J ALLERGY CLIN IMMUNOL

FEBRUARY 2013

AB74 Abstracts

SUNDAY

265 B-Cell Phenotypes in Solid Organ Transplant PediatricPatients with Hypogammaglobulinemia

Jennifer A. Shih,MD1, Lisa J. Kobrynski, MD,MPH, FAAAAI2; 1Emory

University, Atlanta, GA, 2Emory Children’s Center, Atlanta, GA; Emory

University School of Medicine, Atlanta, GA.

RATIONALE: The etiology of persistent hypogammaglobulinemia post

solid organ transplant (SOT) is not clear. T-cell immunosuppressive

medications, the standard of care to prevent rejection of SOT, mainly

affect T-cell function but not B-cell function. We examined patterns of

B-cell differentiation and analyzed B-cell phenotypes in post SOT

pediatric patients with hypogammaglobulinemia.

METHODS: Six patients (<18y) with hypogammaglobulinemia diag-

nosed 2-10 years post SOTwere evaluated. Patient data was retrospectively

collected from the medical records. Peripheral blood mononuclear cells

were isolated from 6 patients and 5 controls. B-cell populations were

analyzed by 4-color flow cytometry using CD19, CD27, IgM, and IgD

monoclonal antibodies.

RESULTS: Four patients received rituximab 1-7 years prior to identifi-

cation of hypogammaglobulinemia. Five patients had higher proportions

(mean577%614) of na€ıve B-cells (CD19+CD27-IgD+) compared to

controls (mean548%616). Three of these had high proportions (80%-

90%) of switched, non-memory B-cells (CD19+CD27-IgM+), 2 of whom

never received rituximab. One patient had normal (77%) switched,

memory B-cells (CD19+CD27+IgM+).

CONCLUSIONS: Hypogammaglobulinemia is an under-recognized

complication of SOT. Five of our patients had significant reduction in

proportion of switched memory B-cells compared to controls, suggesting

impairment of B-cell maturation, regardless of rituximab use. This

preliminary study highlights the importance of monitoring immunoglob-

ulin levels pre and post SOT and B-cell phenotyping to identify patients

who would benefit from replacement immunoglobulin to prevent infec-

tious complications.

266 Respiratory Syncytial Virus (RSV) Infection and Replication inMesenchymal Stem Cells (MSCs)

Michael B. Cheung, MS1,2, Subhra Mohapatra, MS PhD1,2, Shyam S.

Mohapatra, PhD, FAAAAI1,2; 1The University of South Florida Morsani

College of Medicine, Tampa, FL, 2James A Haley VA Hospital, Tampa,

FL.

RATIONALE: Extrapulmonary manifestations of RSV have been

reported but the consequences are poorly understood. Since RSV replicates

readily in human mesenchymal stem cell (MSC) cultures we reasoned that

it might contribute to the chronicity of asthma and investigated whether

RSV infection affects MSC proliferative and immune modulatory

functions.

METHODS: Balb/c mice infected intranasally with 2x106plaque forming

units (pfu) of RSV were sacrificed 5 days post infection (pi). RNA from

their bone marrow cells was isolated and analyzed using RTqPCR to detect

expression of RSVN. Human and mouse MSCs were infected in vitro with

RSVexpressing a fluorochrome and fluorescentmicroscopy, plaque assays,

flow cytometry and RTqPCR of important regulatory cytokines were per-

formed at 24, 48, and 72 hours pi.

RESULTS: RSVN transcript was detected in bone marrow cells of

BALB/c mice infected intranasally with RSV (p50.0097 versus unin-

fected controls). RSV was found to productively infect MSCs from both

humans and mice as analyzed by fluorescent microscopy, flow cytometry,

and plaque assays. RTqPCR analysis of infected human MSCs revealed

that IL6 expression decreased to less than 20% of baseline at 24 and 48

hours pi (p50.0010 and 0.0007, respectively) and later increased to 3-fold

baseline levels at 72 hours pi (p5 0.0007).

CONCLUSIONS: The present study demonstrates the ability of RSV to

affect MSC function via infection of the bone. The reported modulation of

IL6, an important immune regulator, provides evidence of an RSV-

mediated affect on MSC proliferation and immunomodulatory functions

that may be relevant to RSV-associated lung disease.

267 Interactive Effect of Sodium Sulfite and Rhinovirus Infectionin Chemokines Production by Airway Epithelial Cells

Kim Hyun Hee, MD, PhD1, Yoon Hong Chun2, Jong-seo Yoon2, Joon

Sung Lee, MD, PhD2; 1Dept. of Pediatrics Bucheon St. Mary’s Hospital

The Catholic University of Korea, Bucheon-si, South Korea, 2Dept. of Pe-

diatrics The Catholic University of Korea.

RATIONALE: Sulfur dioxide is one of the most important air pollutants

that can adversely affect the respiratory system. Rhinovirus (RV) is a major

risk factor responsible for the exacerbation of asthma. An epidemiological

study suggested that interactions between sulfur dioxide and viral infec-

tions exacerbate respiratory disease. We investigated the effects of sodium

sulfite on the production of RV-induced chemokines such as IL-8,

RANTES, and IP-10 in airway epithelial cells in vitro.

METHODS: A549 cells were pretreated with 2,500 mM sodium sulfite for

6 h at 378C and infected with RV-7 at 1 3 10��u tissue culture infectious

dose 50% (TCID50)/mL for 2 h at 338C. The medium was replaced with a

virus-free medium, and the cells were incubated for 40 h at 338C. Cellculture supernatants andmRNAwere harvested at 24 h and 48 h after sodium

sulfite treatment. Production and mRNA expression of IL-8, RANTES, and

IP-10 in these harvests were assessed by ELISA and real-time PCR.

RESULTS: RV induced the production and mRNA expression of IL-8,

RANTES, and IP-10 in the A549 cells. Sodium sulfite did not affect the

viability of A549 cells or RV replication. When the cells were pretreated

with sodium sulfite prior to RV infection, production and mRNA expres-

sion of RV-induced IL-8, RANTES, and IP-10 were enhanced with no

effect on cell viability or RV replication.

CONCLUSIONS: Our results suggest that sodium sulfite may potentiate

the activity of RV-induced diseases by increasing the production of IL-8,

RANTES, and IP-10.

268 Assessment of Bacterial Colonization in Airways of Childrenwith Asthma and Chronic Cough by Culture and 16S rRNAGene Sequencing

Pia J. Hauk, MD1, Elena Goleva, PhD2, Leisa P. Jackson, BS2, J. Kirk

Harris, PhD3, J. Tod Olin, MD, MSCS4, Susan M. Brugman, MD4, Dave

P. Nichols, MD4, Marzena Krawiec, MD5, Donald Y. Leung, MD, PhD1;1National Jewish Health, Department of Pediatrics, Division Pediatric

Allergy/Immunology, Denver, CO, 2National Jewish Health, Department

of Pediatrics, Denver, CO, 3University of Colorado Anschutz Medical

Campus, Department of Pediatrics, Aurora, CO, 4National Jewish Health,

Department of Pediatrics, Division Pediatric Pulmonology, Denver, CO,5Eastern Carolina University, Pediatric Pulmonology, Greenville, NC.

RATIONALE: Controversy continues concerning the clinical relevance

of bacteria in the lower airways of children with asthma. To determine the

pathogenic role of bacteria, we studied bronchoalveolar lavage (BAL)

from children who underwent clinically indicated bronchoscopies.

METHODS: BAL samples from 29 children, ages 2 to 16 y (median 12 y),

23 with asthma and 6 with chronic cough were analyzed for presence of

bacteria by cultures and 16S rRNA gene sequencing. Bacterial lipopolysac-

charide (LPS) in BAL was measured by chromogenic limulus lysate assay.

RESULTS: Pathogenic bacteria were detected in 79.3% (23/29) of BAL

samples by culture or 16S rRNA sequencing; 87% of those (20/23) were

Gram-negative. Both techniques detected the same bacterium in only

46.4%. Copy numbers of 16S rRNA as measure of bacterial load were

higher in BAL samples with positive compared to negative cultures

(median, 25% - 75% quartiles: 2140, 585 – 5605 versus 545, 401 – 882;

p50.0236). LPS (pg/mg protein) was increased in BAL with Gram-

negative bacteria by culture and sequencing versus sequencing alone

(median, 25% - 75% quartiles: 354, 146 – 597 versus 0, 0 to 60; p50.006).

CONCLUSIONS: Our data suggest that BAL cultures do not detect all

pathogenic bacteria in the lower airways of children who undergo

bronchoscopy. Positive cultures indicate a higher bacterial load and

increased LPS exposure in presence of Gram-negative bacteria. 16S

rRNA gene sequencing may provide further information in the assessment

of bacterial colonization in the airways of children with difficult to manage

asthma and chronic cough.