualsrdquo Cerebral Cortex vol 19 no 3 pp 497ndash510 2009
[2] A Jana and K Pahan ldquoFibrillar amyloid-120573 peptides kill humanprimary neurons via NADPH oxidase-mediated activation ofneutral sphingomyelinase Implications for Alzheimerrsquos dis-easerdquo Journal of Biological Chemistry vol 279 no 49 pp 51451ndash51459 2004
[3] C E Finch and T E Morgan ldquoInflammatory processes ofAlzheimer disease and agingrdquoProceedings of the IndianNationalScience Academy B vol 69 pp 165ndash178 2003
[4] E Zotova J A Nicoll R Kalaria C Holmes and D BocheldquoInflammation in Alzheimerrsquos disease relevance to pathogene-sis and therapyrdquo Alzheimerrsquos Research and Therapy vol 2 no 1article 1 2010
[5] L E Rojo J A Fernandez A A Maccioni J M Jimenezand R B Maccioni ldquoNeuroinflammation implications for thepathogenesis and molecular diagnosis of Alzheimerrsquos diseaserdquoArchives of Medical Research vol 39 no 1 pp 1ndash16 2008
[6] K Jomova D Vondrakova M Lawson and M Valko ldquoMetalsoxidative stress and neurodegenerative disordersrdquo Molecularand Cellular Biochemistry vol 345 no 1-2 pp 91ndash104 2010
[7] V B C Junqueira S B M Barros S S Chan et al ldquoAging andoxidative stressrdquoMolecular Aspects of Medicine vol 25 no 1-2pp 5ndash16 2004
[8] M F Folstein S E Folstein and P R McHugh ldquoMini-mentalstate A practical method for grading the cognitive state ofpatients for the clinicianrdquo Journal of Psychiatric Research vol12 no 3 pp 189ndash198 1975
[9] J C Morris ldquoThe Clinical Dementia Rating (CDR) currentversion and scoring rulesrdquo Neurology vol 43 no 11 pp 2412ndash2414 1993
[10] M B MMontano and L R Ramos ldquoValidity of the Portugueseversion of Clinical Dementia Ratingrdquo Revista de Saude Publicavol 39 no 6 pp 912ndash917 2005
[11] F Tietze ldquoEnzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione appli-cations to mammalian blood and other tissuesrdquo AnalyticalBiochemistry vol 27 no 3 pp 502ndash522 1969
[12] H Buss T P Chan K B Sluis N B Domigan and C CWinterbourn ldquoProtein carbonyl measurement by a sensitiveELISA methodrdquo Free Radical Biology and Medicine vol 23 no3 pp 361ndash366 1998
[13] M K C Brunialti E G Kallas M Freudenberg C Galanosand R Salomao ldquoInfluence of EDTA and heparin onlipopolysaccharide binding and cell activation evaluatedat single-cell level in whole bloodrdquo Clinical Cytometry vol 50no 1 pp 14ndash18 2002
8 Oxidative Medicine and Cellular Longevity
[14] M Hasui Y Hirabayashi and Y Kobayashi ldquoSimultaneousmeasurement by flow cytometry of phagocytosis and hydrogenperoxide production of neutrophils in whole bloodrdquo Journal ofImmunological Methods vol 117 no 1 pp 53ndash58 1989
[15] J Wegiel H Imaki K-C Wang et al ldquoOrigin and turnover ofmicroglial cells in fibrillar plaques of APPsw transgenic micerdquoActa Neuropathologica vol 105 no 4 pp 393ndash402 2003
[16] M R DrsquoAndrea G M Cole and M D Ard ldquoThe microglialphagocytic role with specific plaque types in the Alzheimerdisease brainrdquoNeurobiology of Aging vol 25 no 5 pp 675ndash6832004
[17] J Vina A Lloret ROrtı andDAlonso ldquoMolecular bases of thetreatment of Alzheimerrsquos disease with antioxidants preventionof oxidative stressrdquo Molecular Aspects of Medicine vol 25 no1-2 pp 117ndash123 2004
[18] M C Polidori P Mattioli S Aldred et al ldquoPlasma antioxidantstatus immunoglobulin G oxidation and lipid peroxidation indemented patients relevance to Alzheimer disease and vasculardementiardquo Dementia and Geriatric Cognitive Disorders vol 18no 3-4 pp 265ndash270 2004
[19] C C Conrad P L Marshall J M Talent C A MalakowskyJ Choi and R W Gracy ldquoOxidized proteins in Alzheimerrsquosplasmardquo Biochemical and Biophysical Research Communica-tions vol 275 no 2 pp 678ndash681 2000
[20] J Choi C A Malakowsky J M Talent C C Conradand R W Gracy ldquoIdentification of oxidized plasma proteinsin Alzheimerrsquos diseaserdquo Biochemical and Biophysical ResearchCommunications vol 293 no 5 pp 1566ndash1570 2002
[21] H Heseker and R Schneider ldquoRequirement and supply ofvitamin C E and 120573-carotene for elderly men and womenrdquoEuropean Journal of Clinical Nutrition vol 48 no 2 pp 118ndash1271994
[22] R Stocker and B Frei ldquoEndogenous antioxidant defencesin human blood plasmardquo in Oxidative Stress Oxidants andAntioxidants H Sies Ed Academic Press London UK 1991
[23] K F Gey ldquoPrevention of early stages of cardiovascular diseaseand cancer may require concurrent optimization of all majorantioxidants and other nutrients an update and reevaluationof observational data and intervention trialsrdquo in Free RadicalsLipoprotein Oxidation and Atherosclerosis C Rice-Evans EdRichelieu Press London UK 1995
[24] C Behl and B Moosmann ldquoAntioxidant neuroprotection inAlzheimerrsquos disease as preventive and therapeutic approachrdquoFree Radical Biology and Medicine vol 33 no 2 pp 182ndash1912002
[25] E Esposito D Rotilio V di Matteo C di Giulio M Cacchioand S Algeri ldquoA review of specific dietary antioxidants and theeffects on biochemical mechanisms related to neurodegenera-tive processesrdquoNeurobiology of Aging vol 23 no 5 pp 719ndash7352002
[26] P Mecocci M Cristina Polidori A Cherubini et al ldquoLym-phocyte oxidative DNA damage and plasma antioxidants inAlzheimer diseaserdquoArchives ofNeurology vol 59 no 5 pp 794ndash798 2002
[27] M Sano ldquoDo dietary antioxidants prevent Alzheimerrsquos dis-easerdquoThe Lancet Neurology vol 1 no 6 p 342 2002
[28] S S Chan H P Monteiro F Schindler A Stern and V B CJunqueira ldquo120572-tocopherol modulates tyrosine phosphorylationin human neutrophils by inhibition of protein kinas C activityand activation of tyrosine phosphatasesrdquo Free Radical Researchvol 35 no 6 pp 843ndash856 2001
[29] E M Bulger and R V Maier ldquoAn argument for vitamin Esupplementation in the management of systemic inflammatoryresponse syndromerdquo Shock vol 19 no 2 pp 99ndash103 2003
[30] P P Zandi J C Anthony A S Khachaturian et al ldquoReducedrisk of Alzheimer disease in users of antioxidant vitaminsupplements the Cache County studyrdquo Archives of Neurologyvol 61 no 1 pp 82ndash88 2004
[31] L Kusdra H Rempel K Yaffe and L Pulliam ldquoElevation ofCD69+ monocytemacrophages in patients with Alzheimerrsquosdiseaserdquo Immunobiology vol 202 no 1 pp 26ndash33 2000
[32] J A Casal A Robles and J C Tutor ldquoSerum markers ofmonocytemacrophage activation in patients with Alzheimerrsquosdisease and other types of dementiardquo Clinical Biochemistry vol36 no 7 pp 553ndash556 2003
[33] C K Combs J Colleen Karlo S-C Kao and G E Landrethldquo120573-amyloid stimulation of microglia anti monocytes results inTNF120572-dependent expression of inducible nitric oxide synthaseand neuronal apoptosisrdquo Journal of Neuroscience vol 21 no 4pp 1179ndash1188 2001
[34] Y Beloosesky H Salman M Bergman H Bessler and MDjaldetti ldquoCytokine levels and phagocytic activity in patientswith Alzheimerrsquos diseaserdquo Gerontology vol 48 no 3 pp 128ndash132 2002
[35] F Shalit B Sredni L Stern E Kott and M HubermanldquoElevated interleukin-6 secretion levels by mononuclear cells ofAlzheimerrsquos patientsrdquo Neuroscience Letters vol 174 no 2 pp130ndash132 1994
[36] A Azzi R Gysin P Kempna et al ldquoThe role of 120572-tocopherolin preventing disease from epidemiology to molecular eventsrdquoMolecular Aspects of Medicine vol 24 no 6 pp 325ndash336 2003
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 5
INT
100
101
102
103
104
100
101
102
103
104
INT + LPS
IL-11205721000
0
1000
0
AD
Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0
Forw
ard
scat
ter AD + LPS
IL-1120572-PE IL-1120572-PE
IL-1120572-PE IL-1120572-PE
(a)
IL-6
IL-6-PE IL-6-PE
IL-6-PE IL-6-PE
INT
100
101
102
103
104
100
101
102
103
104
INT + LPS1000
0
1000
0
AD
Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter AD + LPS
(b)
TNF-120572
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter
100
101
102
103
104
100
101
102
103
104
1000
0
1000
0Forw
ard
scat
ter
INT INT + LPS
AD AD + LPS
TNF120572-APC TNF120572-APC
TNF120572-APC TNF120572-APC
(c)
Figure 2 Representative flow cytometric detection ofmonocyte intracellular production of IL-1120572 IL-6 and TNF-120572 Monocytes were selectedin a similar manner as described in Figure 1 LPS-induced production of each cytokine was determined by the percentage of events in theupper right quadrant (URQ) Only upper quadrants are shown INT (sample from cognitively intact patient) and AD (sample from patientwith probable Alzheimerrsquos disease)
species causing oxidative stress which could be reflected bycirculating parameters [17]
In the conditions of this work AD is not associatedwith any evidence of systemic oxidative stress Althoughthe activities of erythrocyte antioxidant enzymes do notshow changes in patients with AD Spearman rank correla-tion test reveals a positive correlation between the mutualactivities of erythrocyte superoxide dismutase catalase andglutathione peroxidase and erythrocyte total glutathionelevels These data confirm the validity of the postulate thatsays that the bodyrsquos antioxidant systems act together inan integrated manner Spearman test has also identified anegative correlation between the levels of lipid peroxidationproducts plasma glutathione peroxidase activity and totalglutathione levels in erythrocytes This fact indicates that
lipid peroxidation is influenced by the activity of antioxidantglutathione peroxidase-glutathione system in this experi-mental model Indeed glutathione-glutathione peroxidasesystem is an important component of antioxidant protectionnetwork to cells [18] As a counterpoint to the measurementof erythrocyte antioxidant activities circulating levels ofoxidation products of macromolecules in the body have beenevaluated To this end we analyzed levels of plasma proteincarbonyls and plasma levels of lipid peroxidation productsPlasma level of protein oxidation products is not altered inAD compared with cognitively intact group This lack ofchange is found in some studies [18] On the other handsome authors have found an increase of oxidized proteinsin plasma from AD patients [19 20] This difference maybe due to difference of sensitivity of the methods employed
6 Oxidative Medicine and Cellular Longevity
Table 3 Correlation between antioxidant erythrocytes parameters and plasmatic oxidation products Superoxide dismutase (SOD) catalase(CAT) glutathione peroxidase (GPx) total glutathione level (TGSH) and thiobarbituric acid reactive substances (TBARS) Analysis wasperformed using the Spearman correlation test
Spearman test SOD CAT GPX GSH TBARSSOD
Coefficient of correlation 1000 0415lowast 0316lowast 0406lowastlowast 0097Significance 0001 0011 0001 0244
CATCoefficient of correlation 0415lowast 1000 0368lowastlowast 0317lowast 0158Significance 0001 0003 0010 0129
GPXCoefficient of correlation 0316lowast 0368lowastlowast 1000 0416lowastlowast 0323lowastlowast
Significance 0011 0003 0001 0009TGSH
Coefficient of correlation 0406lowastlowast 0317lowast 0416lowastlowast 1000 0253lowast
Significance 0001 0010 0001 0034TBARS
Coefficient of correlation 0097 0158 0323lowastlowast 0253lowast 1000Significance 0244 0129 0009 0034
lowastlowastP lt 001 lowastP lt 005
Table 4 Monocyte activation parameters INT (cognitively intactpatients 119899 = 42) and AD (patients with probable Alzheimerrsquosdisease 119899 = 23) GMFI geometric mean fluorescence intensity URQ percentage of events in the upper right quadrant Valuesshown correspond to the means plusmn SEM Comparison betweenaverage results of the groups was performed using Student 119905 test forindependent samples
Parameter INT ADHLADR (GMFI) 10993 plusmn 1059 17774 plusmn 3064
lowast
CD11B (GMFI) 40674 plusmn 3924 41292 plusmn 4897
IL-1120572 (URQ) 6578 plusmn 246 8282 plusmn 652lowast
IL-6 (URQ) 5289 plusmn 431 4971 plusmn 657
TNF-120572 (URQ) 685 plusmn 128 826 plusmn 247
Oxidative metabolism (GMFI) 26632 plusmn 3899 25777 plusmn 6307lowast
119875 lt 005 was considered significant
in the mentioned studies (two-dimensional electrophoresismass spectrometry) in relation to enzyme-immunoassaymethod chosen for this work and the possible differences inquality of care and feeding among the target populations ofeach study Concentration of circulating lipid peroxidationproducts is equivalent in both groups results that agree withthose commonly found in the literature [18] In generalthe population studied seems to enjoy a privileged statusin relation to nutritional populations examined in similarstudies in other locations In addition to the antioxidantprofile outlined above measurements of plasma levels ofnonenzymatic antioxidants of lowmolecularweightwere alsocarried out
Values of vitamin C 120573-carotene and 120572-tocopherolobtained in this study are within the range previouslydescribed for a healthy population group in that age [721] A review of several published papers performed by
Stocker and Frei [22] shows populational values for plasmaconcentrations of vitamin C 120572-tocopherol and 120573-caroteneranging from 30 to 150 120583M 15 to 40 120583M and 03 to 06 120583Mrespectively Other authors report that for cardiovasculardisease and cancer prevention concentrations above 30120583M120572-tocopherol in combination with concentrations greaterthan 50 120583M of vitamin C 04 120583M 120573-carotene and 05120583Mlycopene are desired [23] Thus the two groups of elderlypatients studied have positioned themselves within the limitsfound in several other studies to molecular plasma levels ofantioxidantsThese parameters reflect part of the antioxidantdefense system of the body of patients evaluating compo-nents that can be absorbed directly from the diet so furtherpromoted by the antioxidant defense mentioned above Ofall lowmolecular weight antioxidants studied only vitamin Esignificantly differs between groupsThe INT group has levelsof plasma vitamin E higher than those in AD patients
This apparent homogeneity in antioxidant levels betweenthe various groups opposed to other studies in the literature[24ndash27] This finding as previously mentioned reinforcesthe notion that the oxidative processes that are triggeredduring the course of AD are not always reflected in circu-lating blood and even when that occurs it may be onlyan indication of other underlying mechanisms Of specialinterest is the fact that the establishment of AD expressedby the comparison between the INT and AD groups inour study is accompanied by the decrease of circulatingvitamin E which possesses antioxidant capabilities as wellas recognized anti-inflammatory activities due to its actionon signal transduction mechanisms in phagocytes [28 29]Not surprisingly studies that have obtained the best results inpreventing or treating AD in recent years used 120572-tocopherolas an agent [30]
To complete the picture outlined up to this point circulat-ingmonocytes were analyzed by flow cytometry as indicators
Oxidative Medicine and Cellular Longevity 7
of inflammatory activity involved in AD To this end wequantified the level of expression of surface molecules HLA-DR as an indicator of activation of circulating monocytesand CD-11b an adhesion molecule involved in the processof marginalization and migration of monocytes The resultsshow that AD patients have circulating monocytes thatexpress higher amounts of HLA-DR on the surface evenwithout external stimulus indicating a higher basal activationstate of these cells confirming the data available in theliterature [31] Markers of increased activation of monocytesin the bloodstream are found in patients from a number ofneurodegenerative diseases [32] As previously mentionedthese circulatingmonocytes can cross the blood-brain barrierandmigrate to the brain tissue differentiating into microgliaSince circulating monocytes of AD patients are more activeat baseline than those of INT it can be postulated thatmicroglia derived from these cells react more intensely toinflammatory stimuli producing greater injury to brainLevel of expression of surface protein CD-11b is the same inboth groups indicating that the potential for adhesion andmigration of these cells is not altered in this pathology
Also by flow cytometry monocytes production of IL-1120572 IL-6 and TNF-120572 was measured This measurement wasintended to evaluate the capacity of circulating monocytesto stimulate the inflammatory process by attracting newmonocytes to the inflammatory site The production of IL-1120572is higher in monocytes from AD patients than in INT groupSince IL-1120572 is an important proinflammatory cytokine it isplausible to assume thatmigration anddifferentiation of thesemonocytes produce more active microglia This increasedproduction of IL-1120572 was shown in vitro in human monocyteslineage when stimulated by P120573A by means of mechanismsof signal transduction mediated by tyrosine kinases whichreinforces the above hypothesis [33]
Levels of IL-6 produced by stimulated peripheral mono-cytes are similar in both groups as well as of TNF-120572 agreeingwith Beloosesky et al [34] Other authors detected anincrease in IL-6 production bymonocytes of ADpatients [35]using however a different stimulus (phytohemagglutinin)than that used in this present study (LPS)
From the data shown here it is reasonable to concludethat AD is accompanied by the activation of circulatingmonocytes and a decrease in circulating levels of vitamin ESeveral studies show 120572-tocopherol modulating inflammationactivity [28 36] In our study the group of patients whereplasma 120572-tocopherol is decreased also showed more acti-vated monocytes that respond to stimuli with an increasedproduction of proinflammatory cytokine IL-1120572 This studypoints out that AD is associated with a higher basal activationof circulating monocytes that may be a result of 120572-tocopherolstock depletion
Abbreviations
AD Alzheimerrsquos diseaseAPC AllophycocyaninCDR Clinical dementia ratingFITC Fluorescein isothiocyanateINT Cognitively intact group
GMFI Geometric mean fluorescenceintensity
LPS LipopolysaccharideMMSE Minimental state examinationPE PhycoerythrinPerCP Periidinin chlorophyll proteinROS Reactive oxygen speciesURQ Upper right quadrant
References
[1] B C Dickerson A Bakkour D H Salat et al ldquoThe corticalsignature of Alzheimerrsquos disease regionally specific corticalthinning relates to symptom severity in very mild to mild ADdementia and is detectable in asymptomatic amyloid-positiveindividualsrdquo Cerebral Cortex vol 19 no 3 pp 497ndash510 2009
[2] A Jana and K Pahan ldquoFibrillar amyloid-120573 peptides kill humanprimary neurons via NADPH oxidase-mediated activation ofneutral sphingomyelinase Implications for Alzheimerrsquos dis-easerdquo Journal of Biological Chemistry vol 279 no 49 pp 51451ndash51459 2004
[3] C E Finch and T E Morgan ldquoInflammatory processes ofAlzheimer disease and agingrdquoProceedings of the IndianNationalScience Academy B vol 69 pp 165ndash178 2003
[4] E Zotova J A Nicoll R Kalaria C Holmes and D BocheldquoInflammation in Alzheimerrsquos disease relevance to pathogene-sis and therapyrdquo Alzheimerrsquos Research and Therapy vol 2 no 1article 1 2010
[5] L E Rojo J A Fernandez A A Maccioni J M Jimenezand R B Maccioni ldquoNeuroinflammation implications for thepathogenesis and molecular diagnosis of Alzheimerrsquos diseaserdquoArchives of Medical Research vol 39 no 1 pp 1ndash16 2008
[6] K Jomova D Vondrakova M Lawson and M Valko ldquoMetalsoxidative stress and neurodegenerative disordersrdquo Molecularand Cellular Biochemistry vol 345 no 1-2 pp 91ndash104 2010
[7] V B C Junqueira S B M Barros S S Chan et al ldquoAging andoxidative stressrdquoMolecular Aspects of Medicine vol 25 no 1-2pp 5ndash16 2004
[8] M F Folstein S E Folstein and P R McHugh ldquoMini-mentalstate A practical method for grading the cognitive state ofpatients for the clinicianrdquo Journal of Psychiatric Research vol12 no 3 pp 189ndash198 1975
[9] J C Morris ldquoThe Clinical Dementia Rating (CDR) currentversion and scoring rulesrdquo Neurology vol 43 no 11 pp 2412ndash2414 1993
[10] M B MMontano and L R Ramos ldquoValidity of the Portugueseversion of Clinical Dementia Ratingrdquo Revista de Saude Publicavol 39 no 6 pp 912ndash917 2005
[11] F Tietze ldquoEnzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione appli-cations to mammalian blood and other tissuesrdquo AnalyticalBiochemistry vol 27 no 3 pp 502ndash522 1969
[12] H Buss T P Chan K B Sluis N B Domigan and C CWinterbourn ldquoProtein carbonyl measurement by a sensitiveELISA methodrdquo Free Radical Biology and Medicine vol 23 no3 pp 361ndash366 1998
[13] M K C Brunialti E G Kallas M Freudenberg C Galanosand R Salomao ldquoInfluence of EDTA and heparin onlipopolysaccharide binding and cell activation evaluatedat single-cell level in whole bloodrdquo Clinical Cytometry vol 50no 1 pp 14ndash18 2002
8 Oxidative Medicine and Cellular Longevity
[14] M Hasui Y Hirabayashi and Y Kobayashi ldquoSimultaneousmeasurement by flow cytometry of phagocytosis and hydrogenperoxide production of neutrophils in whole bloodrdquo Journal ofImmunological Methods vol 117 no 1 pp 53ndash58 1989
[15] J Wegiel H Imaki K-C Wang et al ldquoOrigin and turnover ofmicroglial cells in fibrillar plaques of APPsw transgenic micerdquoActa Neuropathologica vol 105 no 4 pp 393ndash402 2003
[16] M R DrsquoAndrea G M Cole and M D Ard ldquoThe microglialphagocytic role with specific plaque types in the Alzheimerdisease brainrdquoNeurobiology of Aging vol 25 no 5 pp 675ndash6832004
[17] J Vina A Lloret ROrtı andDAlonso ldquoMolecular bases of thetreatment of Alzheimerrsquos disease with antioxidants preventionof oxidative stressrdquo Molecular Aspects of Medicine vol 25 no1-2 pp 117ndash123 2004
[18] M C Polidori P Mattioli S Aldred et al ldquoPlasma antioxidantstatus immunoglobulin G oxidation and lipid peroxidation indemented patients relevance to Alzheimer disease and vasculardementiardquo Dementia and Geriatric Cognitive Disorders vol 18no 3-4 pp 265ndash270 2004
[19] C C Conrad P L Marshall J M Talent C A MalakowskyJ Choi and R W Gracy ldquoOxidized proteins in Alzheimerrsquosplasmardquo Biochemical and Biophysical Research Communica-tions vol 275 no 2 pp 678ndash681 2000
[20] J Choi C A Malakowsky J M Talent C C Conradand R W Gracy ldquoIdentification of oxidized plasma proteinsin Alzheimerrsquos diseaserdquo Biochemical and Biophysical ResearchCommunications vol 293 no 5 pp 1566ndash1570 2002
[21] H Heseker and R Schneider ldquoRequirement and supply ofvitamin C E and 120573-carotene for elderly men and womenrdquoEuropean Journal of Clinical Nutrition vol 48 no 2 pp 118ndash1271994
[22] R Stocker and B Frei ldquoEndogenous antioxidant defencesin human blood plasmardquo in Oxidative Stress Oxidants andAntioxidants H Sies Ed Academic Press London UK 1991
[23] K F Gey ldquoPrevention of early stages of cardiovascular diseaseand cancer may require concurrent optimization of all majorantioxidants and other nutrients an update and reevaluationof observational data and intervention trialsrdquo in Free RadicalsLipoprotein Oxidation and Atherosclerosis C Rice-Evans EdRichelieu Press London UK 1995
[24] C Behl and B Moosmann ldquoAntioxidant neuroprotection inAlzheimerrsquos disease as preventive and therapeutic approachrdquoFree Radical Biology and Medicine vol 33 no 2 pp 182ndash1912002
[25] E Esposito D Rotilio V di Matteo C di Giulio M Cacchioand S Algeri ldquoA review of specific dietary antioxidants and theeffects on biochemical mechanisms related to neurodegenera-tive processesrdquoNeurobiology of Aging vol 23 no 5 pp 719ndash7352002
[26] P Mecocci M Cristina Polidori A Cherubini et al ldquoLym-phocyte oxidative DNA damage and plasma antioxidants inAlzheimer diseaserdquoArchives ofNeurology vol 59 no 5 pp 794ndash798 2002
[27] M Sano ldquoDo dietary antioxidants prevent Alzheimerrsquos dis-easerdquoThe Lancet Neurology vol 1 no 6 p 342 2002
[28] S S Chan H P Monteiro F Schindler A Stern and V B CJunqueira ldquo120572-tocopherol modulates tyrosine phosphorylationin human neutrophils by inhibition of protein kinas C activityand activation of tyrosine phosphatasesrdquo Free Radical Researchvol 35 no 6 pp 843ndash856 2001
[29] E M Bulger and R V Maier ldquoAn argument for vitamin Esupplementation in the management of systemic inflammatoryresponse syndromerdquo Shock vol 19 no 2 pp 99ndash103 2003
[30] P P Zandi J C Anthony A S Khachaturian et al ldquoReducedrisk of Alzheimer disease in users of antioxidant vitaminsupplements the Cache County studyrdquo Archives of Neurologyvol 61 no 1 pp 82ndash88 2004
[31] L Kusdra H Rempel K Yaffe and L Pulliam ldquoElevation ofCD69+ monocytemacrophages in patients with Alzheimerrsquosdiseaserdquo Immunobiology vol 202 no 1 pp 26ndash33 2000
[32] J A Casal A Robles and J C Tutor ldquoSerum markers ofmonocytemacrophage activation in patients with Alzheimerrsquosdisease and other types of dementiardquo Clinical Biochemistry vol36 no 7 pp 553ndash556 2003
[33] C K Combs J Colleen Karlo S-C Kao and G E Landrethldquo120573-amyloid stimulation of microglia anti monocytes results inTNF120572-dependent expression of inducible nitric oxide synthaseand neuronal apoptosisrdquo Journal of Neuroscience vol 21 no 4pp 1179ndash1188 2001
[34] Y Beloosesky H Salman M Bergman H Bessler and MDjaldetti ldquoCytokine levels and phagocytic activity in patientswith Alzheimerrsquos diseaserdquo Gerontology vol 48 no 3 pp 128ndash132 2002
[35] F Shalit B Sredni L Stern E Kott and M HubermanldquoElevated interleukin-6 secretion levels by mononuclear cells ofAlzheimerrsquos patientsrdquo Neuroscience Letters vol 174 no 2 pp130ndash132 1994
[36] A Azzi R Gysin P Kempna et al ldquoThe role of 120572-tocopherolin preventing disease from epidemiology to molecular eventsrdquoMolecular Aspects of Medicine vol 24 no 6 pp 325ndash336 2003
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Oxidative Medicine and Cellular Longevity
Table 3 Correlation between antioxidant erythrocytes parameters and plasmatic oxidation products Superoxide dismutase (SOD) catalase(CAT) glutathione peroxidase (GPx) total glutathione level (TGSH) and thiobarbituric acid reactive substances (TBARS) Analysis wasperformed using the Spearman correlation test
Spearman test SOD CAT GPX GSH TBARSSOD
Coefficient of correlation 1000 0415lowast 0316lowast 0406lowastlowast 0097Significance 0001 0011 0001 0244
CATCoefficient of correlation 0415lowast 1000 0368lowastlowast 0317lowast 0158Significance 0001 0003 0010 0129
GPXCoefficient of correlation 0316lowast 0368lowastlowast 1000 0416lowastlowast 0323lowastlowast
Significance 0011 0003 0001 0009TGSH
Coefficient of correlation 0406lowastlowast 0317lowast 0416lowastlowast 1000 0253lowast
Significance 0001 0010 0001 0034TBARS
Coefficient of correlation 0097 0158 0323lowastlowast 0253lowast 1000Significance 0244 0129 0009 0034
lowastlowastP lt 001 lowastP lt 005
Table 4 Monocyte activation parameters INT (cognitively intactpatients 119899 = 42) and AD (patients with probable Alzheimerrsquosdisease 119899 = 23) GMFI geometric mean fluorescence intensity URQ percentage of events in the upper right quadrant Valuesshown correspond to the means plusmn SEM Comparison betweenaverage results of the groups was performed using Student 119905 test forindependent samples
Parameter INT ADHLADR (GMFI) 10993 plusmn 1059 17774 plusmn 3064
lowast
CD11B (GMFI) 40674 plusmn 3924 41292 plusmn 4897
IL-1120572 (URQ) 6578 plusmn 246 8282 plusmn 652lowast
IL-6 (URQ) 5289 plusmn 431 4971 plusmn 657
TNF-120572 (URQ) 685 plusmn 128 826 plusmn 247
Oxidative metabolism (GMFI) 26632 plusmn 3899 25777 plusmn 6307lowast
119875 lt 005 was considered significant
in the mentioned studies (two-dimensional electrophoresismass spectrometry) in relation to enzyme-immunoassaymethod chosen for this work and the possible differences inquality of care and feeding among the target populations ofeach study Concentration of circulating lipid peroxidationproducts is equivalent in both groups results that agree withthose commonly found in the literature [18] In generalthe population studied seems to enjoy a privileged statusin relation to nutritional populations examined in similarstudies in other locations In addition to the antioxidantprofile outlined above measurements of plasma levels ofnonenzymatic antioxidants of lowmolecularweightwere alsocarried out
Values of vitamin C 120573-carotene and 120572-tocopherolobtained in this study are within the range previouslydescribed for a healthy population group in that age [721] A review of several published papers performed by
Stocker and Frei [22] shows populational values for plasmaconcentrations of vitamin C 120572-tocopherol and 120573-caroteneranging from 30 to 150 120583M 15 to 40 120583M and 03 to 06 120583Mrespectively Other authors report that for cardiovasculardisease and cancer prevention concentrations above 30120583M120572-tocopherol in combination with concentrations greaterthan 50 120583M of vitamin C 04 120583M 120573-carotene and 05120583Mlycopene are desired [23] Thus the two groups of elderlypatients studied have positioned themselves within the limitsfound in several other studies to molecular plasma levels ofantioxidantsThese parameters reflect part of the antioxidantdefense system of the body of patients evaluating compo-nents that can be absorbed directly from the diet so furtherpromoted by the antioxidant defense mentioned above Ofall lowmolecular weight antioxidants studied only vitamin Esignificantly differs between groupsThe INT group has levelsof plasma vitamin E higher than those in AD patients
This apparent homogeneity in antioxidant levels betweenthe various groups opposed to other studies in the literature[24ndash27] This finding as previously mentioned reinforcesthe notion that the oxidative processes that are triggeredduring the course of AD are not always reflected in circu-lating blood and even when that occurs it may be onlyan indication of other underlying mechanisms Of specialinterest is the fact that the establishment of AD expressedby the comparison between the INT and AD groups inour study is accompanied by the decrease of circulatingvitamin E which possesses antioxidant capabilities as wellas recognized anti-inflammatory activities due to its actionon signal transduction mechanisms in phagocytes [28 29]Not surprisingly studies that have obtained the best results inpreventing or treating AD in recent years used 120572-tocopherolas an agent [30]
To complete the picture outlined up to this point circulat-ingmonocytes were analyzed by flow cytometry as indicators
Oxidative Medicine and Cellular Longevity 7
of inflammatory activity involved in AD To this end wequantified the level of expression of surface molecules HLA-DR as an indicator of activation of circulating monocytesand CD-11b an adhesion molecule involved in the processof marginalization and migration of monocytes The resultsshow that AD patients have circulating monocytes thatexpress higher amounts of HLA-DR on the surface evenwithout external stimulus indicating a higher basal activationstate of these cells confirming the data available in theliterature [31] Markers of increased activation of monocytesin the bloodstream are found in patients from a number ofneurodegenerative diseases [32] As previously mentionedthese circulatingmonocytes can cross the blood-brain barrierandmigrate to the brain tissue differentiating into microgliaSince circulating monocytes of AD patients are more activeat baseline than those of INT it can be postulated thatmicroglia derived from these cells react more intensely toinflammatory stimuli producing greater injury to brainLevel of expression of surface protein CD-11b is the same inboth groups indicating that the potential for adhesion andmigration of these cells is not altered in this pathology
Also by flow cytometry monocytes production of IL-1120572 IL-6 and TNF-120572 was measured This measurement wasintended to evaluate the capacity of circulating monocytesto stimulate the inflammatory process by attracting newmonocytes to the inflammatory site The production of IL-1120572is higher in monocytes from AD patients than in INT groupSince IL-1120572 is an important proinflammatory cytokine it isplausible to assume thatmigration anddifferentiation of thesemonocytes produce more active microglia This increasedproduction of IL-1120572 was shown in vitro in human monocyteslineage when stimulated by P120573A by means of mechanismsof signal transduction mediated by tyrosine kinases whichreinforces the above hypothesis [33]
Levels of IL-6 produced by stimulated peripheral mono-cytes are similar in both groups as well as of TNF-120572 agreeingwith Beloosesky et al [34] Other authors detected anincrease in IL-6 production bymonocytes of ADpatients [35]using however a different stimulus (phytohemagglutinin)than that used in this present study (LPS)
From the data shown here it is reasonable to concludethat AD is accompanied by the activation of circulatingmonocytes and a decrease in circulating levels of vitamin ESeveral studies show 120572-tocopherol modulating inflammationactivity [28 36] In our study the group of patients whereplasma 120572-tocopherol is decreased also showed more acti-vated monocytes that respond to stimuli with an increasedproduction of proinflammatory cytokine IL-1120572 This studypoints out that AD is associated with a higher basal activationof circulating monocytes that may be a result of 120572-tocopherolstock depletion
Abbreviations
AD Alzheimerrsquos diseaseAPC AllophycocyaninCDR Clinical dementia ratingFITC Fluorescein isothiocyanateINT Cognitively intact group
GMFI Geometric mean fluorescenceintensity
LPS LipopolysaccharideMMSE Minimental state examinationPE PhycoerythrinPerCP Periidinin chlorophyll proteinROS Reactive oxygen speciesURQ Upper right quadrant
References
[1] B C Dickerson A Bakkour D H Salat et al ldquoThe corticalsignature of Alzheimerrsquos disease regionally specific corticalthinning relates to symptom severity in very mild to mild ADdementia and is detectable in asymptomatic amyloid-positiveindividualsrdquo Cerebral Cortex vol 19 no 3 pp 497ndash510 2009
[2] A Jana and K Pahan ldquoFibrillar amyloid-120573 peptides kill humanprimary neurons via NADPH oxidase-mediated activation ofneutral sphingomyelinase Implications for Alzheimerrsquos dis-easerdquo Journal of Biological Chemistry vol 279 no 49 pp 51451ndash51459 2004
[3] C E Finch and T E Morgan ldquoInflammatory processes ofAlzheimer disease and agingrdquoProceedings of the IndianNationalScience Academy B vol 69 pp 165ndash178 2003
[4] E Zotova J A Nicoll R Kalaria C Holmes and D BocheldquoInflammation in Alzheimerrsquos disease relevance to pathogene-sis and therapyrdquo Alzheimerrsquos Research and Therapy vol 2 no 1article 1 2010
[5] L E Rojo J A Fernandez A A Maccioni J M Jimenezand R B Maccioni ldquoNeuroinflammation implications for thepathogenesis and molecular diagnosis of Alzheimerrsquos diseaserdquoArchives of Medical Research vol 39 no 1 pp 1ndash16 2008
[6] K Jomova D Vondrakova M Lawson and M Valko ldquoMetalsoxidative stress and neurodegenerative disordersrdquo Molecularand Cellular Biochemistry vol 345 no 1-2 pp 91ndash104 2010
[7] V B C Junqueira S B M Barros S S Chan et al ldquoAging andoxidative stressrdquoMolecular Aspects of Medicine vol 25 no 1-2pp 5ndash16 2004
[8] M F Folstein S E Folstein and P R McHugh ldquoMini-mentalstate A practical method for grading the cognitive state ofpatients for the clinicianrdquo Journal of Psychiatric Research vol12 no 3 pp 189ndash198 1975
[9] J C Morris ldquoThe Clinical Dementia Rating (CDR) currentversion and scoring rulesrdquo Neurology vol 43 no 11 pp 2412ndash2414 1993
[10] M B MMontano and L R Ramos ldquoValidity of the Portugueseversion of Clinical Dementia Ratingrdquo Revista de Saude Publicavol 39 no 6 pp 912ndash917 2005
[11] F Tietze ldquoEnzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione appli-cations to mammalian blood and other tissuesrdquo AnalyticalBiochemistry vol 27 no 3 pp 502ndash522 1969
[12] H Buss T P Chan K B Sluis N B Domigan and C CWinterbourn ldquoProtein carbonyl measurement by a sensitiveELISA methodrdquo Free Radical Biology and Medicine vol 23 no3 pp 361ndash366 1998
[13] M K C Brunialti E G Kallas M Freudenberg C Galanosand R Salomao ldquoInfluence of EDTA and heparin onlipopolysaccharide binding and cell activation evaluatedat single-cell level in whole bloodrdquo Clinical Cytometry vol 50no 1 pp 14ndash18 2002
8 Oxidative Medicine and Cellular Longevity
[14] M Hasui Y Hirabayashi and Y Kobayashi ldquoSimultaneousmeasurement by flow cytometry of phagocytosis and hydrogenperoxide production of neutrophils in whole bloodrdquo Journal ofImmunological Methods vol 117 no 1 pp 53ndash58 1989
[15] J Wegiel H Imaki K-C Wang et al ldquoOrigin and turnover ofmicroglial cells in fibrillar plaques of APPsw transgenic micerdquoActa Neuropathologica vol 105 no 4 pp 393ndash402 2003
[16] M R DrsquoAndrea G M Cole and M D Ard ldquoThe microglialphagocytic role with specific plaque types in the Alzheimerdisease brainrdquoNeurobiology of Aging vol 25 no 5 pp 675ndash6832004
[17] J Vina A Lloret ROrtı andDAlonso ldquoMolecular bases of thetreatment of Alzheimerrsquos disease with antioxidants preventionof oxidative stressrdquo Molecular Aspects of Medicine vol 25 no1-2 pp 117ndash123 2004
[18] M C Polidori P Mattioli S Aldred et al ldquoPlasma antioxidantstatus immunoglobulin G oxidation and lipid peroxidation indemented patients relevance to Alzheimer disease and vasculardementiardquo Dementia and Geriatric Cognitive Disorders vol 18no 3-4 pp 265ndash270 2004
[19] C C Conrad P L Marshall J M Talent C A MalakowskyJ Choi and R W Gracy ldquoOxidized proteins in Alzheimerrsquosplasmardquo Biochemical and Biophysical Research Communica-tions vol 275 no 2 pp 678ndash681 2000
[20] J Choi C A Malakowsky J M Talent C C Conradand R W Gracy ldquoIdentification of oxidized plasma proteinsin Alzheimerrsquos diseaserdquo Biochemical and Biophysical ResearchCommunications vol 293 no 5 pp 1566ndash1570 2002
[21] H Heseker and R Schneider ldquoRequirement and supply ofvitamin C E and 120573-carotene for elderly men and womenrdquoEuropean Journal of Clinical Nutrition vol 48 no 2 pp 118ndash1271994
[22] R Stocker and B Frei ldquoEndogenous antioxidant defencesin human blood plasmardquo in Oxidative Stress Oxidants andAntioxidants H Sies Ed Academic Press London UK 1991
[23] K F Gey ldquoPrevention of early stages of cardiovascular diseaseand cancer may require concurrent optimization of all majorantioxidants and other nutrients an update and reevaluationof observational data and intervention trialsrdquo in Free RadicalsLipoprotein Oxidation and Atherosclerosis C Rice-Evans EdRichelieu Press London UK 1995
[24] C Behl and B Moosmann ldquoAntioxidant neuroprotection inAlzheimerrsquos disease as preventive and therapeutic approachrdquoFree Radical Biology and Medicine vol 33 no 2 pp 182ndash1912002
[25] E Esposito D Rotilio V di Matteo C di Giulio M Cacchioand S Algeri ldquoA review of specific dietary antioxidants and theeffects on biochemical mechanisms related to neurodegenera-tive processesrdquoNeurobiology of Aging vol 23 no 5 pp 719ndash7352002
[26] P Mecocci M Cristina Polidori A Cherubini et al ldquoLym-phocyte oxidative DNA damage and plasma antioxidants inAlzheimer diseaserdquoArchives ofNeurology vol 59 no 5 pp 794ndash798 2002
[27] M Sano ldquoDo dietary antioxidants prevent Alzheimerrsquos dis-easerdquoThe Lancet Neurology vol 1 no 6 p 342 2002
[28] S S Chan H P Monteiro F Schindler A Stern and V B CJunqueira ldquo120572-tocopherol modulates tyrosine phosphorylationin human neutrophils by inhibition of protein kinas C activityand activation of tyrosine phosphatasesrdquo Free Radical Researchvol 35 no 6 pp 843ndash856 2001
[29] E M Bulger and R V Maier ldquoAn argument for vitamin Esupplementation in the management of systemic inflammatoryresponse syndromerdquo Shock vol 19 no 2 pp 99ndash103 2003
[30] P P Zandi J C Anthony A S Khachaturian et al ldquoReducedrisk of Alzheimer disease in users of antioxidant vitaminsupplements the Cache County studyrdquo Archives of Neurologyvol 61 no 1 pp 82ndash88 2004
[31] L Kusdra H Rempel K Yaffe and L Pulliam ldquoElevation ofCD69+ monocytemacrophages in patients with Alzheimerrsquosdiseaserdquo Immunobiology vol 202 no 1 pp 26ndash33 2000
[32] J A Casal A Robles and J C Tutor ldquoSerum markers ofmonocytemacrophage activation in patients with Alzheimerrsquosdisease and other types of dementiardquo Clinical Biochemistry vol36 no 7 pp 553ndash556 2003
[33] C K Combs J Colleen Karlo S-C Kao and G E Landrethldquo120573-amyloid stimulation of microglia anti monocytes results inTNF120572-dependent expression of inducible nitric oxide synthaseand neuronal apoptosisrdquo Journal of Neuroscience vol 21 no 4pp 1179ndash1188 2001
[34] Y Beloosesky H Salman M Bergman H Bessler and MDjaldetti ldquoCytokine levels and phagocytic activity in patientswith Alzheimerrsquos diseaserdquo Gerontology vol 48 no 3 pp 128ndash132 2002
[35] F Shalit B Sredni L Stern E Kott and M HubermanldquoElevated interleukin-6 secretion levels by mononuclear cells ofAlzheimerrsquos patientsrdquo Neuroscience Letters vol 174 no 2 pp130ndash132 1994
[36] A Azzi R Gysin P Kempna et al ldquoThe role of 120572-tocopherolin preventing disease from epidemiology to molecular eventsrdquoMolecular Aspects of Medicine vol 24 no 6 pp 325ndash336 2003
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 7
of inflammatory activity involved in AD To this end wequantified the level of expression of surface molecules HLA-DR as an indicator of activation of circulating monocytesand CD-11b an adhesion molecule involved in the processof marginalization and migration of monocytes The resultsshow that AD patients have circulating monocytes thatexpress higher amounts of HLA-DR on the surface evenwithout external stimulus indicating a higher basal activationstate of these cells confirming the data available in theliterature [31] Markers of increased activation of monocytesin the bloodstream are found in patients from a number ofneurodegenerative diseases [32] As previously mentionedthese circulatingmonocytes can cross the blood-brain barrierandmigrate to the brain tissue differentiating into microgliaSince circulating monocytes of AD patients are more activeat baseline than those of INT it can be postulated thatmicroglia derived from these cells react more intensely toinflammatory stimuli producing greater injury to brainLevel of expression of surface protein CD-11b is the same inboth groups indicating that the potential for adhesion andmigration of these cells is not altered in this pathology
Also by flow cytometry monocytes production of IL-1120572 IL-6 and TNF-120572 was measured This measurement wasintended to evaluate the capacity of circulating monocytesto stimulate the inflammatory process by attracting newmonocytes to the inflammatory site The production of IL-1120572is higher in monocytes from AD patients than in INT groupSince IL-1120572 is an important proinflammatory cytokine it isplausible to assume thatmigration anddifferentiation of thesemonocytes produce more active microglia This increasedproduction of IL-1120572 was shown in vitro in human monocyteslineage when stimulated by P120573A by means of mechanismsof signal transduction mediated by tyrosine kinases whichreinforces the above hypothesis [33]
Levels of IL-6 produced by stimulated peripheral mono-cytes are similar in both groups as well as of TNF-120572 agreeingwith Beloosesky et al [34] Other authors detected anincrease in IL-6 production bymonocytes of ADpatients [35]using however a different stimulus (phytohemagglutinin)than that used in this present study (LPS)
From the data shown here it is reasonable to concludethat AD is accompanied by the activation of circulatingmonocytes and a decrease in circulating levels of vitamin ESeveral studies show 120572-tocopherol modulating inflammationactivity [28 36] In our study the group of patients whereplasma 120572-tocopherol is decreased also showed more acti-vated monocytes that respond to stimuli with an increasedproduction of proinflammatory cytokine IL-1120572 This studypoints out that AD is associated with a higher basal activationof circulating monocytes that may be a result of 120572-tocopherolstock depletion
Abbreviations
AD Alzheimerrsquos diseaseAPC AllophycocyaninCDR Clinical dementia ratingFITC Fluorescein isothiocyanateINT Cognitively intact group
GMFI Geometric mean fluorescenceintensity
LPS LipopolysaccharideMMSE Minimental state examinationPE PhycoerythrinPerCP Periidinin chlorophyll proteinROS Reactive oxygen speciesURQ Upper right quadrant
References
[1] B C Dickerson A Bakkour D H Salat et al ldquoThe corticalsignature of Alzheimerrsquos disease regionally specific corticalthinning relates to symptom severity in very mild to mild ADdementia and is detectable in asymptomatic amyloid-positiveindividualsrdquo Cerebral Cortex vol 19 no 3 pp 497ndash510 2009
[2] A Jana and K Pahan ldquoFibrillar amyloid-120573 peptides kill humanprimary neurons via NADPH oxidase-mediated activation ofneutral sphingomyelinase Implications for Alzheimerrsquos dis-easerdquo Journal of Biological Chemistry vol 279 no 49 pp 51451ndash51459 2004
[3] C E Finch and T E Morgan ldquoInflammatory processes ofAlzheimer disease and agingrdquoProceedings of the IndianNationalScience Academy B vol 69 pp 165ndash178 2003
[4] E Zotova J A Nicoll R Kalaria C Holmes and D BocheldquoInflammation in Alzheimerrsquos disease relevance to pathogene-sis and therapyrdquo Alzheimerrsquos Research and Therapy vol 2 no 1article 1 2010
[5] L E Rojo J A Fernandez A A Maccioni J M Jimenezand R B Maccioni ldquoNeuroinflammation implications for thepathogenesis and molecular diagnosis of Alzheimerrsquos diseaserdquoArchives of Medical Research vol 39 no 1 pp 1ndash16 2008
[6] K Jomova D Vondrakova M Lawson and M Valko ldquoMetalsoxidative stress and neurodegenerative disordersrdquo Molecularand Cellular Biochemistry vol 345 no 1-2 pp 91ndash104 2010
[7] V B C Junqueira S B M Barros S S Chan et al ldquoAging andoxidative stressrdquoMolecular Aspects of Medicine vol 25 no 1-2pp 5ndash16 2004
[8] M F Folstein S E Folstein and P R McHugh ldquoMini-mentalstate A practical method for grading the cognitive state ofpatients for the clinicianrdquo Journal of Psychiatric Research vol12 no 3 pp 189ndash198 1975
[9] J C Morris ldquoThe Clinical Dementia Rating (CDR) currentversion and scoring rulesrdquo Neurology vol 43 no 11 pp 2412ndash2414 1993
[10] M B MMontano and L R Ramos ldquoValidity of the Portugueseversion of Clinical Dementia Ratingrdquo Revista de Saude Publicavol 39 no 6 pp 912ndash917 2005
[11] F Tietze ldquoEnzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione appli-cations to mammalian blood and other tissuesrdquo AnalyticalBiochemistry vol 27 no 3 pp 502ndash522 1969
[12] H Buss T P Chan K B Sluis N B Domigan and C CWinterbourn ldquoProtein carbonyl measurement by a sensitiveELISA methodrdquo Free Radical Biology and Medicine vol 23 no3 pp 361ndash366 1998
[13] M K C Brunialti E G Kallas M Freudenberg C Galanosand R Salomao ldquoInfluence of EDTA and heparin onlipopolysaccharide binding and cell activation evaluatedat single-cell level in whole bloodrdquo Clinical Cytometry vol 50no 1 pp 14ndash18 2002
8 Oxidative Medicine and Cellular Longevity
[14] M Hasui Y Hirabayashi and Y Kobayashi ldquoSimultaneousmeasurement by flow cytometry of phagocytosis and hydrogenperoxide production of neutrophils in whole bloodrdquo Journal ofImmunological Methods vol 117 no 1 pp 53ndash58 1989
[15] J Wegiel H Imaki K-C Wang et al ldquoOrigin and turnover ofmicroglial cells in fibrillar plaques of APPsw transgenic micerdquoActa Neuropathologica vol 105 no 4 pp 393ndash402 2003
[16] M R DrsquoAndrea G M Cole and M D Ard ldquoThe microglialphagocytic role with specific plaque types in the Alzheimerdisease brainrdquoNeurobiology of Aging vol 25 no 5 pp 675ndash6832004
[17] J Vina A Lloret ROrtı andDAlonso ldquoMolecular bases of thetreatment of Alzheimerrsquos disease with antioxidants preventionof oxidative stressrdquo Molecular Aspects of Medicine vol 25 no1-2 pp 117ndash123 2004
[18] M C Polidori P Mattioli S Aldred et al ldquoPlasma antioxidantstatus immunoglobulin G oxidation and lipid peroxidation indemented patients relevance to Alzheimer disease and vasculardementiardquo Dementia and Geriatric Cognitive Disorders vol 18no 3-4 pp 265ndash270 2004
[19] C C Conrad P L Marshall J M Talent C A MalakowskyJ Choi and R W Gracy ldquoOxidized proteins in Alzheimerrsquosplasmardquo Biochemical and Biophysical Research Communica-tions vol 275 no 2 pp 678ndash681 2000
[20] J Choi C A Malakowsky J M Talent C C Conradand R W Gracy ldquoIdentification of oxidized plasma proteinsin Alzheimerrsquos diseaserdquo Biochemical and Biophysical ResearchCommunications vol 293 no 5 pp 1566ndash1570 2002
[21] H Heseker and R Schneider ldquoRequirement and supply ofvitamin C E and 120573-carotene for elderly men and womenrdquoEuropean Journal of Clinical Nutrition vol 48 no 2 pp 118ndash1271994
[22] R Stocker and B Frei ldquoEndogenous antioxidant defencesin human blood plasmardquo in Oxidative Stress Oxidants andAntioxidants H Sies Ed Academic Press London UK 1991
[23] K F Gey ldquoPrevention of early stages of cardiovascular diseaseand cancer may require concurrent optimization of all majorantioxidants and other nutrients an update and reevaluationof observational data and intervention trialsrdquo in Free RadicalsLipoprotein Oxidation and Atherosclerosis C Rice-Evans EdRichelieu Press London UK 1995
[24] C Behl and B Moosmann ldquoAntioxidant neuroprotection inAlzheimerrsquos disease as preventive and therapeutic approachrdquoFree Radical Biology and Medicine vol 33 no 2 pp 182ndash1912002
[25] E Esposito D Rotilio V di Matteo C di Giulio M Cacchioand S Algeri ldquoA review of specific dietary antioxidants and theeffects on biochemical mechanisms related to neurodegenera-tive processesrdquoNeurobiology of Aging vol 23 no 5 pp 719ndash7352002
[26] P Mecocci M Cristina Polidori A Cherubini et al ldquoLym-phocyte oxidative DNA damage and plasma antioxidants inAlzheimer diseaserdquoArchives ofNeurology vol 59 no 5 pp 794ndash798 2002
[27] M Sano ldquoDo dietary antioxidants prevent Alzheimerrsquos dis-easerdquoThe Lancet Neurology vol 1 no 6 p 342 2002
[28] S S Chan H P Monteiro F Schindler A Stern and V B CJunqueira ldquo120572-tocopherol modulates tyrosine phosphorylationin human neutrophils by inhibition of protein kinas C activityand activation of tyrosine phosphatasesrdquo Free Radical Researchvol 35 no 6 pp 843ndash856 2001
[29] E M Bulger and R V Maier ldquoAn argument for vitamin Esupplementation in the management of systemic inflammatoryresponse syndromerdquo Shock vol 19 no 2 pp 99ndash103 2003
[30] P P Zandi J C Anthony A S Khachaturian et al ldquoReducedrisk of Alzheimer disease in users of antioxidant vitaminsupplements the Cache County studyrdquo Archives of Neurologyvol 61 no 1 pp 82ndash88 2004
[31] L Kusdra H Rempel K Yaffe and L Pulliam ldquoElevation ofCD69+ monocytemacrophages in patients with Alzheimerrsquosdiseaserdquo Immunobiology vol 202 no 1 pp 26ndash33 2000
[32] J A Casal A Robles and J C Tutor ldquoSerum markers ofmonocytemacrophage activation in patients with Alzheimerrsquosdisease and other types of dementiardquo Clinical Biochemistry vol36 no 7 pp 553ndash556 2003
[33] C K Combs J Colleen Karlo S-C Kao and G E Landrethldquo120573-amyloid stimulation of microglia anti monocytes results inTNF120572-dependent expression of inducible nitric oxide synthaseand neuronal apoptosisrdquo Journal of Neuroscience vol 21 no 4pp 1179ndash1188 2001
[34] Y Beloosesky H Salman M Bergman H Bessler and MDjaldetti ldquoCytokine levels and phagocytic activity in patientswith Alzheimerrsquos diseaserdquo Gerontology vol 48 no 3 pp 128ndash132 2002
[35] F Shalit B Sredni L Stern E Kott and M HubermanldquoElevated interleukin-6 secretion levels by mononuclear cells ofAlzheimerrsquos patientsrdquo Neuroscience Letters vol 174 no 2 pp130ndash132 1994
[36] A Azzi R Gysin P Kempna et al ldquoThe role of 120572-tocopherolin preventing disease from epidemiology to molecular eventsrdquoMolecular Aspects of Medicine vol 24 no 6 pp 325ndash336 2003
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Oxidative Medicine and Cellular Longevity
[14] M Hasui Y Hirabayashi and Y Kobayashi ldquoSimultaneousmeasurement by flow cytometry of phagocytosis and hydrogenperoxide production of neutrophils in whole bloodrdquo Journal ofImmunological Methods vol 117 no 1 pp 53ndash58 1989
[15] J Wegiel H Imaki K-C Wang et al ldquoOrigin and turnover ofmicroglial cells in fibrillar plaques of APPsw transgenic micerdquoActa Neuropathologica vol 105 no 4 pp 393ndash402 2003
[16] M R DrsquoAndrea G M Cole and M D Ard ldquoThe microglialphagocytic role with specific plaque types in the Alzheimerdisease brainrdquoNeurobiology of Aging vol 25 no 5 pp 675ndash6832004
[17] J Vina A Lloret ROrtı andDAlonso ldquoMolecular bases of thetreatment of Alzheimerrsquos disease with antioxidants preventionof oxidative stressrdquo Molecular Aspects of Medicine vol 25 no1-2 pp 117ndash123 2004
[18] M C Polidori P Mattioli S Aldred et al ldquoPlasma antioxidantstatus immunoglobulin G oxidation and lipid peroxidation indemented patients relevance to Alzheimer disease and vasculardementiardquo Dementia and Geriatric Cognitive Disorders vol 18no 3-4 pp 265ndash270 2004
[19] C C Conrad P L Marshall J M Talent C A MalakowskyJ Choi and R W Gracy ldquoOxidized proteins in Alzheimerrsquosplasmardquo Biochemical and Biophysical Research Communica-tions vol 275 no 2 pp 678ndash681 2000
[20] J Choi C A Malakowsky J M Talent C C Conradand R W Gracy ldquoIdentification of oxidized plasma proteinsin Alzheimerrsquos diseaserdquo Biochemical and Biophysical ResearchCommunications vol 293 no 5 pp 1566ndash1570 2002
[21] H Heseker and R Schneider ldquoRequirement and supply ofvitamin C E and 120573-carotene for elderly men and womenrdquoEuropean Journal of Clinical Nutrition vol 48 no 2 pp 118ndash1271994
[22] R Stocker and B Frei ldquoEndogenous antioxidant defencesin human blood plasmardquo in Oxidative Stress Oxidants andAntioxidants H Sies Ed Academic Press London UK 1991
[23] K F Gey ldquoPrevention of early stages of cardiovascular diseaseand cancer may require concurrent optimization of all majorantioxidants and other nutrients an update and reevaluationof observational data and intervention trialsrdquo in Free RadicalsLipoprotein Oxidation and Atherosclerosis C Rice-Evans EdRichelieu Press London UK 1995
[24] C Behl and B Moosmann ldquoAntioxidant neuroprotection inAlzheimerrsquos disease as preventive and therapeutic approachrdquoFree Radical Biology and Medicine vol 33 no 2 pp 182ndash1912002
[25] E Esposito D Rotilio V di Matteo C di Giulio M Cacchioand S Algeri ldquoA review of specific dietary antioxidants and theeffects on biochemical mechanisms related to neurodegenera-tive processesrdquoNeurobiology of Aging vol 23 no 5 pp 719ndash7352002
[26] P Mecocci M Cristina Polidori A Cherubini et al ldquoLym-phocyte oxidative DNA damage and plasma antioxidants inAlzheimer diseaserdquoArchives ofNeurology vol 59 no 5 pp 794ndash798 2002
[27] M Sano ldquoDo dietary antioxidants prevent Alzheimerrsquos dis-easerdquoThe Lancet Neurology vol 1 no 6 p 342 2002
[28] S S Chan H P Monteiro F Schindler A Stern and V B CJunqueira ldquo120572-tocopherol modulates tyrosine phosphorylationin human neutrophils by inhibition of protein kinas C activityand activation of tyrosine phosphatasesrdquo Free Radical Researchvol 35 no 6 pp 843ndash856 2001
[29] E M Bulger and R V Maier ldquoAn argument for vitamin Esupplementation in the management of systemic inflammatoryresponse syndromerdquo Shock vol 19 no 2 pp 99ndash103 2003
[30] P P Zandi J C Anthony A S Khachaturian et al ldquoReducedrisk of Alzheimer disease in users of antioxidant vitaminsupplements the Cache County studyrdquo Archives of Neurologyvol 61 no 1 pp 82ndash88 2004
[31] L Kusdra H Rempel K Yaffe and L Pulliam ldquoElevation ofCD69+ monocytemacrophages in patients with Alzheimerrsquosdiseaserdquo Immunobiology vol 202 no 1 pp 26ndash33 2000
[32] J A Casal A Robles and J C Tutor ldquoSerum markers ofmonocytemacrophage activation in patients with Alzheimerrsquosdisease and other types of dementiardquo Clinical Biochemistry vol36 no 7 pp 553ndash556 2003
[33] C K Combs J Colleen Karlo S-C Kao and G E Landrethldquo120573-amyloid stimulation of microglia anti monocytes results inTNF120572-dependent expression of inducible nitric oxide synthaseand neuronal apoptosisrdquo Journal of Neuroscience vol 21 no 4pp 1179ndash1188 2001
[34] Y Beloosesky H Salman M Bergman H Bessler and MDjaldetti ldquoCytokine levels and phagocytic activity in patientswith Alzheimerrsquos diseaserdquo Gerontology vol 48 no 3 pp 128ndash132 2002
[35] F Shalit B Sredni L Stern E Kott and M HubermanldquoElevated interleukin-6 secretion levels by mononuclear cells ofAlzheimerrsquos patientsrdquo Neuroscience Letters vol 174 no 2 pp130ndash132 1994
[36] A Azzi R Gysin P Kempna et al ldquoThe role of 120572-tocopherolin preventing disease from epidemiology to molecular eventsrdquoMolecular Aspects of Medicine vol 24 no 6 pp 325ndash336 2003
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom