Action of mitomycin C on eosinophilicnasal polyposisPaulo Fernando Tormin Borges Crosara1, Evaldo Nascimento2, Anilton César Vasconcelos3, HelenaMaria Goncalves Becker1, Sebastião Cronemberger1, Sandra Letícia Reis Crosara1, Roberto EustáquioSantos Guimaraes1

1 Departamento de Oftalmologia, Otorrinolaringologia e Fonoaudiologia, Universidade Federal de Minas Gerais, Belo Horizonte – Brazil

2 Departamento de Microbiologia, Immunologia e Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte – Brazil

3 Laboratório de Apoptose, Departamento de Patologia, Universidade Federal de Minas Gerais, Belo Horizonte – Brazil

ORIGINAL ARTICLE

325 - Fr ORL - 2007 ; 93

ABSTRACT

Objective: To evaluate the action of mitomycin C (MMC) on the in vitro synthesis of interleukin-5 (IL-5) and gra-nulocyte-macrophage colony stimulating factor (GM-CSF) and the apoptotic index (AI) of eosinophils in eosino-philic nasal polyposis.

Materials and methods: This experimental study was conducted on cultures of eosinophilic nasal polyp fragmentsfrom 22 patients. Two groups of fragments were constituted: samples not treated with MMC and used as controls,and samples treated with 400 µg/mL MMC for five minutes. The supernatant of the two groups was collected at 0,12 and 24 hours for ELISA assay of IL-5 and GM-CSF. The AI was evaluated histologically after haematoxylin-eosin staining.

Results: GM-CSF levels decreased in the MMC group after 24 hours (P≤0.05), but no significant modification ofIL-5 secretion was observed in the group treated with MMC for 24 hours. The AI was increased in the group trea-ted with MMC for 12 hours (P<0.001).

Conclusion: MMC appears to decrease the production of GM-CSF and induces apoptosis of eosinophils.

(Fr ORL-2007;93:325-332)

(Presented on October 2004 at the 111th Congress of the French Society of ORL Head and Neck Surgery, Paris)

Key words: Nasal polyposis, Eosinophils, Mitomycin C, Apoptosis, IL-5, GM-CSF.

Submitted for publication: May 2005Accepted for publication: November 2007Corresponding author: Paulo Fernando TorminBorges CrosaraRua dos Otoni 909/707 Funcionarios Belo Horizonte/MG -Brasil. e-mail: pcrosara@hotmail.com

INTRODUCTION

Eosinophilic nasal polyposis (NP) is a component ofS a m t e r ’s tri a d, allergic fungal sinusitis and Churg -Strauss syndrome. Corticosteroid therapy is the treat-ment of choice for this disease by reducing the inflam-m at o ry process mediated by eosinophilpolymorphonuclear cells [1].

The prominent fe at u re of eosinophilic NP is infi l t ra-tion of nasal polyps by eosinophils, wh i ch helps toexplain the pat h ogenesis of this disease. Tissue eosi-nophilia is secondary to increased migration of eosi-nophils to the site of inflammation and/or by increaseds u rv ival of eosinophils in the tissues, resulting fro minhibition of the mechanisms of apoptosis [2,3].Apoptosis is ch a ra c t e ri zed by a sequence of events, dis-tinct from necrosis, which prevent the release of intra-cellular components and activation of infl a m m ation fa c-tors [4]. Apoptosis is an active process dependent onRNA and protein synthesis [4]. Simon et al. demons-t rated that gra nu l o cy t e - m a c ro p h age colony stimu l at i n gfactor (GM-CSF) [5] and interleukin 5 (IL-5), produ-ced by eosinophils and lymphocytes, are able to pro-long the survival of eosinophils by inhibiting apopto-sis [6-8].

A c t ivation of the Fas re c eptor (CD 95/APO1), memberof the TNF/NGF (neural growth factor) superfa m i ly,can induce apoptosis of eosinophils in normal indivi-duals [9], but does not induce apoptosis of eosinophilsin eosinophilic subjects, while the active Fas receptoris expressed normally on these cells [9]. This suggestst h at other systems are invo l ved in the reg u l ation of ap o p-tosis [10,11].

Whyte et al. [12] showed that inhibition of protein syn-thesis by cy cl o h eximide and actinomycin D led to ap o p-tosis of eosinophils and neutrophils. These authorsc o n cluded that apoptosis is continu a l ly inhibited by thep roduction of certain proteins and induced by theabsence of these proteins [4,13,14].

Mitomycin C (MMC) is an anticancer drug and anti-biotic ex t racted from Strep t o myces caespitosus. Its anti-b a c t e rial mechanism of action is based on selective inhi-bition of DNA synthesis, recombination mechanisms,sister chromatin exchange and DNA repair [15].

Schwartz et al. [16] reported induction of apoptosis inga s t ric tumours tre ated by MMC. Simple ap p l i c ation of0.4 mg/mL MMC for 5 minutes inhibited proliferation

of cultured fibroblasts for 35 days [17]. Kim et al. [18]o b s e rved that MMC, at a dose of 0.4 mg/mL for 5m i nutes, induced in vitro apoptosis in a pri m a ry cultureof human fi b ro blasts from Te n o n ’s cap s u l e. MMC isalso frequently used in laryngology and otology withcomplete safety and good results [19-21].The purpose of this study was to evaluate the action ofm i t o mycin C on cultures derived from eosinophilicnasal polyps by measuring three parameters: GM-CSFsynthesis, IL-5 and the eosinophil apoptotic index.

MATERIAL AND METHODS

The study population was composed of patients withextensive nasal polyposis followed at the Hospital dasClínicas of the Universidade Federal de Minas Gerais(HC/UFMG) Brazil between January and June 2003.Twenty-two patients with eosinophilic NP, not treatedby cort i c o s t e roids or antihistamines for at least 2 monthsand presenting a percentage of eosinophils equal to orgre ater than 40% on histological ex a m i n ation we reselected. Patients with non-eosinophilic nasal polypo-sis, in a context of cystic fi b rosis, Kart age n e r ’s syn-d ro m e, antro - choanal polyp or eosinophilic NP witha c t ive infection we re ex cluded from the study. Th i ss t u dy was ap p roved by the Research Ethics Committeeof the Unive rsidade Fe d e ral de Minas Gerais (No.109/02) (Brazil).

A comparative experimental study was performed on22 samples of eosinophilic polyps. The study gro u pwas composed of cultures of polyp fragments treatedwith MMC and eva l u ated at H0, H12 and H24. Th econtrol group was composed of cultures of polyp frag-ments not treated with MMC and evaluated at the sametime-points (Figure 1).

Nasal polyp biopsies were therefore performed to cha-racterize eosinophilia and for cell culture. Fragmentsobtained for evaluation of eosinophilia were immedia-tely fixed in 10% formalin, stained with haematoxylin-eosin (HE) and submitted to a histological ex a m i n at i o nwith (HE) [22]. For culture, nasal polyp biopsies werep rep a red at room temperat u re immediat e ly after col-lection in the Ear, Nose & Throat outpatients depart-ment of Hospital das Clínicas (HC/UFMG) (Bra z i l ) .Fragments were sectioned into six 3 mm3 portions andone portion was deposited into each well of a six-wellp l at e. Th ree fragments constituted the study groups andthe other three fragments constituted the control gro u p .In the study group, MMC was applied for five minutesat a dose of 400 µg/mL [17]. Cultures were washed in

Fr ORL - 2007 ; 93 : 326

Mitomycin C and nasal polyps

RPMI medium after application of MMC and culturesof the control group we re also washed in RPMImedium. Fragments in the fi rst two compart m e n t s ,control and study at H0, were examined immediatelyby Enzyme Linked Immunosorbent Assay (ELISA) andhistology. The other two pairs of samples, each contai-ning a group control and a study group, were incuba-ted for 12 and 24 hours, respectively, at 37°C in 0.5%CO2, in RPMI 1640 culture medium (Gibco, UK)containing 5% human serum albumin, 2 µM mercap-toethanol, 1 mM L-glutamine, 2 mM sodium pyruvate,10 µg/mL strep t o mycin, 100 U/mL penicillin and 5mg/mL fungizone.

The culture supernatant was removed at H0, H12 andH24 and stored at a temperature of –80°C until analy-sis by ELISA for determination of GM-CSF and IL-5l evels (R&D Systems Kits: Duoset® Human GM-C S F / C at a l ogue: DY215; Duoset® Human IL-5Catalogue: DY205 – Minneapolis, USA) [23].

Fragments of each sample, after removing the super-natant, were fixed in 10% formalin and embedded inp a ra ffin. Prep a rations we re stained with haemat ox y l i n -eosin (HE) and examined by light microscopy. Imagesof fields showing eosinophil infiltration were digitizedand analysed by morphometry software (Kontron KS300, ve rsion 2.0) in the Morp h o m e t ry lab o rat o ry,General Pathology Department, Universidade Federalde Minas Gerais.

For analysis of the apoptotic index (AI), eosinophilswe re counted on each slide with an oil immersion x1,000 objective, by dividing the total number of apop-totic eosinophils by the total number of eosinophils [3,

22]. Apoptosis of eosinophils was defined by loss of cellcontacts, cytoplasmic and nu clear condensation ornuclear extrusion [3,22].

S t atistical analysis used a paired t test to compare gro u p sat each time-point, according to Snedecor and Coch ra n(1977) [24]. A p value ≤ 0.05 was considered signifi-cant. The Minitab, version 13, statistical analysis soft-ware was used.

RESULTS

Figure 2 shows the levels of expression of GM-CSF inthe cultures. A biopsy of sufficient size to also performc u l t u re at H24 was not ava i l able for 3 of the 22 samples.This analysis was there fo re perfo rmed on only 19samples. No significant difference was observed bet-ween the study and control groups at time H0 (P=0.362).At H12, the study group had lower GM-CSF levels thanthe control group, but the diffe rence was not signifi-cant (P<0.570). At H24, a significant diffe rence fo rGM-CSF levels was demonstrated between the twogroups (P<0.05).

A n a lysis of the GM-CSF ex p ression curves in thec o n t rol group did not reveal any stat i s t i c a l ly signifi c a n tdifference between H0 and H12 (P=0.087), but a signi-ficant diffe rence was observed between H0 and H24(P=0.048). No significant diffe rence was observed bet-ween 12 and 24 hours (P=0.409) despite the increasein GM-CSF levels at H24.

A n a lysis of the GM-CSF ex p ression curves in the studygroup revealed a similar behaviour with stat i s t i c a l lys i g n i ficant diffe rences between H0 and H12 (P=0.013),

327 - Fr ORL - 2007 ; 93

Mitomycin C and nasal polyps

Figure 1: Study plan. Fi g u re 2: Curve of GM-CSF ex p ression withand without application of MMC.

Sample of eosinophilic polyp

Culture medium

Control group (0, 12 and 24 hours)

Study group(0, 12 and 24 hours)

ELISA and histology

Assay of GM-CSFand IL-5 expression Apoptotic Index

4035302520151050

Time (hours)

GM-CSF expression

with mitomycin C

without mitomycin C

0 12 24

b o rd e rline significant diffe rences between H0 and H24(P=0.068), and no significant difference between H12and H24 (P=0.606).

Figure 3 shows the levels of expression of IL-5 in thecultures. A biopsy of sufficient size was not availableto also perform culture at H24 for 2 of the 19 samples;this analysis was there fo re perfo rmed on only 17 cases.No significant diffe rence was observed between thes t u dy and control groups at time H0 (P=0.362). The IL-5 ex p ression peak was slightly higher in the study gro u pat H12, but this difference was not significant compa-red to the control group (P=0.281). After 12 hours, am a rked reduction of IL-5 levels was observed in thestudy group, but with no significant difference compa-red to the control group at H24 (P=0.105).

Individual analysis of the control group revealed a sta-t i s t i c a l ly significant diffe rence for IL-5 ex p ression bet-ween H0 and H12 (P=0.046) and between H0 and H24(P=0.027), but no significant difference was observedb e t ween H12 and H24 (P=0.671). Analysis of the studygroup did not reveal any significant diffe rence betwe e nH0 and H12 (P=0.114), H0 and H24 (P=0.635) or H12and H24 (P=0.112).

H i s t o l ogical slides we re prep a red for 9 samples to deter-mine the apoptotic index. A total of 741 digital fieldswere examined at x 1,000 and 67 were examined at x400 for cell counting. The mean cell count (percentageof eosinophils) was 55.7% (ra n ge: 48.2-67.5). AI re s u l t sfor the 9 samples are presented in Figure 4. No signi-ficant diffe rence was observed between the control andstudy groups at H0 (P=0.189). At H12, study culturesp resented a signifi c a n t ly higher apoptotic index than

those of the control group (P<0.001). At H24, no signi-ficant difference was observed between the two groups(P=0.301).I n d ividual analysis of the control group revealed a signi-ficant difference between H0 and H12 (P<0.001), H0and H24 (P<0.001) and H12 and H24 (P<0.001) andsimilar results we re observed for the study group, inwh i ch a significant diffe rence was observed between H0and H12 (P<0.001), H0 and H24 (P<0.001) and H12and H24 (P<0.001).

The method of histological examination was found tobe fairly effective for the detection of apoptotic eosi-nophils, as illustrated in Figures 5 to 7.

DISCUSSION

Several authors have reported that eosinophils are themain cells invo l ved in the pat h ogenesis of these diseases[25]. Similarly, in this study, all types of eosinophilicNP were considered, with no distinction between thevarious cases studied [25].

A s s ay of GM-CSF and IL-5 was justifi e d, as these cy t o-kines are ex t re m e ly important for the diffe re n t i at i o n ,migration, binding, activation and survival of eosino-phils. These cytokines associated with eosinophils the-refore play an essential role in the pathophysiology ofeosinophilic NP [25-27].

Lacy and Moqbel [28] concluded that the importanceof GM-CSF synthesis for eosinophils was related to itsparacrine and autocrine actions that activate and pro-long survival of eosinophils in vivo.

Mitomycin C has a number of potential applications,as it is an antineoplastic and antibiotic agent wh i ch actslike an alkylating agent by inhibiting DNA and proteinsynthesis [20] and the growth of fibroblasts in cell cul-

Fr ORL - 2007 ; 93 : 328

Mitomycin C and nasal polyps

Fi g u re 3: Curve of IL-5 ex p ression with andwithout application of MMC.

Figure 4: Apoptotic Index of cultures withand without application of MMC.

60

50

40

30

20

10

0

Time (hours)

IL-5 expressionwith mitomycin C

without mitomycin C

0 12 24

Time (hours)

Apoptotic Indexwith mitomycin C

without mitomycin C

0 12 24

100806040200

t u re. MMC has alre a dy been used ex p e ri m e n t a l ly toprevent stenosis following maxillary antrotomy, glau-coma and ptery gium surge ry, my ri n gotomies and lary n-geal stenosis [15,17,21].

This study demonstrated significantly lower GM-CSFl evels in the study group after 24 hours of culture(P<0.05). No significant difference was observed at 12h o u rs, pro b ably because of the small sample size(N=22). These results are concordant with those repor-ted in the literat u re concerning decreased GM-CSFexpression in response to corticosteroids for the treat-ment of eosinophilic NP [4,8].

No significant diffe rence was observed between thes t u dy group and the control group in terms of the actionof MMC on IL-5 synthesis at H0, H12 and H24.A n a lysis of the time-course of IL-5 secretion in thestudy group showed an initial increased IL-5 secretionat 12 hours, fo l l owed by a marked reduction at 24 hours ,wh i ch suggests a possible action of MMC in this group.

An action of MMC on IL-5 secretion cannot be fo r-mally excluded on the basis of this small sample sizeand further studies are required.

Apoptosis or programmed cell death is a process thatreg u l ates the infl a m m at o ry re s p o n s e. In eosinophilicnasal polyps, inhibition of apoptosis could delay migra-tion of activated eosinophils, thereby prolonging eosi-nophilia and tissue lesions [5,7].

Va rious studies have shown that high IL-5 and GM-CSF levels inhibit apoptosis. Dibb e rt et al. [14] demons-trated that IL-5 and GM-CSF stimulate the anti-apop-totic Bcl-xL ge n e, while the function of the pro ap o p t o t i cBAX gene is not modified by these cytokines.

By using corticosteroids, Cox [13] and Meagner et al.[4] concluded that inhibition of apoptosis was trigge-red by the production of specific proteins and not by thedirect action of GM-CSF. Whyte et al. [12] used cyclo-h eximide and actinomycin D to induce apoptosis ofeosinophils and neutrophils, demonstrating that pro t e i nsynthesis is necessary for inhibition of apoptosis.

MMC is an antiblastic alky l ating agent that inhibitsD NA synthesis and induces apoptosis in ga s t ric tumourcells [18]. Kim et al. [18] observed that MMC (0.4mg/mL for five minutes) induced apoptosis of humanfibroblasts of Tenon’s capsule in vitro.

Jang et al. [29] studied the action of 400 µg/mL MMCon a culture of tympanic membrane fi b ro blasts and

329 - Fr ORL - 2007 ; 93

Mitomycin C and nasal polyps

Fi g u re 5: Histological section, control gro u p ,at H0.

Eosinophils are mostly viabl e. HE stain x 1,000.Bar = 10 microns.

Fi g u re 6: Histological section, control gro u p ,at H12.

Eosinophils are mostly viabl e, but some alre a dypresent signs of apoptosis (arrow). HE stain x1,000. Bar = 10 microns.

Figure 7: Histological section, with applica-tion of MMC, at H12.

Eosinophils alre a dy show obvious signs of ap o p -tosis. Also note disorga n i z ation of the connec -t ive tissue. HE stain x 1,000. Bar = 10 micro n s .

d e m o n s t rated a stat i s t i c a l ly significant diffe rence ofinhibition of pro l i fe ration compared to the contro lgroup.

A n a lysis of the apoptotic index did not reveal any signi-ficant diffe rence at H0 between MMC-tre ated and non-t re ated groups (P=0.189), but a significant diffe re n c ewas observed between the two groups at H12(P<0.001). This result there fo re supports the action ofMMC as an alky l ating agent, inhibiting protein syn-thesis and tri gge ring the apoptotic cascade, leading tom o rp h o l ogical ch a n ges and cell death. These cellch a n ges we re cl e a rly demonstrated by histological ex a-m i n at i o n .

No significant diffe rence was observed between the twogroups at H24 (P=0.301). AI increased slow ly and pro-gressively in the control group, but increased rapidlyover the first 12 hours in the study group. A more mar-ked progressive cell loss was therefore observed in thestudy group at H12.

Due to its alky l ating action, MMC bl o cks pro t e i nsynthesis in these cells, suspending anti-ap o p t o t i cm e chanisms and tri gge ring pro apoptotic mech a n i s m sinduced by activation of eosinophils [9] but conti-nu a l ly bl o cked by the action of proteins induced bycytokines such as GM-CSF and IL-5 [12].Ap p l i c ation of MMC there fo re induces lower tissuel evels of GM-CSF that inhibit activation of moreeosinophils and tri gger apoptosis of activated eosi-nophils [4,6,14].

A high percentage of apoptotic eosinophils was obser-ved in control cultures at 24 hours, identical to thato b s e rved in the study group at 24 hours, wh i ch can pro-bably be explained by the absence of addition of nutri-tional factors during culture.

HE staining allows clear identifi c ation of eosinophilsand histological ch a n ges re l ated to apoptosis; it is afa i rly simple method with sufficient sensitivity fo ra n a lysis of the phenomena examined in this study[ 3 0 ] .

CONCLUSION

The data of this study indicate that mitomycin C ap p e a rsto inhibit GM-CSF synthesis and increase the apopto-tic index of eosinophils. The present results suggest thatmitomycin C could be used for the treatment of eosi-

nophilic nasal polyposis. Data concerning IL-5 ex p re s-sion suggest that mitomycin C tends to decrease theexpression of this cytokine, but these results need to beconfirmed by other studies.

REFERENCES

1. B e cker HMG, Guimarães RES, Nascimento E, B e cker CG, Gonçalves DU, Cro s a ra PFTB. Pe r fil de citocinas e tipificação de HLA em pacientes com polipose nasossinusal tolerantes e intolerantes a aspirina. Rev Bras Otorri n o l a ri n gol. 2 0 0 3 ; 6 9 : 2 9 6 - 3 0 2 .

2. Settipane GA. Nasal polyps and systemic diseases. In: Sch atz M, Zeiger RS, Settipane GA. Nasal mani-fe s t ations of systemic diseases. Providence: Oceanside Publ. 1991:43-51.

3. Wo o l l ey KL, Gibson PG, Carty K, Wilson AJ, Twaddell SH, Woolley MJ. Eosinophils apoptosis and the resolution of airway infl a m m ation in asthma. Am J Respir Crit Care Med. 1996;154:237-243.

4. M e agher LC, Cousin JM, Seckl JR, Haslett C. Opposing effects of glucocorticoids on the rate of apoptosis in neutrophilic and eosinophilic granulocytes. J Imunnol. 1996;156:4422-4428.

5. Simon HU, Yo u s e fi S, Sch ranz C, Sch ap owal A, B a ch e rt C, Blaser C. Direct demonstration of d e l ayed eosinophil apoptosis as a mechanism causing tissue eosinophilia. The J Immunol. 1997;158:3902-3908.

6. Simon HU. Reg u l ation of eosinophil and neutrophil apoptosis – Similarities and differences. Immunol Rev. 2001;179:156-162.

7. Kowalski ML, Grzgo rczyc J, Pawliczak R, Ko rn at owzki T, Wagow z k a - D a n i l ewicz M, D a n i l ewicz M. Deceased apoptosis and distinct p ro file of infi l t rating cells in the nasal polyps of p atients with aspirin hy p e rs e n s i t iv i t y. Allergy 2002;57:493-500.

8. B j o rnson BN, Harvey JM, Rose L. Diffe rential e ffects of hy d ro c o rtisone on eosinophil and neu-t rophil pro l i fe ration. J Clin Invest. 1985;76:924-929.

9. D avidsson A, Anderson T, Hellquist HB. Apoptosis and phago cytosis of tissue-dwelling eosinophils in sinonasal polyps. Lary n go s c o p e. 2 0 0 0 ; 1 1 0 : 1 1 1 -116.

10. H eb e s t reit H, Yo u s e fi S, Balatti M et al. E x p ression and function of FAS on human blood and tissue eosinophils. Eur J Immunol. 1 9 9 6 ; 2 6 : 1 7 7 5 - 1 7 8 0 .

Fr ORL - 2007 ; 93 : 330

Mitomycin C and nasal polyps

11. Hebestreit H, Dibert B, Balatti I et al. Disruption of fas receptor signaling by nitric oxide in eosino-phils. J Exp Med. 1998;187:415-425.

12. Whyte MKB, Meagher L, Haslett C. C y cl o h eximide and actinomicin-d programmed cell d e ath (apoptosis) in neuthophils. Clin Sci.1991;80:85.

13. Cox, G. Glucocorticoids treatment inhibits apop-tosis in human neutrophils, separation of survival and activation outcomes. J. Immunol. 1995;154:4719-4725.

14. D i bb e rt B, Daigle I, Braun D, et al. Role of Bcl -xL in delayed eosinophil apoptosis mediated by gra nu l o cy t e - m a c ro p h age colony - s t i mu l ation factor and interleukin-5. Blood. 1998;92:778-783.

15. Carrano AV, Thompson LH, Stretka DG, Minkler JL, Frong S. DNA crosslinking sister ch ro m atid exchange and specific locus mutation. Mutat Res. 1979;63:175-188.

16. Schwartz GK, Haimovtz-Friedman A, Dhupar SK et al. Potentiation of apoptosis by treatment with p rotein kinase-c specific inhibitor safi n gol in mito-mycin c treated gastric cancer cells. J Nat Cancer Inst. 1995;87:1394-1399.

17. Khaw PT, Doyle JW, Sherwood MB, Grierson H, Shultz G, Megorray S. Prolonged localized tissue e ffects from 5-minute ex p o s u res to fl u o ro u racil and m i t o mycin C. Arch Ophthalmol. 1994;72:155-161.

18. Kim JW, Kim SK, Song IH, Kim, IT. Mitomycin C-induced apoptosis in cultured human Tenon's c apsule fi b ro blasts. Ko rean J Ophthalmol. 1999;13:7-15.

19. C o rrea AJ, Reinish L, Sanders DL, Huang S, Deriso W, Duncavage JA. Inhibition of subglottic steno-sis with mitomycin C in the canine model. Ann Otol Rhinol Laryngol. 1999;108:1053-1060.

20. Rahbar R, Valdez TA, Shapshay SM. Preliminaryresults of intraoperative mitomycin-C in the treat-ment and prevention of glottic and subglottic stenosis. J Voice. 2000;14:282-286.

21. B e cker CG, Silva AL, Guimarães RES, Becker HM, Barra IM, Olive i ra WD. Tratamento cirúrgico da otite media com efusão: tubo de ventilação versus aplicação tópica de mitomicina C. Rev Bras Otorrinolaringol. 2003;69:513-519.

22. Ingels K, Durez JP, Cuvelier C, Cauwenberge PV.Nasal biopsy is superior to nasal smear for finding eosinophils in nonallergic rhinitis. Allergy. 1997;52:338-341.

23. Te ram LM, Pa rk HS, Djukanovic R, Roberts K, Holgate S. Cultured nasal polyps from nonatopic and atopic patients release RANTES spontaneouslyand after stimu l ation with phy t o h e m agglutinin. J Allergy Clin Immunol. 1997;100:499-504.

24. Snedecor GW and Coch ran WG. Statistical Methods AMES: Iowa State Unive rsity Press, 1977.

25. Jankowski R. Eosinophils in the Pathophysiologyof Nasal Po lyposis. Acta Otolary n gol (Stockh). 1996;116:160-163.

26. Mullol J, Xaubet A, Gaya A, et al. Cytokine gene ex p ression and release from epithelial cells. A c o m p a rison study between healthy nasal mucosa and nasal polyps. Clin Exp Allergy. 1 9 9 5 ; 2 5 : 6 0 7 - 6 1 5 .

27. Vo egels RL, de Melo Pádua FG. Study of inter-leukins 1, 3, 4, 5 befo re and after surge ry of patients with nasal polyposis. Otolary n gol Head Neck Surg. 2005;132:613-619.

28. L a cy P, Moqbel R. Eokines: Synthesis, Storage and Release from human Eosinophils. Mem Inst Oswaldo Cruz. 1997;92:125-133.

29. Jang CH, Song CH, Pak SC. Effect of exposure to m i t o mycin c on cultured tympanic membrane fi b ro blasts. Int J Pe d i atr Otorhinolary n gol. 2003;67:173-176.

30. Walsh GM, Dewson G, Wardlaw AJ, Lebi-Shaffer F, Moqbel R. A comparat ive study of diffe rent methods for the assessment of apoptosis and n e c rosis in human eosinophils. J. Immunol. Methods. 1998;217:153-163.

331 - Fr ORL - 2007 ; 93

Mitomycin C and nasal polyps

Fr ORL - 2007 ; 93 : 332

Mitomycin C and nasal polyps