• Users Online: 345
  • Print this page
  • Email this page


 
 
Table of Contents
ORIGINAL ARTICLE
Year : 2012  |  Volume : 14  |  Issue : 2  |  Page : 81-85

Linking extraesophageal gastric reflux ( EEGR) to chronic rhinosinusitis (CRS) in children: Pepsinogen Study of paranasal sinus and adenoid tissue.


Departments of Otolaryngology-Head and Neck Surgery, King Saud University and McGill University, Montreal, Canada

Date of Web Publication3-Jan-2020

Correspondence Address:
M A Al Essa
King Abdulaziz University Hospital PO box : 245, Riyadh
Canada
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1319-8491.274780

Rights and Permissions
  Abstract 


Objectives: The primary objective of this study was to determine the association between chronic rhinosinusitis in children (CRS) and extraesophageal laryngopharyngeal reflux (EEGR).
Methods: The proposed study was done in Montreal Children Hospital (Montreal, Canada)) from April 2009 till January 2010. Tissue from the paranasal sinus and adenoids were obtained from 14 patients with CRS (age 2-18 years) undergoing endoscopic sinus surgery (ESS) and 18 matched control subjects with no history of CRS (only adenoid tissue) were obtained during adenoidectomy (+/- tonsillectomy). We have used Immunocytochemistry (ICC) to evaluate the presence of pepsinogen in the paranasal sinus tissue of children with CRS group.We have also examined the presence of pepsinogen in adenoid tissue of CRS group and compared them with adenoid tissue of control group.
Results: Immunoreactivity for pepsinogen was identified in the epithelial layer of adenoid tissue (50%) and paranasal sinus tissue (65%) in the CRS group. Only occasional staining was observed in the epithelial layer of the adenoid tissue of the control group.
Conclusions: The detection of immunoreactivity of pepsinogen in paranasal sinus tissue suggested a possible role of EEGR in CRS in children .

Keywords: extraesophageal gastric reflux, EEGR, chronic rhinosinusitis , CRS, pepsinogen, adenoids, paranasal sinuses


How to cite this article:
Al Essa M A. Linking extraesophageal gastric reflux ( EEGR) to chronic rhinosinusitis (CRS) in children: Pepsinogen Study of paranasal sinus and adenoid tissue. Saudi J Otorhinolaryngol Head Neck Surg 2012;14:81-5

How to cite this URL:
Al Essa M A. Linking extraesophageal gastric reflux ( EEGR) to chronic rhinosinusitis (CRS) in children: Pepsinogen Study of paranasal sinus and adenoid tissue. Saudi J Otorhinolaryngol Head Neck Surg [serial online] 2012 [cited 2023 Jan 30];14:81-5. Available from: https://www.sjohns.org/text.asp?2012/14/2/81/274780




  Introduction Top


Chronic rhinosinusitis (CRS) is a common disorder with an incompletely understood pathophysiology. Affecting approximately 15% of the general population, CRS has an even higher prevalence among children [1]. Several studies have demonstrated associations between extraesophageal gastric reflux (EEGR) and respiratory tract disease. In a prevalence study of children under investigation for gastroesophageal reflux (GER), EEGR documented by dual-probe pH testing was found to be associated with laryngeal and pulmonary abnormalities [2]. In a similar prevalence study of pediatric patients under investigation for CRS, dual-probe pH testing demonstrated higher than expected rates of GER (32%) and EEGR (63%) when compared to the general population. [3] Similarly, in a pediatric study of nearly 2000 cases and 8000 controls, GER disease (GERD) was demonstrated to be a significant risk factor for sinusitis (odds ratio, 2.3; 95% confidence intervals 1.73.2) [4].

It has been theorized that reflux of gastric contents to the level of the nasopharynx and nasal cavity triggers mucosal inflammation leading to impaired mucociliary clearance, obstruction of sinus ostia, and the subsequent signs and symptoms of sinusitis [3]. In addition to being acidic, gastric secretions contain pepsinogen, a proteolytic zymogen produced by gastric chief cells. In acidic environments, pepsinogen is converted to pepsin, its active form. Upper respiratory epithelium exposed to pepsin has been shown to take it up by means of receptor-mediated endocytosis [5]. Subsequently, the presence of pepsinogen and/or pepsin within the upper respiratory in amounts exceeding serum concentration may be used as a diagnostic marker of EEGR. This method was recently used to suggest a causal relationship between EEGR and otitis media with effusion (OME) [6]. In this study, the presence of pepsinogen in concentrations higher than serum was detected in 84% of middle ear effusions and in statistically significant higher proportions of adenoid tissues in children with OME undergoing adenoidectomy versus controls.

In addition to being a marker of EEGR, pepsin itself is believed to play an important role in mucosal dysfunction. It is believed that mucosal uptake of pepsin results in down-regulation of mucosa-protective carbonic anhydrase isoenzyme expression, predisposing mucosa to reflux mediated inflammatory damage. This has been shown for laryngeal mucosa in a study of patients with laryngopharyngeal reflux disease (LPRD) [7]. Carbonic anhydrase isoenzyme expression is also present in sinonasal mucosa [8], and decreased expression has recently been linked to nasal polyposis [9].

Antacids, such as H2 receptor antagonists and proton pump inhibitors (PPIs), are commonly used in the management of GERD and LPRD. In reducing gastric acidity, they function to protect esophageal and extraesophageal mucosa directly from the harmful effects of low pH and possibly indirectly by decreasing the conversion of pepsinogen to pepsin. If indeed a causal relationship between EEGR and CRS exists, the protective effects of antacids on sinonasal mucosa could have an important role in the future management of this entity. At least one retrospective study has already demonstrated positive results in avoiding surgery through the use of PPIs in treating pediatric CRS refractory to conventional medical therapy [10].

To date, no prospective controlled studies linking pathology proven EEGR to CRS have been published. EEGR associated CRS remains controversial. The primary purpose of this study wss to determine the association between EEGR and CRS by the presence of pepsinogen in adenoidal tissue in pediatric CRS patients versus non-CRS patients undergoing adenoidectomy. The secondary purpose was to determine the association between EEGR and CRS by the presence of pepsinogen in paranasal sinus tissue in CRS patients with and without a positive skin allergy test.


  Methods Top


In this prospective, case-control, single blinded study, patients aged 2-18 with refractory CRS scheduled for surgery at the Montreal Children’s Hospital ( Montreal ,Canada) from April 2009 till January 2010 were consecutively enrolled. Participation was by parental informed consent & ethical approval was obtained too. The study received prior institutional review board ethical approval. Refractory CRS was defined as two or more of the following symptoms present for 12 weeks or more despite medical treatment: anterior or posterior mucopurulent drainage, nasal obstruction, daytime cough, halitosis, irritability, low grade fever, facial-pain, pressure or fullness. Medical treatment consisted of 12 weeks of nasal corticosteroid sprays with an initial concurrent course of antibiotic therapy for 14 days. Refractory CRS was confirmed prior to surgery by standard non-infused computer tomography scan of the paranasal sinuses. Surgery consisted of either endoscopic sinus surgery (ESS) with adenoidectomy or simple adenoidectomy. All CRS patients underwent allergic skin testing prior to surgery.

During the same period, patients aged 2-18 without symptoms of CRS scheduled for adenoidectomy with or without tonsillectomy, septoplasty, or turbinoplasty were identified as controls. Exclusion criteria for controls included any history of asthma, chronic cough, and atopy.

Exclusion criteria for case and control groups included patients with cystic fibrosis, neurological disorders, maxillofacial anomalies, trisomy 21, documented GERD, congenital anomalies of gastrointestinal tract, immunodeficiency, antacid use within past month, systemic steroid use within past six months, and recent acute respiratory tract infection.

Adenoid tissue was obtained from all CRS patients and controls. Paranasal sinus tissue from the uncinate process was also obtained from CRS patients undergoing concurrent ESS. All specimens were immediately fixed in 10% neutral-buffered formalin (Sigma Chemical Co., St. Louis, MO) overnight. Tissues were processed using an automatic tissue processor (Lieca TP 1020, Lieca, Hiedelburg, Germany) before being embedded in paraffin wax. Slides with 5 μm formalin-fixed, paraffin-embedded tissue sections were placed in an incubator at 60°C overnight to fix the tissue section on the slide. Tissue sections were deparaffinized in xylene, dehydrated in various grades of alcohol, and then stained with hematoxylin and eosin to confirm the tissue type of the epithelium lining (respiratory or stratified squamous) and pathologic features. Pathologists were blinded with regard to specimen group origin.

Monoclonal mouse antihuman antibody to pepsinogen I (Cat. No. 610055, Biohit, Helsinki, Finland) was used as the primary antibody. Slides with 5 μm formalin-fixed, paraffin-embedded tissue sections were deparaffinized in xylene and dehydrated in various grades of alcohol for 5 minutes each. Endogenous peroxidase activity was blocked by incubating the sections in 5% hydrogen peroxide for 30 minutes at room temperature. Antigen retrieval was preferred by placing the sections in an ethylenediaminetetraacetic acid solution puffer (pH 8.0) and heating them in a pressure cooker for approximately 19 minutes. Slides were allowed to cool in distilled water before further treatment. Nonspecific antigen sites were blocked by incubating the sections in a universal blocker (Dako, Canada Inc.) for 30 minutes at room temperature. Primary antibody antipepsinogen I (Biohit Diagnostics) was then applied to each section overnight in a 4° humid chamber. A biotinylated rabbit antimouse secondary antibody (Dako, Canada Inc.) was then applied to each slide for 45 minutes. After washing with phosphate-buffered saline solution, the slides were incubated with avidin-biotin-horseradish peroxidase conjugate (Dako, Canada Inc.) for 45 minutes. Subsequent development of a coloured reaction product was achieved by treating the slides with 3,3-diaminobenzidine for 1 minute. Sections were counterstained with hematoxylin before mounting for microscopic examination. Normal gastric tissue sections were used as positive control samples, and sections of lymphatic tissue were used as negative control samples. The specificity of the required tissue was further confirmed by omitting the primary antibody (7.11) The level of immunostaining for the percentage of pepsinogen in the epithelium and subepithelium was assessed by a previously described scoring system [6],[11],[12]. The percentage of epithelial cells exhibiting immunoreactivity for pepsinogen was estimated and expressed in grades from 0 to 8, where 0 represents no staining, 1 represents positive staining in 0 to 12.5% of the epithelia, 2 represents 12.5 to 25%, 3 represents 25 to 37.5%, 4 represents 37.5 to 50%, 5 represents 50 to 67.5%, 6 represents 67.5 to 75%, 7 represents 75 to 87.5%, and 8 represents 87.5 to 100%.

The primary outcome measure was the degree of pepsinogen staining in adenoidal tissue in pediatric CRS patients versus non-CRS patients undergoing adenoidectomy. The secondary outcome measure was the degree of pepsinogen staining in paranasal sinus tissue in CRS patients with and without a positive skin allergy test. The Fisher exact test was used with α = 0.05. Data was analysed using SPSS 11.0.2 (Chicago, IL).


  Results Top


Thirty-two patients were recruited, fourteen CRS patients and 18 controls. Among the CRS group, 9 (64%) were male and 5 (36%) female, with a median age of 7.5 years (range 6-18 years). Among controls, 11 (61%) were male and 7 (39%) female, with a median age of 5 years (range 3-8) years.

Positive staining of gastric mucosa samples (within chief cells and submucosal glands) and negative staining of lymphatic tissue samples confirmed antigen sensitivity and specificity. Strong pepsinogen staining was identified in the epithelial layer of approximately 50% of the adenoid and 65% of the paranasal sinus tissues among the CRS patients [Figure 1]. Only occasional staining was observed in the epithelial layer of the adenoid tissue of controls. Each specimen was graded from 0 to 8, and an average staining grade was calculated for CRS and control groups [Figure 2]. In addition, average staining grades were also calculated for the paranasal sinus tissue among CRS patients with positive and negative skin allergy tests [Figure 3]. Statistically significant higher amounts of pepsinogen were found in adenoid tissue samples of CRS patients compared to controls (p < 0.001). In addition, statistically significant higher amounts of pepsinogen were also found in paranasal sinus tissue samples of CRS patients with a negative skin allergy test compared to CRS patients with a positive skin allergy test (p <0.001).
Figure 1: Immunohistochemical detection of pepsinogen indicated by the arrows. (A) and (B). Arrows indicating Strong staining within the cytoplasm of surface epithelium (A) and weak staining within the subepithelium (B) of adenoid tissue from CRS patients (×200 original magnification). (C). arrow indicating Detection of pepsinogen immunoreactivity in the paranasal sinus mucosa of a CRS patient with a negative skin allergy test.

Click here to view
Figure 2: Average pepsinogen immunostaining grades (0–8) of adenoid epithelium among CRS and control patients.

Click here to view
Figure 3: Average pepsinogen immunostaining grade (0–8) of paranasal sinus mucosa among CRS patients with and without a positive skin allergy test.

Click here to view



  Discussion Top


This prospective, case-control, blinded study is the first to demonstrate the presence of pepsinogen in statistically higher average amounts in adenoid tissue of patients with CRS compared to controls. In addition, it has also demonstrated higher levels of pepsinogen in paranasal sinus tissue of CRS patients with negative skin allergy tests compared to CRS patients with positive skin allergy tests. These results strengthen the evidence linking EEGR as a causative factor in CRS in non-atopic patients, and may suggest a role for antacid therapy in selected subsets of CRS patients. In the future, positive adenoidal pepsinogen staining post-adenoidectomy may become a useful diagnostic tool for EEGR, and may guide the treating physician towards implementation of post-operative anti-reflux therapy. Further studies are needed to establish whether antireflux treatment post adenoidectomy in patients with positive pepsinogen staining prevents the recurrence of CRS symptoms.


  Conclusions Top


Our data supports the theory of EEGR as a causative factor in CRS among pediatric patients. Antacid therapy may be indicated in the treatment of pediatric CRS.



 
  References Top

1.
Van Cauwenberge P, Watelet JB. Epidemiology of chronic rhinosinusitis. Thorax. 2000;55 Suppl 2:S20-21.  Back to cited text no. 1
    
2.
Little JP, Matthews BL, Glock MS, et al. Extraesophageal pediatric reflux: 24-hour double-probe pH monitoring of 222 children. Ann Otol Rhinol Laryngol Suppl. 1997;169:1-16.  Back to cited text no. 2
    
3.
Phipps CD, Wood WE, Gibson WS, Cochran WJ. Gastroesophageal reflux contributing to chronic sinus disease in children: a prospective analysis. Arch Otolaryngol Head Neck Surg. 2000;126(7):831-836.  Back to cited text no. 3
    
4.
El-Serag HB, Gilger M, Kuebeler M, Rabeneck L. Extraesophageal associations of gastroesophageal reflux disease in children without neurologic defects. Gastroenterology. 2001;121(6):1294-1299.  Back to cited text no. 4
    
5.
Johnston N, Dettmar PW, Lively MO, et al. Effect of pepsin on laryngeal stress protein (Sep70, Sep53, and Hsp70) response: role in laryngopharyngeal reflux disease. Ann Otol Rhinol laryngol. 2006;115(1):47-58.  Back to cited text no. 5
    
6.
Al-Saab F, Manoukian JJ, Al-Sabah B, et al. Linking laryngopharyngeal reflux to otitis media with effusion: pepsinogen study of adenoid tissue and middle ear fluid. J Otolaryngol Head Neck Surg. 2008;37(4):565-571.  Back to cited text no. 6
    
7.
Johnston N, Knight J, Dettmar PW, Lively MO, Koufman J. Pepsin and carbonic anhydrase isoenzyme III as diagnostic markers for laryngopharyngeal reflux disease. Laryngoscope. 2004;114(12):2129-2134.  Back to cited text no. 7
    
8.
Tarun AS, Bryant B, Zhai W, Solomon C, Shusterman D. Gene expression for carbonic anhydrase isoenzymes in human nasal mucosa. Chemical Senses. 2003;28(7):621-629.  Back to cited text no. 8
    
9.
Kim TH, Lee HM, Lee SH, et al. Down-regulation of carbonic anhydrase isoenzymes in nasal polyps. Laryngoscope. 2008;118(10):1856-1861.  Back to cited text no. 9
    
10.
Bothwell MR, Parsons DS, Talbot A, Barbero GJ, Wilder B. Outcome of reflux therapy on pediatric chronic sinusitis. Otolaryngology--head and neck surgery. J Ann Acad Otolaryngol Head Neck Surg. 1999;121(3):255-262.  Back to cited text no. 10
    
11.
Tulic MK, Fiset P-O, Manoukian JJ, et al. Role of toll-like receptor 4 in protection by bacterial lipopolysaccharide in the nasal mucosa of atopic children but not adults. Lancet. 2004;363(9422):1689-1697.  Back to cited text no. 11
    
12.
Hamid Q, Springall DR, Riveros-Moreno V, et al. Induction of nitric oxide synthase in asthma. Lancet. 1993;342(8886-8887):1510-1513.  Back to cited text no. 12
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Methods
Results
Discussion
Conclusions
References
Article Figures

 Article Access Statistics
    Viewed993    
    Printed58    
    Emailed0    
    PDF Downloaded60    
    Comments [Add]    

Recommend this journal