1Medical Institute, College of Computer Science and Business Administration, Lomza, Poland2Department of Anatomy, Poland3Research Laboratory of Cosmetology, Poland4Department of Forensic Medicine, Poland5Medical College of the Universal Education Society, Lomza, Poland6Department of Radiology, Medical University Children Hospital, Bialystok, Poland7The Children's Memorial Health Institute, Warsaw, Poland8Department of Lung Diseases and Tuberculosis, Medical University of Bialystok, Poland9Department of the Emergency Medicine and Disasters, Medical University of Bialystok, Poland
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* Corresponding author at: Medical Institute, College of Computer Science and Business Administration, Akademicka Str. 14, 18-400Łomża, Poland. Tel.: +48 86 215 59 53; fax: +48 86 215 66 01.
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Introduction
Nasal polyps (gr. polýpous – polypod, i.e. many pads) [1] aresmooth, grapes shape outgrowths of the nasal mucosa,projecting into nasal cavity, reducing nasal air flow, andcreating danger of extra- and intra-cranial complications [2–4]. Nasal polyps affect from 0.2–5% to 28% of the worldpopulation, mostly men in Europe, after 20 years of life witha mean age 38–39 years [5–9]. Nasal polyps accompanyingby eosinophilia consist 80–90% of all nasal polyps, and therest is accompanied by neutrophilia [10–12].
Local application of glucocorticosteroids is effective inrelieving symptoms of the eosinophilic nasal polyps, decrea-sing their size and usually preventing against relapse, butneutrophilic polyps did not answer corticosteroid treatment[13, 14]. Recently new therapies are being tested for nasalpolyps treatment: antiallergic drugs with monoclonal antibo-dies against IgE and antibiotics directed against bacterialbiofilm [4, 15–19]. In the nasal polyps treatment, the best resultsobtained by polypectomy, particularly in large polyps andpolyps occupying nasal sinuses [20, 21]. However in up to 87%of polypectomised patients recurrence of was observed [3, 18].
Majority of physicians believe that nasal polyps are finalstage of a chronic inflammatory process in the nasal mucosalmembrane [22–24]. In 1994 Bernstein proposed inflammatory-bioelectric theory of nasal polyps formation based on abnor-mal chloride channels favoring increased absorption ofsodium cations, changed composition of nasal mucus, increa-sed water penetration to nasal mucosa intracellular matrix,swelling of mucosal tissue [9, 27]. The Bernstein theory is inagreement with reported higher (in comparison to healthynasal mucosa) ability of the nasal polyps tissue to absorb Na+
[27, 28]. Recently attempts to correlate nasal polyps incidencewith eosinophilia and eosinophilic inflammation of mucosawere reported [10, 11]. However, it was reported that only 5%of persons with nasal polyps suffer from allergy, withexception of fungal allergy (allergic fungal rhinosinusitis –
AFRS) where frequency of nasal polyps exceeded 85% [2, 7, 25,26]. It is generally accepted that in rhinosinuitis with nasalpolyps formation following mechanisms are involved: exces-sive fibroblast proliferation after injury, degranulation ofmastocytes, histamine release, eosinophiles infiltration andedema with exudation [7, 28].
It appears that, independently of etiology nasal polypsformation should be related to changes in structure, concen-tration and metabolism of substances building nasalmucosa and among them glycoconjugates (glycoproteins,glycolipids, and proteoglycans) constituting cell membranes,extracellular matrix and biofilm covering nasal mucosa [17,19]. In catabolism of glycoconjugates saccharide chainslysosomal exoglycosidases are involved [37]. Thus, the aimof our work was an evaluation of selected lysosomalexoglycosidases in pathogenesis of the nasal polyps.
Materials and methods
Patients. Nasal polyps were obtained during polypectomyfrom 40 patients (10 women and 30 men, aged 23–84 years)
with diagnosed chronic rhinosinusitis with nasal polyps.Control tissues were derived from mucosa of lower nasalconchas obtained during mucotomy operations from 20patients (10 women and 10 men, aged 23–61 years).
Biological material processing. To perform histopatholo-gical examination, fragments of polyps and normal nasaltissue were immersed in 10% formalin and subsequentlystained with H + E. For biochemical investigations fragmentsof polyps and normal nasal mucosa were homogenized inhomogenizer (Ultra-Turrax T8, Germany). Homogenateswere centrifuged at 12 000 � g for 20 min at 4 8C beforefurther procedures.
Glycosidases activity. Activity of exoglycosidases insupernatants of centrifuged homogenates was determinedby Chatterjee et al. method [29] in modification of Zwierzet al. [30] and Marciniak et al. [31] with 4-nitrophenyl-derivatives of the appropriate sugars. Total protein concen-tration was determined by the Lowry et al. method [32].
Ethical issues. Informed written consent was obtainedfrom all the participants after explanation of the nature,purpose, and potential risk of the study. The study wasapproved by the Bioethical Committee of the MedicalUniversity of Białystok, Poland (R-I-002/46/2007).
Statistical analysis. All variables were checked for nor-mality. Results were expressed as the mean � SD. P valuesless than 0.05 were considered significant. Statistical analy-sis was performed using packet Statistica 6.0 (StatSoft,Poland).
Results
Normal nasal mucosa and nasal polyp tissue with patholo-gical findings (submucosal edema, inflammatory infiltration,superficial hyperplasia) are presented in Fig. 1.
We observed significant lower values of GAL, FUC andtendency to decrease MAN and GLU concentration in nasalpolyps (P) in comparison to controls (C) (Fig. 2). In nasalpolyp tissue (P) no differences of GAL, MAN and FUC specificactivity in comparison to control mucosa were observed (C)(Fig. 3). It is worthy to note a similar decrease in concen-tration of activity for GAL, MAN, and FUC (from 0.71 for FUCto 0.78 for MAN) in polypous tissue in comparison tocontrols (Table I) and the same level of tendency to decreasein specific activity, i.e. from 0.79 to FUC to 0.92 for MAN(Table I).
Discussion
In pathological situations connected with tissue remodelingsuch as colorectal cancer, pancreatic cancer, renal cancer,salivary gland tumors [36], alcoholism [37], Lyme disease[50] significant increase in lysosomal exoglycosidases acti-vity was observed. Decrease in concentration of the activityof lysosomal exoglycosidases in nasal polyps tissue iscontraindicatory to active inflammatory process in nasalpolyps.
In nasal polyps we observed significant decrease inconcentration the activity of GAL, FUC, and tendency to
[(Fig._1)TD$FIG]
Fig. 1 – (A) Normal mucosa (H + E, magnification 20T). (B). Submucosal edema of polypous tissue (H + E, magnification 10T). (C)Polypous tissue with inflammation foci (H + E, magnification 10T). (D) Stratified squamous epithelium hyperplasia on thesurface of a polyp (H + E, magnification 20T)
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decrease MAN concentration (Table I, Fig. 2). In polypoustissue, in comparison to control nasal mucosa, specificactivity of GAL, MAN, and FUC had tendency to decrease(Table II, Fig. 3). Decrease in concentration of GAL and FUCin association with tendency to decrease in concentration ofMAN and all exoglycosidases specific activities is in agree-ment with accumulation of water in polypous tissue. Our
[(Fig._2)TD$FIG]
Fig. 2 – Lysosomal exoglycosidases concentrations in nasalpolyps and control tissues; c – control, p – polyp, *p < 0.05,**p < 0.01
results are contradictory to existence of full symptomaticeosinophilic [11, 33] or neutrophilic [21, 33] inflammation innasal polyps, as inflammatory state is connected withsignificant increase in activity of lysosomal exoglycosidases[37]. It is worthy to note that level of decrease in exoglyco-sidases concentration at nasal polyps tissue was similar forall exoglycosidases tested, e.g. from 0.71 for FUC to 0.78 for
[(Fig._3)TD$FIG]
Fig. 3 – Lysosomal exoglycosidases specific activities innasal polyps and control tissues; c – control, p – polyp
Table I – Proportions of lysosomal exoglycosidases concentrations in nasal polyps and control tissues
Exoglycosidase Tissue N Concentration of activity (pKat/1 g tissue) p
GAL control 20 259.1 � 93.3 0.017polyp 40 195.7 � 89.9
MAN control 20 185.2 � 84.3 0.072polyp 40 143.9 � 74.4
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MAN (Table I), which suggest similar dilution of the exoglyco-sidases activity in the nasal polyps tissue. Of exoglycosidasestested in nasal polyps only concentration activity of MAN(taking part in degradation of N-linked oligosaccharides) didnot significantly decrease, which suggest weaker involvementof N-linked glycoproteins in nasal polyps pathology.
Full inflammatory response includes production of theinflammation mediators such as prostaglandins [34] andcytokines [35] responsible for local inflammatory reaction.Decrease in concentration of exoglycosidases and presenceof some inflammatory cells in nasal polyps, i.e. acidophilicand neutrophilic granulocytes suggest deregulation of themechanisms of inflammation. In full symptomatic inflam-matory processes, e.g. tonsillitis [38] was reported increasein tissue remodeling expressed by increase in lysosomalcatabolism of glycoconjugates, reflected by increase inconcentration of activity the lysosomal exoglycosidases.Decrease in concentration of exoglycosidases activity parti-cipating in the catabolism of oligosaccharide chains ofglycoconjugates in nasal polyps tissue (Figs. 2 and 3) is anargument against recognition nasal polyps as a fully inflam-matory disease. Our results confirm inflammatory-bioelec-trical theory of nasal polyp pathogenesis [17, 27, 28]. Ourresults corroborate the decrease in secretion of cytokines,expression of adhesion molecules, amount of eosinophilesand mastocytes observed in polyps tissue [39–43]. Glucocor-ticosteroid application in prevention of nasal polyps recur-rence suggests involvement of chronic inflammatory immu-nological mechanism deregulation in pathogenesis of thenasal polys [44, 45].
Our results agree with results of Metzler et al. [46], thatinflammation precedes nasal polyps, by accumulation offluids and albumins in subepithelial layer. Rostkowska-Nadolska et al. [45, 47] reported that cytokeratines 4,13, and19 expressions suggest involvement in nasal polyps ratherstroma than epithelium. Our results as well as lack of cellsedema in nasal polyps tissue [18], and excessive hydratationof nasal polyps extracellular matrix are in agreement with
Table II – Proportions of lysosomal exoglycosidases specific ac
Exoglycosidase Tissue N
GAL control 20polyp 40
FUC control 20polyp 40
MAN control 20polyp 40
Bernstein and Yankaskas suggestion [48, 49], that one of thefactors responsible for nasal polyps creation are disturban-ces in transport of water and electrolytes in nasal mucosa.Bernstein and Yankaskas proposed the abnormal CysticFibrosis Transmembrane Regulator (CFTR) protein, regula-ting sodium channels (ENaC), for increase in amount ofopen sodium channels on the surface of nasal mucosaendothelial cells and stromal swelling [48]. For absorption ofsodium cations in stroma also Major Basic Proteins may beresponsible, secreted by eosinophils, which decrease secre-tion of mucus and increase absorption of sodium [17, 22].Higher ability to absorb sodium and chloride ions by nasalpolyps mucosa supports the opinion of significant role ofderegulation water and mineral metabolism in pathogenesisof nasal polyps [27]. In extracellular matrix main substancesresponsible for water absorption are hyaluronate and pro-teoglycans (e.g. chondroitin sulfate), because of their nega-tive charged polyanion and hydrophilic –OH groups [50]. It isworthy of note that only small part of water molecules aredirectly bound to the proteoglycans by hydrogen bounds,but majority of water molecules is mechanically trappedinside of heteropolysaccharide structure of hyaluronate [51,52]. Therefore it would be a valuable information oncomposition and distribution of glycosaminoglycans andproteoglycans in nasal polyps tissue in comparison tonormal nasal mucosa.
In conclusion, our research supports bioelectrical theory ofnasal polyps pathogenesis and directs attention at researchon glycoconjugates (particularly glycosaminoglycans) of thenasal mucosa extracellular matrix, the structural elementsresponsible for absorption the excessive amounts of water.
Authors' contributions/Wkład autorów
SC – study design, data collection and interpretation, accep-tance of final manuscript version, literature search. AM –
study design, data collection and interpretation, acceptance
o t o l a r yn go l o g i a p o l s k a 6 7 ( 2 0 1 3 ) 1 9 2 – 1 9 7196
of final manuscript version. MK, AN-J – data collection andinterpretation, acceptance of final manuscript version. PK –
statistical analysis, data interpretation, acceptance of finalmanuscript version. BZ-S – data collection, acceptance offinal manuscript version. AK, NW – data interpretation,acceptance of final manuscript version. ŁM – statisticalanalysis, data interpretation, literature search. KZ – studydesign, data interpretation, acceptance of final manuscriptversion, funds collection. SDS – study design, data interpre-tation, acceptance of final manuscript version.
Conflict of interest/Konflikt interesu
None declared.
Financial support/Finansowanie
The study was financed from the grant of Medical Universityof Bialystok No. R-I-002/46/2007.
Ethics/Etyka
The work described in this article have been carried out inaccordance with The Code of Ethics of the World MedicalAssociation (Declaration of Helsinki) for experiments invol-ving humans; EU Directive 2010/63/EU for animal experi-ments; Uniform Requirements for manuscripts submitted toBiomedical journals.
r e f e r e n c e s / p i �s m i e n n i c t w o
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