UNIVERSITY OF HASANUDDIN

TNF- AND IL-10 LEVEL IN RECURRENT APHTHOUS

STOMATITIS ULCER BY SWAB TECHNIC

Will submit to RDM&E USU Medan,

November 11-12 2011

This study aimed to examine validity of swab technic in cytokine level measurement
of RAS with promising result.

TNF- AND IL-10 LEVEL IN RECURRENT APHTHOUS

STOMATITIS ULCER BY SWAB TECHNIC
E.Marlina*, P. Hadi**, M. Jusri**
*

Faculty of Dentistry, University of Hasanuddin, Makassar, South Sulawesi,

90245,Indonesia
** Faculty of Dentistry, University of Airlangga, Surabaya, East Java, 60132, Indonesia

Corresponding author : drg. Erni Marlina, Sp.PM, tel.:+62(0411)-882103,319225 HP:

+62-813-5552-1711, e-mail:e.marlina@yahoo.co.id
Abstract
Recurrent Aphthous Stomatitis (RAS) are among the most common problem
seen by clinician. The aetiology remain uncertain but possible predispotition factors
were systemic condition, hereditary, and autoimmun. Some study focused on cytokine
using invasive technic such as vena puncture for blood peripheral, and biopsy. But
others used swab mucosal to examine cytokine. This study aimed to examine validity
of swab technic in cytokine level measurement of RAS. The result by the paired Ttest showed statistically significant level of TNF- with p = 0,006 and IL-10 with p =
0,041 with each interval confidence 95%. Swab technic could use for cytokine
measurement for ulser and mucosal RAS patient.
Introduction
Recurrent Aphthous Stomatitis (RAS) are among the most common problem
seen by clinician. (Field et al, 2004; Greenberg, 2003 ). The aetiology remain
uncertain (Field et al,2004; Scully, 2004; Regezy et al, 2003), but possible
predispotition factors were systemic condition, hereditary, and autoimmun. Some
study focused on TNF- and IL-10 as cytokine which plays a major role in
pathogenesis of RAS (Natah, 2004). This studies use pheripheral blood mononuclear
cell as a cytokine source but this assumed that the cytokine production and
distribution not representative as local action rather than other stimulus. And it was an
invasive examination even though it was stabile (Buno, 1998; Lin 2005; Yamamoto,
1994). Some other study using exicional biopsy of RAS ulcer to compared cytokine

with normal side.

This technic representative enough for local production and

distribution even still invasive. (Natah, 2001; Natah, 2000).

Gerber and partners (2003) study IL-1 gene polymorphisme using
vestibulum vaginal or buccal mucosal swab to examine cytokine roles in vulvar
vestibulitis syndrome. It could applied in RAS lesion because non invasive nature,
and because RAS ulcer is a superficial tissue necrosis with fibrinopurulent exudate
consisting of clotted fibrin, and numerous red blood cells forming haemorrhagic
focci. Neutrophils and cellular debris cover the necrotic area. The epithelium is
infiltrated with variable numbers of intraepithelial lymhocytes and some neutrophils.
Neutrophils predominate in the immediate ulcerated area, although peripheral areas
surrounding the ulcer remain mononuclear in nature. (Lehner 1969a; Mills et al.
1980; Schroeder et al. 1983; Hyrinen-Immonen et al. 1991).

Neutrophil,

Lymphocytes, and fibrin clothing-contained pseudomembrane are sources for

cytokine. Swabbing this pseudomembrane could show TNF- that found in burden
level, and low level of IL-10 as a basic examination to demonstrate the validity of
swabbing technic in cytokine level of RAS.
This study aimed to examine validity of swab technic in cytokine level
measurement of RAS.
Material and Methode
All reccurent ulcer patients (men) age range from 18 to 45 visited to Oral
Medicine Departement of Dentistry Faculty of Airlangga University asked to
participate in this study. Participant follow the anamnesis, clinical examination, and
full blood count to eliminate anemis condition, vitamin B12 and folic acid deficiency.
Participant with gastrointestinal or other systemic condition excluded. Diagnosis of
RAS was made from clinical presentation which show discrete, painful, shallow,
recurrent ulcer, covered by a yellow-grey pseudomembrane and surrounded by an
erythenatous halo.
Entry requirements were that the patient should have RAU ulser on nonkeratinized mucosal with at least 3 episode per year on average, RAS lesions studied
3

were 2-3 days old and located at simetrical sided such as the right buccal with left
buccal as normal mucosal.

Patients should have not systemic condition or other

immunodefisiensi, treated their current ulcer with any form of topical or systemic
medication.
The study protocolwas approved by The Ethics Committee of Faculty of
Dentistry, University of Airlangga, Surabaya and informed cosent was obtained from
each participant of the study. Seven aphthous ulcers were swabs from 7 patients
RAS, all man (mean age 21 years old, ranging from 19 to 22 years). In addition,
specimens from the clinically unaffected area at the corresponding site opposite to the
ulcer were obtained from all patients to demonstrate cytokine diffrences between
clinically unaffected mucosa and RAS lesions. Cottol roll sterile used to swab RAS
lesions and the opposite site, and cotton roll tip drawn on effendorf tube contain
MEM and stored at -20oC until ELISA tes done.
ELISA Kit AssayPro Cat No. ET2010-1 used as Kit assay, and the result
measure in pikogram (pq). One Sample Komlogorof Semirnov test used for sample
normality distribution. The means of the two groups were compared by using the
paired-T with considered statistically significant 0,05 or less. SPSS 13.5 for windows
was used for all calculations.
Result
Kolmogorov Smirnov as normality distribution test used for each group and
showed normal distrbution with p > 0,770 for TNF- level of normal mucosal and p
= 0,998 for TNF- ulser. IL-10 also showed normal distribution with p = 0,134 for
IL-10 normal mucosal and p = 0,180 for IL-10 ulcer.

Tabel. TNF- and IL-10 on Recurrent Aphthous Stomatitis (RAS) ulcers and
normal mucosal RAS patiens
Subject
1
2
3
4
5
6
7
Mean

TNF-
N mucosal (pg/ml)
ulcer (pg/ml)
140
296
0
230
62
185
170
250
215
270
0
185
0
380
83,85 (SD 90,68)
256,57 (SD 68,2)

IL-10
N mucosal (pg/ml)
78
88
76
87
88
93
211
103 (SD 48)

ulser (pg/ml)
65
67
65
71
72
65
119
74 (SD 19,68)

Table 1, showed result of the paired T-test to compare TNF- normal

mucosal dan TNF- ulcers with statistically significant level of p = 0,006 and
interval confidence 95%. Different level of IL-10 normal mucosal and IL-10 RAS
ulcer showed significant level with p = 0,041 and interval confidence 95%.
DISCUSSION
Commonly, cytokine measurement using vena puncture technic for serum
specimen or both of Fine Needle Aspiration biopsy (FNAB) and exicional biopsy
technic (Lewkowicz, 2005; Natah,2000). Our knowledge and assay tecnology support
the measurement of cytokine for research and clinical approach (Hill, 2000;
OGorman, 2008). One of this approach was swab technic using cotton strip sterile or
brush previously used for mucosal microorganism. This technic applied by a stroke of
mucosal 3 to 5 times to carry microorganisme or cell exfoliation (Spafford, 2010).
This technic also used for cytokine gene polymorphisme on vaginal, gut and oral
mucosal (Greber et al, 2005; Guimares, 2007). Also for immunobloth analisi and
densitometry optica for D1 cyclin biomarker expression change and as blood
examination alternative for them with contraindication or reject to cytokine gene
polymorphisme for Hodgkin Limfoma (Lan qing, 2006). This approach also used to
collected servical cells and cervicovaginal mucous (Hill, 2000), for urethral cytokine
examination (Pate, 2001), cytokine of vaginal fluid (agnew et al, 2008), and the

validity measuremen studied with comparing IL-6 cytokine concentration between

swab aplicator used by the professional dan by subject and concluded that this technic
valid for both of them (Faro et al, 2006).
Swab technic for mucosal organ could demonstrate cytokine level by
lymphosite, macrophage, and dendritic cell as cell source of cytokine which burden in
Mucosal associated lymphoid tissues (MALT) covered oral mucosal.
In the current study we observed that swabs technic on oral mucosal could
show TNF- and IL-10 level. Some sample show zero value but it was a normality
fenomena because TNF- commonly not found in epithelium. However this does not
mean that these cells are not producing and secreting this cytokine. It is ossible that
such cell types may contain insufficient TNF- to be demonstrate by our assay
(Natah, 2001).
TNF- test in normal distribution with differences of TNF- level between
normal mucosal and ulcers RAS significant at p = 0,028. This value similiar to that
previously reported by Natah,2003 and Buno 1998 which TNF- significantly
increased in RAS. Elevated levels of this cytokine related to the mechanism of
proinflamation cytokine as a respon for oral stimulus such as bacteri, minor trauma,
chemical, or thermis as a triggering factor for RAS.
IL-10 level show expected value which decrease significantly with p = 0,47.
This previously reported by Lewkowicz 2005 studied this cytokine in PBMC of RAS
patients. This decrease possible because abnormality balance of cytokine profile as a
cause of RAS ulcers. And this decrease assumed as immune failure for suppress oral
inflamation reaction (Buno,1998).
In conclusion, swab technic could use to measure proinflamation and
antiinflamation cytokine especially TNF- and IL-10 level. Our study need further to
continue as our result did not show specifity of this cytokine because of we do not
compare this result with PBMC or biopsy technic which relativly stabile and more
representative and with small sample size.

ACKNOWLEDGMENT
This research was supportd by South Sulawesi goverment and our gratefull to
Prof.Savage and Natah S for the inspiring study and written. Not metion for Mrs.
Rahayu at Tropical Diseases Centre of Airlangga University that conduted all
laboratories procedurs.
RFFERENCES
Agnew JK et al., 2008., effect of semen on vaginal fluid cytokines and secretory
leukocyte protease inhibitor., Inf dis in obs and gyne., vol 2008, 1-4.
Avery KJ., 2002., Oral development and histology., 3 rd eds., Goerge thieme Verlag.,
New York.,p.243-62.
Brodie SJ., 2002., Determinants of mucosal HIV replication and shedding., available
at www.PRN.org December;7:4,p.11-15.
Buno IJ, Huff JC, Weston WL, Cook DT, Brice SL. 1998., Elevated levels of
interferon gamma, tumor necrosis alpha, interleukin 2, 4 and 5, but not
interleukin 10, are present in recurrent aphthous stomatitis. Arch dermatol; 134
: 827-31
Cibas SE, Ducatman BS., 2009., Cytology : Diagnostic principles and clinical
correlates., 3rd eds., Saunders-Elsevier., Philadelphia.,p.1-10.
Dissanayake WHV., 2008., Ethics review committee guidelines ; risk benefit
analysis., Forum ethics review committee. Sri Lanka
Dragnev KH, Petty JW, Shah S, Biddle a, Desai NB, Memoli V., 2005., Bexarotene
and erlotinib for arodigestive tract cancer., J Clin Oncol;23;p.8757-64.
Faro CJ, Holler LM, Bishop K., 2006., Comparison of vaginal cytokine collection
methods., am J Reprod Immunol, May;55(5):315-20.
Field A, Longman L. 2003., Tyldesleys oral medicine., 5th ed., Oxford university
press., p.3-9.
Gerber S, Bongiovanni AM, Ledger WJ, Witkin SS, 2003, Interleukin-1 gene
polymorphism in women with vulvar vestibulitis syndrome. Eur Jour Obst
Gine Repro Bio 107 : 74-77
Gestner A, Thiele a, Tarnok a, Machlitt J, Ocken J, Tannapfel A, Weber A, Boot
ZF.,Preoperative detection of laryngeal cancer in mucosal swabs by slide based
cytometry., Eur J Cancer., Feb; 41(3):445-52.
Greenberg SM., 2003., Ulserative, vesicular and bullous lesions., in Lynch A.M.,
Brightman J.Vernon., Greenberg S. M., Greenberg : Ilmu Penyakit Mulut., 8th
ed. Bina Rupa Aksara.,Jakarta., P. 52- 54.
Guimaraes SLA, Correia-Silva FDJ, et al. 2007., Investigation of functional gene
polymorphisms IL-1, IL-6, IL-10, and TNF- in individuals with recurrent
aphthous stomatitis. Arch of Oral Bio; 52 : 268-72.

Hayrinen-Immonen R, 1992., Immune-activation in recurrent oral ulcer ROU. Scand

J Dent Res: 100 : 222-7.
Hyrinen-Immonen R, Nordstrm D, Malmstrm M, Hietanen J, Konttinen YT.
2003., Immune-inflammatory cells in recurrent oral ulcers (ROU). 1991. Scand
J Dent Res 99:510-8.
Herdani PRA.2004., Identifikasi protein spesifik pada RAU. Tesis pascasarjana
UNAIR; 15-39.
Hill JA., 2000., Cytokines in human reproduction., Wiley-Liss., Kanada.p.80-81
Kliewer MA, Shearfor DH, Paulson EK, Helsper RS, Hertzberg bS, Nelson RC.,
1999., Percutaneous liver biopsy : a cost-benefit analysis comparing
sonographic and CT guidance., am J Roent., 173.p.1199-1202.
Lehner T. 1969. Pathology of recurrent oral ulceration and oral ulceration in Behets
syndrome: light,electron and fluorescence microscopy. J Pathol 97: 481-93.
Lewkowicz N, Lewkowicz P, Banasik M, Kurnatowska A, Tchorzewski., 2005.,
Predominance of Type 1 cytokines and decreased number of CD4+CD25+high
T regulatory cells in peripheral blood of patients with recurrent aphthous
ulcerations : 99 ; 57-62.
Lighdale CJ.,2010., Advance imaging of barretts : implication for surveillance and
ablation., Gastroenterological endoscopy., 2nd eds., thieme., New york.,p.2135.
Lin SS, Chou YM, Ho CC. 2005., Study of the viral infection and cytokines
associated with recurrent aphthous ulceration. Micro and Inf; 7: 635-44.
Melhus A., 2001., Effects of amoxicillin on the expression of cytokines during
experimental acute otitis media caused by non-tupeable harmophilus
influenzae., j Antimic chemo; 48.,p.397-402.
Mestecky J, Lamm EM. 2004., Immunology of diseases of the oral cavity. 3 rd ed.
Elsevier Academic Press: 1518-46.
Mills MP, Mackler BF, Nelms DC, Peavy DL. 1980., Quantitative distribution of
inflammatory cells in recurrent aphthous stomatitis. J Dent Res 59: 562-6.
Miyamoto NT, Borra RC, Abreu M, Weckx LM, Franco M., 2008., Immuneexpression of HSP27 and IL-10 in recurrent aphthous ulceration.,J Oral Pathol
Med 37(8) : 462-7.
Murphy K, Travers P, walport M., 2008., Janeways immunobiology., 7th ed., Garland
Science., New York., p.762-772.
Natah SS, Immonen HR, Hietanen J, Patinen P, Malmstrom M, Savilahti E,et al.,
2000., Increased density of lymphocytes bearing / T-ce;p; receptors in
recurrent aphthous ulceration (RAU)., Int J Oral Maxillofac Surg. 29 : 375380.
Natah SS, Konttinen YT, Enattah NS, et al. 2004., Recurrent aphthous ulcers today : a
review of the growing knowledge. Int J Oral Maxillofac Surg; 33 : 221-34.
Natah SS., 2001., Recurrent aphthous ulceration : imuno-pathological aspects.,
academic dissertation, University of Helsinki. p.30-60

OGorman RGM, Donnenberg DA., eds. 2008., Handbook of Human Immunology.,

2nd ed., CRC Press., p.495-525.
Pate SM, Hedge SP, Sibley DA, Russel MW, Hook III EW, Mestecky J., 2001.,
urethral cytokine and immune responses in Chlamydia trachomatis-infected
males., inf n immune., vol nov 2001. p.7178-7181.
Pinchuk G., 2002., Theory and problems of immunology.,McGraw-Hill., New York.,
p.158-81.
Porter SR, Scully C, Pedersen A. Recurrent aphthous stomatitis. Crit Rev Oral Biol
Med 9: 306-21, 1998
Regezi JA, Sciubba J, Jordan RCK.,2003., Oral pathology clinic : clinical pathologic
correlation., 4th ed., Saunders California., p.38-41
Remirc GD, Friedland SJ.,eds., 1997., Cytokines in health and disease., 2 nd ed.,
Marcel dekker, Inc., p.29-240.
Roitt IM, Delves PJ, Roitts essential immunology.10 th ed. Blackwell publishing,
Massachusetts. P.177-89.
Savage WN, Boras VV. 2007., Recurrent aphthous ulcerative disease : presentation
and management. Aus Dent J; 52(1): 10-15.
Schroeder HE, Mller-Glauser W, Sallay K. 1983., Stereologic analysis of leukocyte
infiltration in oral ulcers of developing Mikulicz aphthae. Oral Surg Oral Med
Oral Pathol 56: 629-40.
Scully C, 2002. Aphthous stomatiitis. eMedicine journal vol.3 No.6
Scully C. 2003. The Diagnosis and Management of Reccurent Aphthous Stomatitis.
JADA, February Vol.134:200-7.
Silverman S., Eversole R.L., Truelove L.E., Essentials of Oral Medicine., 1 st edition.,
B.C. Decker Inc. Hamilton.London. hal 67-70.2001
Spafford MF et al., 2000., Detection of head and neck squamous cell carcinoma
among exfoliated oral mucosal cells by microsatellite analysis.,available at
compuserve.com November.
Sun JD, Weatherly RA, Koopmannjr CF., Carey TE., 2000., Mucosal swabs detect
HPV in laryngeal papilomatosis patients but not family members., Inter J
Pedia Otprhino.,June; 95-103
T. Yamamoto K, K. Yoneda, E. Ueta, T.Osaki, 1994, Serum cytokines, interleukin-2
receptors, and soluble intercellular adhesion molecule-1 in oral disorders, Oral
Surg. Oral Med. Oral Pathol. Oral Radiol. Endodon.78 ; 737-35
Thomson WA, Lotze TM., eds., 2003., The cytokine handbook., 4 th ed., Academic
Press., Amsterdam., p.167-853.
Yu Lu S, 2004. Initial diagnosis of anemia from sore mouth and improved
classification of anemias by MCV and RDW in 30 patients. Oral SurgOral
Med Oral Pathol Oral Radiol Endod ;98:679-85