J ALLERGY CLIN IMMUNOL
VOLUME 132, NUMBER 5
From athe Department of Pediatrics, Graduate School of Medicine, Chiba University,
and bthe Department of Allergy and Rheumatology, Chiba Children’s Hospital, Chiba,
Japan. E-mail: yuzaburo@chiba-u.jp.
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1. Detoraki A, Granata F, Staibano S, Rossi FW, Marone G, Genovese A. Angiogen-
esis and lymphangiogenesis in bronchial asthma. Allergy 2010;65:946-58.
2. Lee CG, Link H, Baluk P, Homer RJ, Chapoval S, Bhandari V, et al. Vascular endo-
thelial growth factor (VEGF) induces remodeling and enhances TH2-mediated
sensitization and inflammation in the lung. Nat Med 2004;10:1095-103.
3. Asai K, Kanazawa H, Kamoi H, Shiraishi S, Hirata K, Yoshikawa J. Increased
levels of vascular endothelial growth factor in induced sputum in asthmatic
patients. Clin Exp Allergy 2003;33:595-9.
4. Hossny E, El-Awady H, Bakr S, Labib A. Vascular endothelial growth factor
overexpression in induced sputum of children with bronchial asthma. Pediatr
Allergy Immunol 2009;20:89-96.
5. Yoo Y, Choi IS, Byeon JH, Lee SM, La KS, Choi BM, et al. Relationships of
methacholine and adenosine monophosphate responsiveness with serum vascular
endothelial growth factor in children with asthma. Ann Allergy Asthma Immunol
2010;104:36-41.
6. Global Initiative for Asthma (GINA). The Global Strategy for Asthma Manage-
ment and Prevention. 2012. Available from http://www.ginasthma.org/. Accessed
June 1, 2013.
7. Marek N, Mysliwiec M, Raczynska K, Zorena K, Mysliwska J, Trzonkowski P.
Increased spontaneous production of VEGF by CD41 T cells in type 1 diabetes.
Clin Immunol 2010;137:261-70.
8. Sun CY, She XM, Qin Y, Chu ZB, Chen L, Ai LS, et al. miR-15a and miR-16 affect
the angiogenesis of multiple myeloma by targeting VEGF. Carcinogenesis 2013;
34:426-35.
9. Dejean E, Renalier MH, Foisseau M, Agirre X, Joseph N, de Paiva GR, et al.
Hypoxia-microRNA-16 downregulation induces VEGF expression in anaplastic
lymphoma kinase (ALK)-positive anaplastic large-cell lymphomas. Leukemia
2011;25:1882-90.
10. Lal A, Thomas MP, Altschuler G, Navarro F, O’Day E, Li XL, et al. Capture of
microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator
of growth factor signaling. PLoS Genet 2011;7:e1002363.
11. Yin KJ, Olsen K, Hamblin M, Zhang J, Schwendeman SP, Chen YE. Vascular
endothelial cell-specific microRNA-15a inhibits angiogenesis in hindlimb
ischemia. J Biol Chem 2012;287:27055-64.
12. Yuan Y, Kasar S, Underbayev C, Vollenweider D, Salerno E, Kotenko SV, et al.
Role of microRNA-15a in autoantibody production in interferon-augmented
murine model of lupus. Mol Immunol 2012;52:61-70.
13. Lorenzi JC, Brum DG, Zanette DL, de Paula Alves Souza A, Barbuzano FG, Dos
Santos AC, et al. miR-15a and 16-1 are downregulated in CD41 T cells of multiple
sclerosis relapsing patients. Int J Neurosci 2012;122:466-71.
Available online August 15, 2013.
http://dx.doi.org/10.1016/j.jaci.2013.06.041
Regional differences in the expression of innate
host defense molecules in sinonasal mucosa
To the Editor:
Nasal mucosal epithelial cells form the first line of defense by
maintaining a barrier to restrict potentially harmful airborne
substances and pathogens. To aid in host defense, specialized
epithelial cells in submucosal glands as well as on the mucosal
surface secrete mucous that helps to immobilize pathogens and
other harmful substances. Beneath this mucous blanket resides an
aqueous surface liquid layer that allows proper ciliary function
to clear the mucous and entrapped pathogens. In concert with
the maintenance of a physical barrier, airway epithelium secretes
dozens of antimicrobial peptides and proteins that become
incorporated into the mucous and aqueous lining fluids.1,2 It has
become clear that proper functioning of the epithelium is essential
for suppressing the growth of pathogenic organisms and promot-
ing healthy upper airway physiology. Dysfunction in innate im-
mune expression of host defense molecules has been linked to
many airway diseases.3-8 Surprisingly, the regional distribution
LETTERS TO THE EDITOR 1227
of important epithelial-derived antimicrobial proteins secreted
into upper airways has not been studied in detail. In this study,
we analyzed the expression of select antimicrobial proteins that
are known to be secreted into the lining fluids of the upper airways
in 2 different anatomical sites of the sinonasal mucosa in healthy
human subjects, namely, the inferior region (inferior turbinate
[IT]) and the superior region (uncinate tissue [UT]). We chose
IT, as it is the proximal point of contact of inhaled air containing
particulate matter and potential pathogens. Moreover, studies of
chronic rhinosinusitis (CRS) and other diseases of the upper air-
ways routinely use IT tissue as a control tissue to make compar-
isons with nasal polyps. UTwas chosen for its important role in the
drainage pathways of maxillary, frontal, and anterior ethmoid si-
nuses. We chose to study the expression of S100A7 (S100 family),
hBD2 (b-defensin family), SPLUNC1 (PLUNC family), and lac-
toferrin as they represent broad families of antimicrobial proteins,
which are either constitutively present or induced by inflammation
triggered by pathogens and collectively have antimicrobial effects
against a variety of pathogens (bacteria, fungi, and viruses).
Sinonasal tissue samples from IT and UT were collected from
subjects undergoing surgery to correct noninflammatory condi-
tions, including facial anatomical defects (deformity, trauma, etc),
to improve airflow, and during the course of skull base surgery to
remove tumors. The subjects were not diagnosed with any upper
or lower airway diseases at the time of sample collection. Detailed
patient characteristics are provided in Table E1 in this article’s On-
line Repository at www.jacionline.org. The Investigational Re-
view Board of Northwestern University approved all methods
for the present study, and all patients provided informed consent.
At the time of surgery, tissues and nasal epithelial scraping cells
were collected and stored for further analysis. Tissue samples
and epithelial cells were analyzed for mRNA expression by using
real-time PCR. Protein expression and localization were analyzed
by using ELISA and immunohistochemistry, respectively. Alcian
blue/periodic acid-Schiff staining was performed to characterize
the glandular differences between IT and UT. Details of the
methods used can be found in the Online Repository and our other
publications.5,7
Earlier studies of S100A7 expression in CRS by our group
indicated that S100A7 was differentially expressed in IT and UT
tissues.7 To determine regional variations in mRNA expression of
this and other innate immune molecules in sinonasal tissues, we
performed real-time PCR in a total of 21 samples (10 UT and 11
IT). We found that the mRNA expression of S100A7 and hBD2
was considerably higher in IT tissue than in UT (400- and
80-fold, respectively, Fig 1, A and B). In stark contrast, UT had
a substantially higher mRNA expression of SPLUNC1 than did
IT tissue (8-fold, Fig 1, E—note log scale). Lactoferrin mRNA
expression did not differ between UT and IT (Fig 1, F). Next,
we attempted to confirm the mRNA findings at the protein level
by using ELISA. In concordance with our mRNA data, the expres-
sion of S100A7 and hBD2 proteins was significantly higher in IT
than in UT, by 40- and 21-fold, respectively (P <.05) (Fig 1, C and
D). Likewise, SPLUNC1 protein was elevated in UT when
compared with IT (8-fold, P < .05) (Fig 1, G). In contrast to the
mRNA data, lactoferrin protein was 3-fold higher in UT than in
IT (Fig 1, H). Because it is well known that sinuses contribute a
majority of the respiratory NO production, and because NO plays
an antimicrobial role, we also evaluated the expression of
various nitric oxide synthases (NOS1, NOS2, and NOS3) in
IT and UT (see Fig E1 in this article’s Online Repository at
1228 LETTERS TO THE EDITOR J ALLERGY CLIN IMMUNOL
NOVEMBER 2013

FIG 1. Evaluation of expression of antimicrobial mRNA and protein in uncinate and inferior turbinate
tissues of healthy human subjects. Total RNA and protein were extracted from uncinate and inferior
turbinate tissues of healthy subjects. Expression of mRNA was analyzed by using real-time PCR. Expression
of proteins was analyzed by using ELISA. AB/PAS staining was used to observe differences in the presence
of glands. Data for panels D (Tieu et al7) and E to H (UT only-Seshadri et al5) are taken from earlier publica-
tions as indicated. AB, Alcian blue; NEC, nasal scraping cells; NS, not significant; PAS, periodic acid-Schiff.
*P < .05, **P < .01, and ***P < .001 (Mann-Whitney U test).

www.jacionline.org). We confirmed the previous studies by Lund-
berg et al,9 showing that the expression of mRNA for NOS2, the
major form of NOS in the sinuses involved in the production of
NO, was much higher in UT than in IT (22-fold, 292.3 6 82.8
vs 13.3 6 9.4 copies/ng total RNA, P 5 .0006, n 5 7-8). Interest-
ingly, we observed that NOS1 (4.7-fold) and NOS3 (1.6-fold)
were elevated in IT compared with UT. Taken together, these
observations indicate that there is a differential expression of
molecules involved in innate host defense in sinonasal tissues
taken from different anatomical sites of healthy human subjects.
We confirmed our previous observation that S100A7 was
primarily expressed in the mucosal epithelium with modest
staining in submucosal glands, whereas SPLUNC1 was highly
expressed in the submucosal glands with minimal presence in the
mucosal epithelium (see Fig E2 in this article’s Online Repository
at www.jacionline.org).5,7 To further confirm the immunohisto-
chemistry data indicating high expression levels of S100A7 in
the surface epithelium, we performed ELISA with isolated nasal
epithelial cells from IT and UT tissues. In agreement with our
ELISA data in whole tissue homogenates, we found that IT
epithelial cells had a 12-fold higher S100A7 protein expression
than did UT epithelial cells (Fig 1, I). In contrast, the expression
of SPLUNC1 in epithelial cells from IT and UT was not different
(Fig 1, J). This suggests that the regional variation of SPLUNC1
expression in tissue samples may be caused by variability in
expression in glands rather than in mucosal epithelium (see
below). These findings also suggest that the underlying molecular
mechanism responsible for the regional variation of S100A7 may
differ from the differential transcriptional expression mechanism
of SPLUNC1.
To further understand the regional variations between IT and
UT tissues in the expression of host defense molecules that are
largely derived from submucosal glands, specifically SPLUNC1
and lactoferrin, we assessed the relative expression of these
molecules in both serous and mucous cells within glands from
both locations. In initial studies, we stained both tissues with
mucous (alcian blue) and serous (periodic acid-Schiff) stains. We
failed to observe any appreciable differences in the number or
proportion of serous and mucous cells in IT when compared to UT
tissues (Fig 1, K and L; see Fig E3 in this article’s Online Repos-
itory at www.jacionline.org). These experiments indicate that the
regional variation of host defense molecules is not explained by
regional variation in either submucosal gland density or propor-
tions of serous and mucous cells within glands. We must caution
J ALLERGY CLIN IMMUNOL LETTERS TO THE EDITOR 1229
VOLUME 132, NUMBER 5

FIG 2. Regional differences in the expression of innate host defense molecules in sinonasal mucosa.

that while there is no appreciable difference in the number of to speculate that the increased expression of host defense mole-
glands based on microscopic observation, it remains possible cules such as SPLUNC1, lactoferrin, and MUC5B in UT may
that there are differences in the volume of glands between the be important for additional host defense in sinus drainage path-
IT and UT tissues. ways. In support of this hypothesis, there is emerging evidence
A pictorial summary of our findings is shown in Fig 2. Our indicating that SPLUNC1 has surfactant properties and may be
finding of differential expression of innate immune proteins be- involved in biophysical aspects of mucociliary clearance.11 Our
tween IT and UT is unexpected because these tissues have been data also suggest that there may potentially be different micro-
used interchangeably to study gene expression levels and as con- biota and/or susceptibilities for microbial colonization in the
trol tissue to make comparisons with diseased tissues such as nasal proximal (IT) and distal (UT) areas of the sinonasal mucosa,
polyps in the case of CRS. A recent report from Laudien et al10 with UT having a more profound expression of submucosal
compared the expression of host defense molecules between the gland–derived antimicrobial proteins.
nasal vestibule (anterior part of the nasal cavity) and the IT region In conclusion, our findings indicate anatomic site-specific
of the proper nasal mucosa. The findings of differential expression expression of antimicrobial proteins in sinonasal tissues, suggest-
of some of the molecules tested in their study between the nasal ing the possibility of specialized regional functions of these
vestibule and IT are interesting, though not surprising, because proteins within the sinonasal cavity. Also, our findings of different
it is known that epithelial cells of the nasal vestibule are similar intrinsic baseline levels of gene expression in IT and UT suggest
to keratinocytes (stratified squamous), which are very different that a careful selection of the site of control tissue is needed when
from respiratory epithelial cells (pseudostratified columnar). studying host defense molecules.
This study demonstrates that the expression of some innate host
defense molecules in various regional tissues is quite variable, We thank Ms Jacqi Schaffer for assistance in producing Fig 2.
with expression of some glandular proteins (lactoferrin and Sudarshan Seshadri, PhDa
SPLUNC1) greater in UT than in IT. Interestingly, we initially Mariel Rosati, BSa
used the mucin MUC5B as a marker of mucous cells, but found a David C. Lin, MDa
differential expression similar to that of SPLUNC1—that is, high Roderick G. Carter, BSa
levels in UT and much lower levels in IT (6181 6 1993 vs 2147 6 James E. Norton, MSa
411 copies/ng of total RNA, P 5 .03). This supports the concept Andrew Wonho Choi, BSa
that submucosal glands near the UT are a much richer source of a Lydia Suh, BSa
number of host defense molecules than are corresponding glands Atsushi Kato, PhDa
Rakesh K. Chandra, MDb
in the IT region. The apparent differences that we observe could
Kathleen E. Harris, BSa
be due to the different embryonic origins of these tissues (maxil- Hong Wei Chu, MDc
loturbinal [IT] vs ethmoturbinal [UT]). As a result of different Anju T. Peters, MDa
embryonic origins, these tissues could be governed by ontologi- Bruce K. Tan, MDb
cally different gene regulation pathways. Alternatively, region- David B. Conley, MDb
ally dependent environmental exposures may also be a cause or Leslie C. Grammer, MDa
factor in the differential expression observed. Finally, as UT is Robert C. Kern, MDb
located at a point of drainage of various sinuses, it is tempting Robert P. Schleimer, PhDa,b
1230 LETTERS TO THE EDITOR
From athe Division of Allergy-Immunology, Department of Medicine, and bthe
Department of Otolaryngology, Northwestern University Feinberg School of Medi-
cine, Chicago, Ill; and cthe Department of Medicine, National Jewish Health, Denver,
Colo. E-mail: rpschleimer@northwestern.edu.
This research was supported in part by National Institutes of Health grants RO1 HL
078860, R37 GK068546, and RO1 AI072570 and by the Ernest S. Bazley Trust.
Disclosure of potential conflict of interest: R. P. Schleimer has received grants from the
National Institutes of Health (NIH); has consultant arrangements with Intersect ENT,
GlaxoSmithKline, Allakos, and Aurasense; and has stock/stock options in Allakos. A.
Kato and R. K. Chandra have received grants from the NIH. A. T. Peters has received
payment for lectures, including service on speakers’ bureaus, from Baxter. K. E. Har-
ris is employed by Northwestern University. B. K. Tan has received grants from the
NIH (K23DC012067) and the Triological Society-American College of Surgeons;
has consultant arrangements with Acclarent, Inc; and has received travel/accommo-
dations/meeting expenses from the Foundation for Innovation, Education, and
Research in Otorhinolaryngology. L. C. Grammer has received grants and support
for travel to meetings for study or other purposes from the NIH; has received funding
for other purposes from the Bazley Foundation Grant; has consultant arrangements
with Astellas Pharmaceuticals; is employed by Northwestern University and the
Northwestern Medial Faculty Foundation; has grants/grants pending from the NIH,
the Food Allergy Network, and S&C Electric; has received payment for lectures,
including service on speakers’ bureaus, from the AAAAI, Mount Sinai, New York;
and has received royalties from Lippincott, UpToDate, BMJ, and Elsevier. The rest
of the authors declare that they have no relevant conflicts of interest.
REFERENCES
1. Kato A, Schleimer RP. Beyond inflammation: airway epithelial cells are at the
interface of innate and adaptive immunity. Curr Opin Immunol 2007;19:711-20.
2. Ooi EH, Wormald PJ, Tan LW. Innate immunity in the paranasal sinuses: a review
of nasal host defenses. Am J Rhinol 2008;22:13-9.
3. Baines KJ, Simpson JL, Gibson PG. Innate immune responses are increased in
chronic obstructive pulmonary disease. PLoS One 2011;6:e18426.
4. Chu HW, Thaikoottathil J, Rino JG, Zhang G, Wu Q, Moss T, et al. Function and
regulation of SPLUNC1 protein in Mycoplasma infection and allergic inflamma-
tion. J Immunol 2007;179:3995-4002.
5. Seshadri S, Lin DC, Rosati M, Carter RG, Norton JE, Suh L, et al. Reduced expres-
sion of antimicrobial PLUNC proteins in nasal polyp tissues of patients with
chronic rhinosinusitis. Allergy 2012;67:920-8.
6. Avila PC, Schleimer RP. Airway epithelium. In: Allergy and allergic diseases. 2nd
ed. Hoboken, NJ: Wiley-Blackwell; 2008.
7. Tieu DD, Peters AT, Carter RG, Suh L, Conley DB, Chandra R, et al. Evidence for
diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.
J Allergy Clin Immunol 2010;125:667-75.
8. Ramanathan M Jr, Lee WK, Spannhake EW, Lane AP. Th2 cytokines associated
with chronic rhinosinusitis with polyps down-regulate the antimicrobial immune
function of human sinonasal epithelial cells. Am J Rhinol 2008;22:115-21.
9. Lundberg JO, Farkas-Szallasi T, Weitzberg E, Rinder J, Lidholm J, Anggaard A, et al.
High nitric oxide production in human paranasal sinuses. Nat Med 1995;1:370-3.
10. Laudien M, Dressel S, Harder J, Glaser R. Differential expression pattern of
antimicrobial peptides in nasal mucosa and secretion. Rhinology 2011;49:
107-11.
11. McGillivary G, Bakaletz LO. The multifunctional host defense peptide SPLUNC1
is critical for homeostasis of the mammalian upper airway. PLoS One 2010;5:
e13224.
Available online July 26, 2013.
http://dx.doi.org/10.1016/j.jaci.2013.05.042
National burden of antibiotic use for adult
rhinosinusitis
To the Editor:
Rhinosinusitis (RS) is among the most common conditions
encountered in medicine, affecting approximately 15% of the
adult population annually.1,2 According to major consensus
guidelines, antibiotics are not recommended for most patients
with uncomplicated cases of acute RS (ARS).1-5 The role of
antibiotics for chronic RS (CRS) is controversial,2 and recent
authors recommend that objective evidence by endoscopy or
computed tomography should be obtained if a prolonged course
of antibiotics is to be given for CRS.6 However, previous studies
J ALLERGY CLIN IMMUNOL
NOVEMBER 2013
show that antibiotics are prescribed extensively to treat RS, in
approximately more than 80% of ARS7 and 50% of CRS8 patient
visits.
Excessive antibiotic use is associated with consequences
including allergic reactions, adverse effects, unnecessary costs,
and increasing bacterial resistance. With the clinical and
economic tolls of inappropriate antibiotic prescribing in mind,
the goal of this study was to perform a contemporary analysis of
the overall antibiotic burden of RS on a national level.
We analyzed data from the National Ambulatory Medical Care
Survey and National Hospital Ambulatory Medical Care Survey
Outpatient Department component from 2006 to 2010. These
surveys are conducted annually by the US Department of Health
and Human Services to provide data from a national sample of
outpatient visits. These data are weighted to produce national
estimates that describe the use of ambulatory medical care
services in the United States.
We identified visits by adults aged 18 years or older in
which an antibiotic was prescribed (antibiotic visits). The
total number and percentage of primary diagnoses, identified
by International Classification of Diseases, Ninth Revision,
Clinical Modification (ICD-9-CM) code, associated with
each antibiotic visit were tabulated. Primary diagnoses
commonly grouped together9 were also analyzed in groups.
The number and percentage of antibiotic visits associated
with a primary diagnosis of ARS (ICD-9-CM 461.x) and
CRS (ICD-9-CM 471.x or 473.x) were tabulated. Statistical
analyses were performed by using STATA (version 12.0;
STATA Corp, College Station, Tex). Descriptive data are pre-
sented as mean with 95% CIs. Accounting for the survey’s
sampling design, statistics were derived by a multistage esti-
mation procedure designed by the National Center for Health
Statistics to produce essentially unbiased national estimates:
(1) inflation by reciprocals of the probabilities of selection,
(2) adjustment for nonresponse, (3) a ratio adjustment to fixed
totals, and (4) weight smoothing.10
Over the 5-year study period, there were 21.4 million (95% CI,
17.2-25.7 million) estimated visits associated with a primary
diagnosis of ARS and 47.9 million (95% CI, 41.2-54.7 million)
estimated visits associated with a primary diagnosis of
CRS. Overall, antibiotics were prescribed in 85.5% (95% CI,
80.7%-89.2%) of ARS visits and 69.3% (95% CI, 64.8%-73.5%)
of CRS visits.
Over the 5-year study period, RS (ARS and CRS combined)
accounted for 11.0% (95% CI, 10.0%-12.1%) of all primary
diagnoses for ambulatory care visits with antibiotic prescriptions,
more than any other diagnosis or commonly grouped diagnoses
(Fig 1). When comparing individual diagnoses, a primary
diagnosis of unspecified CRS (ICD-9 473.x) accounted for 7.1%
(95% CI, 6.2%-8.0%) of antibiotic visits, still more than any
other primary diagnosis. Analyses by individual year are
presented in Table I. There was no significant linear time trend
in overall antibiotic burden in RS groups (P 5 .98 for all RS,
P 5 .16 for CRS, P 5 .19 for ARS). Because CRS with nasal
polyps may be coded as 473.x (CRS) and 471.x (nasal polyp),
we have shown that a diagnosis of nasal polyp did not make a
meaningful contribution to the overall antibiotic burden of RS
(Table I). In 2010, unspecified CRS (ICD-9 473.x) accounted
for 5.57% of antibiotic visits and ranked second to unspecified
urinary tract infection, which accounted for 5.59% of antibiotic
visits.
1230.e1 LETTERS TO THE EDITOR J ALLERGY CLIN IMMUNOL
NOVEMBER 2013

METHODS were spun at 5000g for 5 minutes to remove debris. Clarified cell lysates
were used for S100A7 and SPLUNC1 ELISA.
Real-time PCR
Total RNA from sinonasal tissue was extracted by using QIAzol (Qiagen,
Valencia, Calif) using RNeasy purification system according to manufac-
turer’s instructions. DNA contamination was eliminated by treatment of RNA
Immunohistochemistry
Immunohistochemistry was performed as described in Seshadri et alE2
with DNase I during purification. Purified RNA was assessed with Agilent
(SPLUNC1) and Tieu et alE3 (S100A7). Sections stained for alcian blue/peri-
2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif) by using a RNA
odic acid-Schiff reagents were deparaffinized, rehydrated, and stained sequen-
6000 Nano LabChip (Agilent Technologies). One microgram of RNA was
tially with alcian blue (Sigma-Aldrich; St Louis, Mo), periodic acid (Electron
converted to single-strand cDNA by using SuperScript II reverse transcriptase
Microscopy Sciences; Hatfield, Pa), and Schiff reagent (Richard-Allan Scien-
system (Invitrogen, Carlsbad, Calif) using random primers. Semi-quantitative
tific, Kalamazoo, Mich). Glandular quantitation of alcian blue/periodic acid-
real-time RT-PCR was performed with a TaqMan method by using an Applied
Schiff sections was performed by 2 independent observers in 10 randomly
Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster
chosen fields by using an Olympus IX71 Inverted Microscope at a magnifica-
City, Calif) in 15 mL reactions (7.5 mL of 23 TaqMan Gene Expression
tion of 2003 and a MicroFire AR digital microscope camera (Optronics,
Master mix [Applied Biosystems], 0.75 mL of 203 primer/probe mix, and 5
Goleta, Calif). Each section was blinded, and subject diagnosis was unknown
mL of 2 ng/mL cDNA). All primers/probes were purchased from Applied
to the observers.
Biosystems. Primers for S100A7 have been described previously.E1 Median
expression of GUSB (Human b-glucuronidase endogenous control, PN;
4326320E) was used for normalizing gene expression. Exact copy number
of the target genes was determined by running serial dilutions of quantified
ELISA
ELISA for S100A7 (Tieu et alE3) and SPLUNC1 and lactoferrin (Seshadri
PCR product of the target gene as standards in the PCR reaction. Target
el alE2) has been previously described. ELISA for human beta defensin-2 was
gene expression was expressed as copies/ng of total RNA.
purchased from Phoenix Pharmaceuticals (Burlingame, Calif) and used ac-
cording to manufacturer’s recommendations. According to the manufacturer,
the minimum detection limit of the ELISA is 7.815 pg/mL. ELISA data were
Extraction of proteins from sinus tissue and nasal
normalized to total tissue protein content. Protein concentration was deter-
scraping cells mined by using BCA protein assay (Thermo Fisher Scientific, Rockford, Ill).
Tissue proteins were extracted as described in Seshadri et al.E2 Briefly, tis-
sues were cut into small pieces and resuspended in PBS containing 0.05%
Tween 20 supplemented with a protease inhibitor cocktail (Sigma-Aldrich,
St Louis, Mo). Tissues were lysed in bullet blender (Next Advance, Averill REFERENCES
Park, NY) with 1.6 mm stainless steel beads for 4 minutes twice at setting E1. Richer SL, Truong-Tran AQ, Conley DB, Carter R, Vermylen D, Grammer LC,
et al. Epithelial genes in chronic rhinosinusitis with and without nasal polyps.
7. Tissue lysates were centrifuged at 1968g to clear the debris. Tissue lysates
Am J Rhinol 2008;22:228-34.
were stored at 2208C until further use. Nasal scraping cells were collected by
E2. Seshadri S, Lin DC, Rosati M, Carter RG, Norton JE, Suh L, et al. Reduced expres-
scraping tissues with Rhinoprobe. Nasal scraping cells were suspended in sion of antimicrobial PLUNC proteins in nasal polyp tissues of patients with
RPMI1640 medium. Cells were centrifuged at 5000g for 5 minutes. Pelleted chronic rhinosinusitis. Allergy 2012;67:920-8.
cells were washed with PBS and centrifuged again at 5000g for an additional E3. Tieu DD, Peters AT, Carter RG, Suh L, Conley DB, Chandra R, et al. Evidence for
5 minutes. Pelleted cells were lysed by vortexing in PBS supplemented with diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.
0.05% Tween 20, protease, and phosphatase inhibitor (Sigma). Cell lysates J Allergy Clin Immunol 2010;125:667-75.
J ALLERGY CLIN IMMUNOL LETTERS TO THE EDITOR 1230.e2
VOLUME 132, NUMBER 5

FIG E1. Evaluation of expression of nitric oxide synthase mRNA in uncinate and inferior turbinate tissues of
healthy human subjects. Expression of mRNA of NOS1 (nNOS), NOS2 (iNOS), and NOS3 (eNOS) was
evaluated by using real-time PCR.
1230.e3 LETTERS TO THE EDITOR J ALLERGY CLIN IMMUNOL
NOVEMBER 2013

FIG E2. Immunohistochemical staining for SPLUNC1 and S100A7 in sinonasal tissue. SPLUNC1 (A) and
S100A7 (B) immunostaining in uncinate of a control subject. Representative of at least 3 donors per group.
J ALLERGY CLIN IMMUNOL LETTERS TO THE EDITOR 1230.e4
VOLUME 132, NUMBER 5

FIG E3. Alcian blue/periodic acid-Schiff (AB/PAS) staining in sinonasal tissues. AB/PAS staining in uncinate
(A) and inferior turbinate (B) of a control subject. Representative of 5 donors per group.
1230.e5 LETTERS TO THE EDITOR J ALLERGY CLIN IMMUNOL
NOVEMBER 2013

TABLE E1. Subject characteristics

PCR Uncinate IT

No. of subjects 10 (3 M) 11 (9 M)
Age (y), median (range) 42 (16-62) 37 (27-77)

Tissues-ELISA Uncinate IT

S100A7
No. of subjects 9 (4 M) 9 (7 M)
Age (y), median (range) 46 (16-59) 40 (25-59)
Beta defensin-2
No. of subjects 11 (7 M) 16 (13 M)
Age (y), median (range) 45 (16-59) 40 (19-62)
SPLUNC1
No. of subjects 10 (4 M) 12 (8 M)
Age (y), median (range) 52.5 (16-72) 36 (17-72)
Lactoferrin
No. of subjects 7 (5 M) 10 (7 M)
Age (y), median (range) 45 (19-62) 40 (17-62)

NECs-ELISA Uncinate IT

S100A7
No. of subjects 14 (10 M) 16 (13 M)
Age (y), median (range) 52 (19-72) 48 (19-72)
SPLUNC1
No. of subjects 8 (7 M) 7 (5 M)
Age (y), median (range) 50.5 (19-72) 52 (19-72)

Alcian blue/PAS Uncinate IT

No. of subjects 5 (4 M) 5 (3 M)
Age (y), median (range) 49 (19-62) 35 (16-62)
IHC, Immunohistochemistry; M, male; NECs, nasal epithelial cells; PAS, periodic
acid-Schiff.