Post on 02-Jan-2017
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.