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Correlating matrix metalloproteinase-9 sinus secretion levels with tissue biopsy levels.

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ORIGINAL ARTICLE
Correlating matrix metalloproteinase-9 sinus secretion levels with tissue
biopsy levels
Matt A. Wilson, MD, Richard R. Orlandi, MD, Matthew A. Firpo, PhD
Background: Collecting mucosal biopsies is invasive and
creates additional inflammation, hampering a beer understanding of nasal and sinus disease evolution and response
to treatment. We examine whether sinus secretion collection can replace tissue biopsy for protein determination.
Conclusion: Secretion collection can replace tissue biopsy
for MMP-9 determinations, reducing morbidity. Furthermore, secretion collection allows sequential sampling from
C 2011 ARS-AAOA, LLC.
the same location. Methods: Prior to surgical intervention for chronic rhinosinusitis (CRS), a piece of gelatin foam was used to collect secretions from the ethmoid mucosa. A tissue biopsy
was then taken from the same location. Matrix metalloproteinase-9 (MMP-9) protein levels were measured in each
sample.
Key Words:
Results: MMP-9 protein levels in secretions and tissues
were significantly correlated (p = 0.0033, r = 0.52, by Pearson correlation).
A
llergic nasal and sinus inflammation affects millions
Americans, more individuals than arthritis or hypertension. The effect of nasal and sinus inflammation on quality of life rivals those of diabetes and congestive heart failure. Notwithstanding this high prevalence and significant
morbidity, the cause (or causes) of chronic rhinosinusitis
(CRS) is unknown. A principal barrier in understanding sinus inflammation is a simple and reliable way to increase
human sampling of inflammatory mediators found in the
sinuses.
Despite the prevalence of CRS, its causes remain largely
unknown. Inflammation of the sinus has been thought to
be related to fungal allergy,1,2 superantigens,3,4 biofilms,5,6
and underlying bone inflammation.7,8 A firm understandDepartment of Surgery, Division of Otolaryngology–Head and Neck
Surgery, University of Utah, Salt Lake City, UT
Correspondence to: Matt A. Wilson, Department of Surgery, Division of
Otolaryngology–Head and Neck Surgery, University of Utah, 50 North
Medical Dr. Rm #3C120, Salt Lake City, UT 84132; e-mail:
matthew.wilson@hsc.utah.edu
Funding sources for the study: American Academy of Otolaryngic Allergy.
Potential conflict of interest: None provided.
Podium presentation at the American Academy of Otolaryngic Allergy
(AAOA) Annual Meeting, September 24, 2010, Boston, MA.
Received: 9 September 2010; Revised: 23 November 2010; Accepted: 7
December 2010
DOI: 10.1002/alr.20043
View this article online at wileyonlinelibrary.com.
allergic sinusitis; chronic sinusitis; ELISA; inflammatory
markers; MMP-9; nasal secretions; sinus biopsy; sinus mucosa; sinus sampling; wound healing
How to Cite this Article:
Wilson MA, Orlandi RR, Firpo MA. Correlating matrix
metalloproteinase-9 sinus secretion levels with tissue
biopsy levels. Int Forum Allergy Rhinol, 2011; 1:106–108
ing of the underlying pathology of allergic rhinitis (AR)
and CRS is necessary to develop innovative and more effective treatment plans. Understanding the interplay of allergic and immunologic inflammatory mediators is the first
step in effective treatment strategies. Metalloproteinase9 (MMP-9) is found to be active in the inflamed sinus
mucosa. It has been found to be a reliable way to monitor inflammation and healing in the sinus.9 Transforming growth factor-beta 1 (TGF-β1) is another mediator
of inflammation that has been identified. Interleukin (IL)4, IL-5, and IL-13 have been shown to be active in patients with allergic rhinosinusitis in response to fungal
antigens.2
Methods are currently available to sample the sinus
for diagnostic, research, and treatment purposes. These
methods include biopsy, aspiration, irrigation, and swab
sampling.10 Biopsy of the sinus is an effective way to sample
the sinus and is currently the most reliable way to sample inflammatory mediators. However, it is difficult for a patient
to tolerate in the office, is invasive, and cannot be repeated
in the same location with any amount of frequency due to
the confounding effect of the wound created by the biopsy.
Aspiration is likely the gold standard in sampling the sinus
for culturing purposes, but like a biopsy it is invasive and
not well-tolerated.
Limited amounts of secretions are obtained with aspiration. Irrigation, while effective in the symptomatic
International Forum of Allergy & Rhinology, Vol. 1, No. 2, March/April 2011
106
MM-9 sinus secretion and tissue biopsy levels
treatment of CRS, has not been shown to be an effective
way to sample the sinuses. Vogen et al.11 demonstrated that
swab sampling is an effective way of sampling bacteria and
fungus from the sinus in 90% of patients. This procedure is
less invasive, accurate, and can be done on multiple occasions. It is not clear if secretions can be obtained for protein
or messenger RNA (mRNA) analysis using swabbing.
In 2005, Branski et al.12 demonstrated the feasibility
of analyzing inflammatory mediators collected from secretions of the rabbit larynx using gelatin foam. Successful
application of such a technique to the human nose and
sinuses would lead to an improved understanding of the inflammatory process in chronically inflamed sinus mucosa.
A noninvasive procedure would decrease discomfort and
thereby facilitate sampling of greater numbers of individuals as well as the same individuals at multiple time points
without results skewed by the inflammation due to the sampling itself.
We perform a cross-sectional study comparing MMP9 protein levels collected via biopsy with MMP-9 protein levels collected via noninvasive secretion sampling
of the anterior ethmoid. We aim to correlate MMP9 levels from biopsy samples and secretion samples in
hopes of developing a noninvasive way to sample sinus inflammatory/healing markers, remove the effect of
additional inflammation from tissue biopsy, and enable
physicians to track medical and surgical therapy through
time.
Patients and methods
A total of 43 individuals 18 years of age or older undergoing surgical management of CRS at the University of
Utah Medical Center participated. An Institutional Review
Board (IRB)-approved research consent was signed by all
participants; this research was approved by the University
of Utah IRB office. Each patient was placed under general
anesthesia and a 6-mm2 piece of gelfoam was placed at the
anterior ethmoid for 5 minutes. Immediately following, a
biopsy was taken from the same location. The same instrument was used to take the biopsy every time in order to
acquire samples of equal size. Samples that appeared large
were trimmed to match the other samples taken. The samples were immediately placed into microcentrifuge tubes
and stored at −80◦ C for later analysis.
The gelfoam samples were prepared by adding 1.0 mL of
normal saline to each tube. The sample was then mixed by
vortex for 2 minutes. The sample was then allowed to set for
5 minutes for complete saturation of the normal saline into
the gelfoam. We then mixed the sample by vortex again for
2 minutes. Following this the samples were centrifuged at
10,000 rpm for 10 minutes. The supernatant was then extracted for enzyme-linked immunosorbent assay (ELISA).
All steps were performed cold.
The tissue samples were prepared by adding 1.0 mL
of normal saline to each tube. The samples were then
107
International Forum of Allergy & Rhinology, Vol. 1, No. 2, March/April 2011
homogenized using a tissue homogenizer until all tissue
had been thoroughly homogenized. The sample was then
mixed by vortex for 2 minutes. Following this, the samples
were centrifuged at 10,000 rpm for 10 minutes. The supernatant was then extracted for ELISA. Again, all steps were
performed cold.
We then preformed MMP-9 ELISA per the supplier’s recommend protocol on all samples (R&D Systems, Inc.).
Statistical analysis of the samples was performed using a
2-tailed Pearson’s correlation test.
Results
Detectable levels of MMP-9 were found in all samples. A
total of 13 of 43 patients had highly concentrated levels
of MMP-9 in both the secretions and tissue biopsies. The
samples were so concentrated that the ELISA was saturated
requiring additional dilution of the samples. Unfortunately,
we did not have enough sample remaining to perform this
dilution so these samples were excluded from the analysis.
A 2-tailed Pearson’s correlation test was used to analyze
the remaining 30 patients. There was a positive correlation
between MMP-9 collected in secretions from the anterior
ethmoid with that of tissue biopsies from the same location
(p = 0.0033, r = 0.52) (Fig. 1).
Discussion
The annual cost of CRS exceeds $4.3 billion a year in the
United States alone.13 Understanding of healing and inflammation in the sinus is still not well understood, making
treatment difficult. One of the hindrances to studying sinus
inflammation and healing is a simple and accurate way to
study inflammatory markers. Sample sizes are often limited
to patients going to the operating room or those patients
FIGURE 1. Correlation of MMP-9 protein levels in tissues dissolved in
1.0 mL saline and secretions extracted in 1.0 mL of saline. MMP-9 = matrix
metalloproteinase-9.
Wilson et al.
willing to undergo biopsy in the office, which can sometimes be quite uncomfortable. Another limiting factor is
the effect of biopsies. Biopsies cause added inflammation,
making it impossible to follow inflammation and wound
healing through time in the same patient. The ideal sample would be comfortable, easy to obtain, and cause no
additional inflammation.
One limitation to our study was not weighing each of
the samples. Tissue samples were standardized by using the
same biopsy instrument, with samples larger than the instrument trimmed appropriately. We also used the same size
of gelfoam for each sample. Despite these approximations,
our data showed highly significant correlations. Standardizing the samples by weighing may be helpful in improving
the accuracy of the secretion method even further in the
future.
We have found MMP-9 levels in tissue biopsy samples
to correlate with secretion samples taken from the same
location. Sampling localized areas of the sinus mucosal secretions provides a safe, comfortable, and accurate method
for following healing and inflammation through time.
Conclusion
MMP-9 levels collected from sinus secretions correlated
with MMP-9 levels collected via tissue biopsy. Collection of
sinus secretions is an accurate, noninvasive way of assessing
MMP-9 levels. It also provides a technique that does not
increase inflammation, allowing one to follow a patient’s
inflammation through time without confounders. In the
future this could provide physicians with a way to track
medical and postsurgical therapies in the clinic setting.
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International Forum of Allergy & Rhinology, Vol. 1, No. 2, March/April 2011
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