J ALLERGY CLIN IMMUNOL Abstracts AB231

VOLUME 125, NUMBER 2

903 Cord Blood Eosinophil Progenitor Expression of Major Basic

Protein mRNA is Associated with Maternal Atopic
Sensitization
A. K. Ellis, K. Fraser, L. Crawford, J. A. Denburg; Queens University,
Kingston, ON, CANADA.
RATIONALE: We recently developed and validated multiplex Q-PCR
analyses of key eosinophil-lineage gene_including that of major basic pro-
tein-1(MBP1)_in cord blood(CB) progenitor populations stimulated with
IL-5. Our current objective was to determine if differences exist in kinetic
expression patterns of MBP1 mRNA by Q-PCR in IL-5 stimulated CB non-
adherent mononuclear cells(NAMNC) between infants born to atopic(>1
positive skin prick tests(SPTs) vs. non-atopic(negative SPTs) mothers.
METHODS: CB NAMNC were isolated from 10 atopic and 10 non-atopic
mothers participating in a prospective birth cohort study. Cells were incu-
bated with 1 ng/mL rhIL-5; at 24, 48 and 72h post-stimulation, RNA was
isolated, reverse transcribed, and expression of MBP mRNA determined,
utilizing multiplex Q-PCR with GAPDH and beta-actin as housekeeping
genes. Relative expression ratios of stimulated and un-stimulated cells
were calculated using the DDCt method.
RESULTS: Stimulation of CB NAMNC with IL-5 resulted steady up-reg-
ulation of MBP1, as previously demonstrated in random CB donors.
Differences were noted in MBP1 mRNA expression levels between infants
born to atopic vs. non-atopic mothers: the mean fold increase at 48h was
1.7 for non-atopics, vs. 8.1 for atopics(p50.032), and at 72h 9.9 for the
non-atopics vs. 18.2 for the atopics(p 5 0.046) on independent samples
t-testing.
CONCLUSION: Multiplex Q-PCR analyses of MBP1 mRNA from CB
eosinophil progenitors demonstrate enhanced sequential expression of crit-
ical eosinophil lineage-specific genes, in subjects born to atopic vs. non-
atopic mothers. These findings suggest upregulation of eosinophilopoiesis
as a result of maternal allergen sensitisation, and could be applied to pro-
vide CB molecular biomarkers of the atopic diathesis.
905 Effect of Soybean Isoflavones on the Pathogenesis of Murine
Peanut Allergy
M. Masilamani, M. Paul, B. OKeefe, H. A. Sampson; Mount Sinai
School of Medicine, New York, NY.
RATIONALE: Soybeans are rich in anti-inflammatory isoflavones such as
genistein, daidzein and glycitein, and are the most common source of iso-
flavones in the human food supply. We hypothesize that the active isofla-
vones in the gut milieu are capable of modulating immune responses to
dietary antigens by regulating dendritic cell (DC) function. We tested
this hypothesis in human and murine dendritic cells (DCs) and in a murine
model of peanut allergy.
METHODS: Human monocyte derived DCs (MDDC) and mouse bone
marrow derived DCs (BMDC) were activated with lipopolysaccharide
(LPS) or cholera toxin (CT) in the presence of isoflavones. The surface ex-
pression levels of DC activation markers were analyzed by flow cytometry.
The effect of dietary isoflavones on the pathogenesis of peanut allergy was
investigated in C3H/HeJ mice fed with a soy-free diet. The anaphylactic
symptoms in these mice were compared to those fed on a regular diet
that contained soy.
RESULTS AND CONCLUSIONS: We observed a significant reduction
(>50%) in the activation-induced cell-surface expression of CD83,
CD80 and CD86 in both human MDDC and mouse BMDC in the presence
of isoflavones in vitro. The expression of MHC class II molecules on DCs
was not significantly affected by isoflavone treatment. Absence of dietary
isoflavones in mice worsened the anaphylactic symptoms and increased the
number of degranulated mast cells (by 30-40%) in the ear specimens, when
compared to mice fed with regular soy-based diet. These results suggest
that soybean isoflavones could modulate DC function and protect against
the development of food allergy.

904 Ultraviolet C-Induced DNA Repair: A Potential Mechanism for

Immunomodulation of Circulating Lymphocytes
T. Watkins1, S. L. Gao1, S. Maynard2, S. A. Hudson1, C. Cheadle1, K. C.
Barnes1, D. N. Grigoryev1; 1Division of Allergy and Clinical Immunol-
ogy, Johns Hopkins University, Baltimore, MD, 2Laboratory of Molecular
Gerontology, National Institute on Aging, Baltimore, MD.
RATIONALE: The beneficial clinical effects of ultraviolet C (UVC) treat-
ment of peripheral blood mononuclear cells (PBMCs) have been demon-
strated for chronic infection and allergies. However, the precise
mechanism of this therapy is unknown. We hypothesize that UVC exert ep-
igenetic immunomodulating effect on PBMCs.
METHODS: DNA damage was assessed by measuring the degree of re-
laxation of supercoiled pGEM-vector. Human PBMCs (106 cells/ml)
were exposed to therapeutic dose of UVC (254 nm, 151J/m2) n53 or vis-
ible light (controls) n53. After 6 hours healthy PBMCs were detected by
flow cytometry using double staining (Annexin/PI) and DNA repair was
evaluated by SCGE (Comet) assay. Gene expression profiling was con-
ducted using Illuminas Sentrix HumanRef-8_V3_0_R1 BeadChip
(24,500 probes) and analyzed by BeadStudio_1.5 and SAM_2.0. Genes
with fold change (FC)>2 and false discovery rate (FDR) <5% were consid-
ered significantly affected by UVC.
TUESDAY
RESULTS: Despite the finding that the applied UVC dose damaged >95%
of tested DNA, 18.263.3 (% of control) cells were successfully repaired 6
hours later and remained viable without signs of apoptosis. At the mid-
point of repair (3 hours) we identified 36 upregulated genes, 10 (28%) of
which encode histones and are overrepresented by histone cluster 1: H2B
(FC55.40), H3A (FC54.95), H4B (FC53.80), H1C (FC52.10), H4E
(FC52.02). Changes in immunoregulators were also detected, including
the genes CXCL2 (FC53.42) and CCL20 (FC53.15).
CONCLUSIONS: We demonstrate the ability of PBMCs to repair and sur-
vive UVC treatment. Our data suggest that the repacking of repaired DNA
with newly synthesized histones might remove previous epigenetic infor-
mation, thus transforming cells towards their nave state.