Blood was collected from
healthy elderly (age 65-90 years) and young volunteer (age 20-35 years) donors.
Elderly subjects belong to middle-class socio-economic status and are living
independently. A week prior to the study, they were asked to discontinue any
vitamins, minerals and antioxidants that they may have been taking. The
Institutional Review Board of the University of California, Irvine, approved
of human monocyte derived dendritic cells.
DCs were prepared essentially as described [43
]. Briefly, peripheral
blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density
gradient centrifugation. Monocytes were purified from the PBMCs by positiveselection with anti-CD14 microbeads (Stemcell Sep, Vancouver, BC). The purity
of the isolatedCD14+
monocytes was >90% as determined
by flow cytometry.For the induction of DC differentiation, purified
monocytes were cultured in a humidified atmosphere of 5%CO2
at 37°C in RPMI 1640 supplemented with10%FBS, 1 mMglutamine, 100
U/ml penicillin, 100 μg/ml streptomycin,50 ng/ml human rGM-CSF
(PeproTech, Rocky Hill, NJ), and 10 ng/ml human rIL-4(Peprotech).
Half of the medium was replaced every2 days and DCs (CD14-
cells) were collected after 6 days. The purity of the DC was >95% as
determined by the expression of CD14, CD11c and HLA-DR.
Self DNA Preparation.
isolated from the blood of aged and young subjects using Qiamp DNA Blood Midi
Kit from Qiagen (Valencia, CA). RNase was added to remove any contaminating
RNA. Purity and yield of DNA was measured by UV spectrophotometer. Preparations
with 260/280 ratio above 1.9 were used in all experiments. DNA obtained was
free of endotoxin contamination as determined by Limulus amoebocytelysate
(LAL) assay (Lonza Inc, Allendale, NJ).
Transfection reagent Lipofectamine 2000 (Invitrogen,
Carlsbad, CA) was used to deliver self DNA to DCs. DNA was mixed with
lipofectamine (lipo) in 100 μl of Opti-MEM for 20 minutes at
room temperature, according to the protocol recommended by the manufacturer and
added to 4×105 DCs in 300 μl of complete
medium. Final concentration of the DNA was 1μg/ml. Cell viability
was unaffected by this treatment. Unstimulated and Lipofectamine-stimulated DCs
were used as controls. After 24 hours, cells were harvested and stained for
surface markers CD80, CD86 and CD83, using directly conjugated antibodies and
isotype controls (BD Pharmingen, San Jose, CA). 10,000 CD11c+HLA-DR+
cells per condition were acquired using a FACSCalibur (BDPharmingen,
San Jose, California). Analysis was performed using the Flow jo software
(Treestar Inc, Ashland, OR).
IFN-α in the supernatants was measured by verikine IFN-α measuring kit (PBL Biomedicals, Piscatway, NJ) as per the
of DNA methylation.
methylation of DNA from aged and young subjects was determined using the Methylamp™ Global DNA Methylation Quantification Kit
from Epigentek (Brooklyn, NY), as per the manufacturer's protocol. In this kit
the methylated fraction of DNA is recognized by 5-methylcytosine
antibody and quantified through an ELISA-like reaction.
1μg of DNA was methylated in GC Reaction Buffer containing 60 units of
GpC Methyltransferase (M.CviPI) and 160 μM
S-adenosylmethionine from New England Biolabs (Ipswich, MA) at 37oC
for either 4 or 18 hours. The GC Methyltransferase methylates all cytosine
residues within the double stranded recognition sequence of 5'..GC..3'. Percent
methylation was determined using the global DNA methylation kit.
of DNA damage.
PBMCs from young
donors were treated with 10uM hydrogen peroxide for 1 h at 37oC. DNA
was extracted as described from both treated and untreated PBMCs. Damage was assessed
using the DNA damage quantification kit.
of DNA damage.
DNA damage from aged
and young subjects was quantified using DNA damage Quantification kit from
BioVision (Mountain View, CA) following the manufacturer's protocol. This kit
determines the number of abasic sites in purified DNA samples utilizes the ARP
(Aldehyde Reactive Probe) reagent that reacts specifically with an aldehyde
group, which is the open ring form of the Apurinic/apyrimidinic
Statistical analysis was performed using GraphPad
Prism™ 4.00 software (GraphPad Software, San Diego, USA). Unpaired data were
analyzed with the Mann-Whitney test. Wilcoxon signed ranked test was used for
paired analyses. Statistical significance was acknowledged when the P-value