J ALLERGY CLIN IMMUNOL

FEBRUARY 2012

AB366 Abstracts

L8 A Distinct Proteome Expression Profile and Extensive Changesin Cysteinyl S-Nitrosylation (SNO) in Eosinophilic Esophagitis

C. M. Davis1, J. E. Wiktorowicz2, K. V. Soman2, C. Straub2, C. Nance1,

M. D’Souza1, M. Lester1, S. Stafford2, K. Pazdrak2, K. Thakkar1, A. P. Ol-

ive1, A. Kurosky2; 1Baylor College of Medicine, Houston, TX, 2The Uni-

versity of Texas Medical Branch, Galveston, TX.

RATIONALE: Inflammatory mediators have been discovered in eosin-

ophilic esophagitis (EoE) through mRNA expression, but protein expres-

sion and post-translational modifications have not been explored.

Cysteinyl S-nitrosylation (SNO) is a major mechanism through which

nitric oxidemodulates protein activity. Herewe describe our results using a

novelmethod (SNOFlo) to quantify SNO1 in tissues of an EoE patient com-

pared to normal.

METHODS: Proteins from biopsy samples of EoE and controls were

assayed for total cysteine and half-cystine content by amino acid analysis,

and fluorescent labeled before and after ascorbate treatment using the

SNOFlo method. Labeled proteins were separated by 2D electrophoresis,

imaged, and analyzed by SameSpots software (Nonlinear Dynamics).

S-nitrosylation was determined for each protein spot, and selected spots

were picked, trypsin digested, and analyzed by MALDI-TOF (AB 4800).

Protein identification utilized MASCOT applied to the Swiss-Prot

database.

RESULTS: Applying SNOFlo to the EoE proteome, we detected differ-

entially S-nitrosylated proteins between EoE and normal samples. Proteins

from distal esophageal (DE) samples had more striking post-translational

modifications than proximal (PE) tissues. Compared to controls, DE

samples had 33 increased and 38 decreased SNO proteins, and PE samples

had 22 increased and 27 decreased SNO proteins. Types I and II keratin,

annexin, glutathione S-transferase, peroxiredoxin 1 and 2, epidermal fatty

acid binding protein, and cystatin A were significantly differentially

nitrosylated in EoE (MS expectation scores <_0.001).

CONCLUSIONS: Evidence of unique protein expression patterns in EoE

with widespread involvement of SNO implicates a possible aberrant nitric

oxide regulation in disease pathogenesis.1Wiktorowicz, J.E., et al. Biochemistry 50:5601-5614, 2011.

L9 Th17 Related Cytokines Promote The Production of ProfibroticCytokines from Human Eosinophils

R. Halwani1, S. Al-Muhsen1, Q. Hamid2; 1King Saud University, Riyadh,

SAUDI ARABIA, 2Meakins-Christie Laboratories, McGill University,

Montreal, QC, CANADA.

RATIONALE: Asthma is a chronic inflammatory disorder of the lung

airways that is associated with airway remodeling and hyperresponsive-

ness. One of the most critical structural changes that affect airway

functionality is fibrotic tissue deposition within the airway wall.

Eosinophils have been proposed in different studies to contribute to the

production of several mediators and cytokines, including the profibrotic

cytokines, TGF-b and IL-11. In this study, we hypothesize that cytokines

prevailing in asthmatic tissue such as Th1, Th2, and Th17 cytokines, may

induce eosinophils to produce pro-fibrotic cytokines.

METHODS: Eosinophils were isolated from peripheral blood of 6 mild

asthmatics and 6 normal control subjects. Eosinophils were stimulatedwith

Th1, Th2 and Th17 cytokines and production of pro-fibrotic cytokines,

TGF-b and IL-11, were determined using Intra-cellular cytokine detection

and FACS analysis, immunohistochemistry, as well as real time PCR.

RESULTS: The level of basal expression of TGF-b and IL-11 was

significantly upregulated in asthmatic patients compared to healthy

individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not

induce expression of eosinophils derived pro-fibrotic cytokines. However,

stimulating eosinophils with IL-17 resulted in the enhancement of the

expression TGF-b and IL-11 in asthmatic individuals.

CONCLUSIONS: The regulation of expression of pro-fibrotic cytokines

within eosinophils is Th1/Th2 independent. However, IL-17 seems to

regulate eosinophl profibrotic cytokine release in asthmatic patients and

hence contributing to the accumulation of fibrotic tissue in asthmatic

airways.

L10 L-selectin Deficiency as a Novel Primary ImmunodeficiencyCauses Recurrent Infections

D. M. Al-Zahrani1, M. K. AL-Sayegh2; 1Allergy and Immunolgy, King

Abdulaziz Mdical City, Jeddah, SAUDI ARABIA, 2King Abdulaziz On-

cology Center, Jeddah, SAUDI ARABIA.

RATIONALE: Recruitment of leukocytes into sites of infection /inflam-

mation involves leukocyte-to-leukocyte and leukocyte-to-endothelium

interactions that required adhesion molecules and their ligands. We

describe a case with L-selectin (CD62L) deficiency as a novel primary

immunodeficiency disease.

METHOD: One month old girl with history of fever for 5-days treated

with ceftrixone intramuscularlly in private clinic. She was admitted with

fever, seizure, and mild DIC. Ampicillin, cefotaxim and acyclovir were

used for partially treated sepsis. She had fever recurrence with significant

neutrophilia and several antibiotics were used for recurrent sepsis includ-

ing anti-fungal. History of sibling death at 6-months of age due to infection

and recurrent sepsis raised possibility of underlying immunodeficiency.

RESULTS: Complete blood count showed: leucocytes 61,690 cells/cmm;

absolute neutrophils 38,865 cells/cmm and lymphocytes 11,721 cells/

cmm, haemoglobin 15.3 g/dl and thrombocytes 27,000/cmm. Erythrocyte

sedimentation rate 76mm/hr. Her coagulopathy resolved. Bone marrow

biopsy: showed no malignancy. Septic screen, viral and metabolic screen

were negative. Immunoglobulin levels, Tand B-Lymphocyte markers were

normal. Patient’s neutrophils showed absence of L-selectin (CD62L)

expression and migration failure through cell culture inserts towards

concentration gradient of the chemoattractant (fMLP) compared to normal

control by flowcytomerty. CD11/18 and CD 15 were normal.

CONCLUSION: The adhesion molecules and their ligands are all

required for immune defense against microbes. Isolated L-selectin

(CD62L) deficiency causing primary immunodeficiency was not previ-

ously described. We described isolated L-selectin deficiency confirmed by

absence of CD62L molecule on leukocytes as a novel primary immuno-

deficiency that caused recurrent sepsis. L-selectin deficiency should be

considered in the differential diagnosis of recurrent infections.

L11 Transplacental Passage of IgG anti-IgE/IgE ImmuneComplexes (ICs)

A. P. Matson1,2, E. Rafti1; 1Univ of CT Health Ctr, Farmington, CT, 2CT

Children’s Med Ctr, Hartford, CT.

RATIONALE: Maternal IgG enhances the placental transfer of several

antigens; however, it is unclear if maternal IgG may facilitate the

transplacental passage of IgE as IgG anti-IgE/IgE ICs.

METHODS: Mother/infant dyads were prenatally recruited and maternal

allergic status was determined by questionnaire. Maternal blood was

collected prior to delivery and cord blood (CB) was collected immediately

after birth. Serum concentrations of IgG anti-IgE/IgE ICs were determined

using a modified ELISA. The relationship betweenmaternal and infant ICs

was determined by Spearman’s rank correlation.Maternal and CB total IgE

levels were determined by Phadia (Thermo Fisher Scientific) ImmunoCAP.

IgAwas measured to detect maternal blood contamination of CB.

RESULTS: Serum concentrations of IgG anti-IgE/IgE ICs were deter-

mined in 30 allergic mother/infant dyads and 73 non-allergic mother/infant

dyads. There was no difference in concentrations of IgG anti-IgE/IgE ICs

in mothers or infants based onmaternal history of allergic disease. Overall,

maternal and CB concentrations of IgG anti-IgE/IgE ICs were highly

correlated (rho50.89, p<0.001), suggesting placental transmission. A

similar relationship was observed for the allergic (rho50.90, p<0.001) and

non-allergic (rho50.88, p<0.001) groups. IgE concentrations were higher

in allergic mothers compared to non-allergic mothers (p<0.01). Maternal

and infant concentrations of IgG anti-IgE/IgE ICs correlated poorly with

CB IgE concentrations.

CONCLUSIONS: Fetal acquisition of maternal IgE in the form of IgG

anti-IgE/IgE ICs likely occurs via the transplacental route. The ability of

maternal IgG anti-IgE/IgE ICs to influence fetal and/or neonatal immunity

remains unclear.