Clin. exp. Immunol. (1992) 89, 94-99

Distribution of retroviral pl5E-related proteins in neopiastic andnon-neoplastic human tissues, and their role in the regulation

of the immune response

R. A. SCHEEREN*, R. A. J. OOSTENDORPt. S. VAN DER BAAN*. R. M. J. KEEHNENt, R. J. SCHEPERt& C . J. L. M. M E I J E R t Departments of *OtorhinokiryngologyjHead and Neck Surgery and ^Pathology.

Free University Hospital. Amsterdam. The Netherlands

(Acceptedfor publication 19 February 1992)

SUMMARY

In patients with head ;ind neck carcinomas and in palients with chronic purulent upper airwayinfections, low molecular weight retroviral p 15E-like factors are found. These factors are responsiblefor partial defects in the cellular immune response. We studied the distribution of these p 15E-relatedproteins in neoplaslic. inflamed and normal human tissues and related these findings with thepresence of p!5E-like factors in patients' sera. Demonstration of pl5E-like proteins in sera ofpatients with upper airway infections and of patients with head and neck carcinomas correlatedexclusively with the presence of pi 5E in normal and pathologic epithelium ofthe upper respiratorytract, p 15E was not demonstrated in epithelia of other localizations. Our results suggest that chronicstimulation or neopiastic transformation ofthe epithelia ofthe upper respiratory tract stimulates theproduction of pl5E-like proteins leading to their reported immunosuppressive actions.

Keywords serum factors pl5E inimunosuppression human tissues

INTRODUCTION

Retroviral infection can result in suppression of the immuneresponse. This has been well documented in leukaemias causedby murine and feline leukaemia viruses il-4]. The retroviraltransmembrane envelope protein pl5E plays an important rolein this phenomenon [5], Immunosupprcs.sive proteins isolatedfrom human [6] and murine tumours [5], human malignantpleura! effusions [6] and serum from patients with head and neckcarcinomas [7] and patients with chronic purulent rhinosinusitis[8] have been shown to have antigenic similarities with pl5E.and exert an inhibitory effect on IL-2 production, lymphocyteproliferation and chemota.i(is of mononuclear phagocytes. Thein vitro chemotactic responsiveness of monocytes has beenshown to be very sensitive to these pl5E-Iike proteins [6-8].

Since tissue-specific and age-dependent expression of retro-viral transcripts has been noted in mice and in humans [9,10]and sequences have been found in human DNA [11.12] thatshare homology with pl5E, an endogenous origin of pl5E-likeproteins is suggested. However, a detailed analysis of thedistribution of pl5E-!ike proteins in human tissues has neverbeen reported. Furthermore, the role of these proteins in theregulation ofthe normal immune response still remains unelear.

Correspondence; R. A. Scheeren, Department orOtorhino!aryngo-logy/Head and Neck Surgery. Free University Hospital, De Boelelaan1117, 1081 HV AnLsterdam. The Netherlands.

We therefore studied the expression of pl5E-like proteins innormal, malignant and inflamed human tissues. In addition, werelated this expression to pI5E-mediated immunosuppressivcactivity in serum.

MATERIALS AND METHODS

Tis.'iuesNeopiastic and non-neoplastic tissues were obtained fromsurgical procedures. Tissues also were derived from autopsiesperformed within 6 h after death. Tissues were snap-frozen andstored in liquid nitrogen.

SeraSera were obtained from the following seven groups of patients:

(i) Healthy controls (13 women, 12 men, age range 22-40years, mean 29) without known diseases.

(ii) Patients with carcinoma ofthe cervix uteri {n= 10, agerange 32-75 years, mean 57; clinico-pathologica! stage rangefrom I'^-IV'^).

(iii) Patients with morbus Crohn. based on clinico-patho-iogical, radiological and endoscopical criteria as previouslydescribed [13] {seven women, three men. age range 22-46 years,mean 32).

(iv) Patients with primary ciliary dyskinesia, based onabnormal ciliary motility [14] and abnormal ciliary uUrastruc-

94

Refroviral p }5E-relatedfactors in human tissues 95

ture [15] (14 male and eight female patients, age range 2-58years, mean 23).

(v) Patients with polyposis nasi. i.e. recurrent upper airwayinfections and the presence of nasal polyps at tnc moment ofsurgery or endoscopic examination (16 women. 12 men. agerange 18-66 years, mean 42).

(vi) Patients with chronic purulent rhinosinusitis, i.e. recur-rent upper airway infections not responding to adequateantibiotic treatment or surgical procedures, and without thepresence of nasal polyps al the time of surgery or endoscopicexamination (27 female and 14 male subjects, age range 7-71years, mean 34).

No patient with chronic purulent rhinosinusitis or polyposisnasi had abnormal ciliary motility or abnormal ciliary ultra-structure, Kurthcrmore, the leucocyte counts were withinnormal range and no defects in humoral immunity were present.

(vii) Patients with head and neck squamous cell carcinoma(seven men, three women, age range 55-81 years, mean 65;TNM classification ranging from T2NO-T3N3).

Monoclonal antibodiesThe MoAbs used were: 4F5 (IgG2a) [16] and 19F8 (IgC.2b) [17],both specific for pl5E but recognizing dirfcrent cpitopes [16];control MoAbs were an lgG2a and lgG2b, secreted by themouse myeloma PI. 17 and MPCI1 .OU A cell lines, respectively,both obtained from the American Type Culture Collection(ATCC; Rockville. MD).

Immunoperoxidase stainingSections of frozen tissue blocks (4-/(m thick) were prepared with;i cryostat (Jung-Reichert. Nussloch. Germany), mounted onpoly-L-lysine-coated glass slides, air dried, and acetone-fixedduring 10 min at room temperature. The sections were incu-bated for 60 min at room temperature with MoAb. washed, andincubated for 30 min at room temperature with horseradish-peroxidase-conjugated rabbit anti-mouse IgG (Dakopatts.Glostrup. Denmark) in the presence of 5'"., normal humanserum. Subsequently, the sections were weakly counter-stainedwilh haematoxylin.

Tissue reactivity was scored for the percentage of positivestaining cells and the intensity of staining ( — . negative; ± . veryweak; -I-. weak; and + + . strong). In the negatively scoredtissue samples, there was no reactivity present with the anti-pl 5E MoAbs or there were no differences in reactivity betweenIhe anti-pl5E MoAbs and the respective control MoAbs.

Monocyte isolation /rom healthy donorsPeripheral blood monocytes were isolated from four healthydonors who gave their informed consent to participation in thestudy. Buffy coais from 500 ml of human blood were obtainedfrom healthy donors. The monocytes were purified by successiveisopycnic centrifugation and clutriator centrifugation as pre-viously described [18] with minor modifications [19]. Thesehealthy monocytes were used to test the effects of patients"serum fractions.

Determination in patient serum of low molecular weight factors(LM WFl inhibiting the polarization of healthy donor monocytesSera were collected from patients by venepuncture and diluted1.1 in saline. These dilutions were subjected to ultrafiitrationI lirough Amicon CF25 Centriflo cones (Amicon, Danvers, MA)

for 15 min al 700 j? (mol. wt cut-olT point 25 kD). The residues,the LMWF. were dissolved in PBS and stored at —70 C untilfurther use.

The capability of the serum fractions to inhibit fMLP-induced polarization of healthy donor monocytes (elutriatorpurified) was determined by incubating ihe monocytes (1 x 10**/ml) for 15 min at 37 C, either with fMLP alone or with f MLP incombination with a serum fraction (final dilution I;60). Thepercentage of inhibition was calculated according to the for-mula:

( P —P

P

where Po= "̂> spontaneous polarization; P| = %polarizationafter incubation with f M LP alone; and P: = '^.polarization afterincubation with fMLP and LMWF.

Serum fractions were tested in triplicate. Addition of serumfractions to non-stimulated (fMLP) donor monocytes did notaffect the spontaneous polarization.

Determination of the pl5E-like character of patient LMWFTo validate the pl5E-like character of the LMWF in humanserum, adsorption experiments were carried out by neutralizingthe serum fractions before testing in the monocyte polarizationassay with a pl5E-specific MoAb (I9F8) in a final dilution of1:200 (25 mg/ml) at 4 C for 16 h, followed by Amiconultrafiitration lo remove formed immunecomplexes. Thisadsorption/neutralizing procedure was carried out twice [7].Adsorption experiments carried out wilh the MoAb 4F5 or with4F5and I9F8 together did not show any difference in neutraliz-ing the serum fractions as with 19F8 alone. Control experimentswere earried out with the isotype matched control MoAbs.

Statistical analysisStatistical analysis was performed by Student's r-test.

RESULTS

Reactivity ofanti-pI5E MoAbs with normal human tissueImmunohistochemical staining was performed on frozen sec-tions. Among the normal human tissues tested, the respiratoryepithelium cells of nasal mucosa reacted with the anii-pl5EMoAb. Furthermore, staining was seen in the squamousepithelium surrounding the tonsils at places where histologicallykeratinization could easily be observed. There was no differenceof staining pattern between the anti-pl5E MoAbs 4F5 andI9F8. although the reactivity with the 4F5 MoAb was moreintense. No reactivity was found with epithelium from skin,digestive tract or urogenital tract, nor in any other tissue tested,including immunocompetent cells in Ihe lymphoid organs(Table 1).

Reactivity ofanti-pl5E MoAbs with human neopla.sms and tissuewith chronic inflammationOf all human neopiastic tissues tested, only well-differenliatedareas of squamous cell carcinoma ofthe head and neck showedreactivity with the anti-pl5E MoAbs (Table 2. Fig. I). Again, nodifference was detected in binding pattern between 4F5 andI9F8. In contrast with normal human nasal mucosa, morebinding of the anti-pl5E MoAbs was seen in squamous cellcarcinoma from the head and neck region. Squamous cell

96 R. A. Scheeren et al.

Table 1. Frozen (issue reacliviiy ofMoAbs directed against pISE wilh nor-

mal human lissue specimens

Table 2. Frozen tissue reactivity of MoAbsdirected against pl5E with human neoplastic

tissues

Organ

Nasal mucosaConcha inferiorConcha media

LungBronchusMuscleSkinBrainKidneyLiverHeartStomachBladderGall bladderLymph nodeThymusSpleenAdenoidTonsilEsophagusVaginaLarynxColonCervixPlacentaThyroidAdrenal glandParolid glandPancreasOvaryTestes

Positive/tested

10/12*6/12*0/20/20/20/20/20/20/20/20/20/20/20/50/50/60/22/2t0/20/10/40/20/20/20/20/20/30/20/2()''2

In negative scored tissue nol a singleeel! was labelled by 4F5/I9F8, or therewas no significant diPFerence with thecontrol MoAb.

• Positive staining in basal cell layerof respiratory epithelium. More than75'! .̂ of the cells showed a stainingintensity from 4- to -(--)-.

t Positive staining ( + / + + ) insquamous epithelium surrounding thetonsils especially at places where kerati-nization could be observed easily.

carcinoma from other regiotis, other type of carcinoma includ-ing adenocarcinoma, and small cell carcinoma, melanomas.Hodgkin's lymphoma and non-Hodgkin's lymphoma did notshow any positive staining.

Reactivity of both anti-pI5E MoAbs was seen with respira-tory and squamous epithelium in inflammatory nasal polyps(Fig. 2) and purulent bronchitis and with epithelium coveringinflammation of the middle car (Table 3), Again, more intensestaining ofthe anti-pI5E MoAbs was seen with the ehronieallyinflamed mucosa of the upper airways than with normal humanupper airway mucosa. Inflamed lissue from the skin, gut andcervix did not show any binding of the MoAbs.

Neoplastic tissue

Squamous cell carcinomaHead and neckCervixLungEsophagus

AdenocarcinomaColonBreastOvaryLung

Small ceil carcinomaLung

MelanomasHodgkin's lympKomaNon-Hodgkin's lympboma

Positive/tested

13/13*0/20/20/2

0/40/60/50/1

o/i0/40/20/2

* Positive staining ( + / + + ) focally in well-differentia led areas of the tumour.

Influence of retrotiral-like LM WF on fMLP-induced monocytepolarizationThe results of the inhibition of the fM LP induced polarizationof healthy donor monoeytes by LMWF of patients withsquamous ceil carcinoma of the head and neek, patientswith squamous cell carcinoma of the cervix uteri, patients withmorbus Crohn,, patients with primary ciliary dyskinesia.patients with polyposis nasi, patients with chronic recurrentpurulent rhinosinusitis and of healthy individuals arc shown inFig. 3, The LMWFof patients with squamous cell carcinoma ofthe head and neck and of patients with recurrent upper airwayinfections (polyposis nasi. primary ciliary dyskinesia andehronic recurrent purulent rhinosinusilis) all showed clearsignificant (/'<()OOI) inhibition of fMLP-indticed monoeyteehemotaxis. The optimal inhibiting efleet of fMLP-inducedmonocyte chemotaxis was seen with the LMWF in a linaldilution of 1:60, A limited number of serum samples couldadditionally be assayed at 1:30 and 1: 120 dilutions, and similardifferences in inhibition were seen (data not shown). In all testedconcentrations., the inhibitory activity observed in serum sam-ples from patients with squamous eell carcinoma was morepronounced in comparison to those observed from non-neopiastic disease. Since no inhibition of fMI.P-induced mono-cyte chemotaxis could be detected after neutralizing the serumfractions with a pl5E-specific MoAb (19F8) prior to testing inthe monocyte polarization, the inhibition was due to thepresence of pl5E in the LMWF, In contrast, no significantinfluence of LMWF of patients with squamous cell carcinomaofthe cervix uteri and morbus Crohn on the fMLP-inducedchemotaxis of healthy donor monocytes was detected (Fig. 3).In addition, there was also no influence of LMWF of patientswith adenocarcinoma ofthe breast and patients with soft tissuesarcoma in ITVlLP-induccd chemotaxis of monocytes fromhealthy donors (data not shown).

The inhibition data of the LMWF of the diflferent patientgroups on monocyte ehemotaxis correlated well witli the

Retroviral pi5E-related factors in human tissues 97

Fig. 1. (a) Reactivity wilh the anti-plSE MoAb 4F5 within the well-differentiated areas of squamous cell carcinoma of Ihe head andneck: (b) no binding delectable wilh the control MoAb in the same area of squamous cell carcinoma. Original magnilicaiion, x 400.

a

Kiy. 2. (aj Retel l \it> wLtli lliL-aLili p l ^ L Mu. ' \b41 > in i l icbusal lajct uUhccpi t l tc l iuniol iia.sitl pol jps , (b) Ihc cptlhulium of nasal polypsshows no binding with the control MoAb. Original magnification, x400.

outcome of the immunohistochcmicai slainlng. pl5E-Iikcserum factors could only be detected in patients with pathologi-eai conditions in which the tissue specimens showed activitywith MoAbs directed against pi 5E. The low expression of pi 5E-like faetors in "normar human nasal mueosa is apparently notsufficient to produce detectable serum levels, since pI5E-!ikefactors were not present in serum of healthy controls.

DISCUSSION

In humans, several studies have shown thiu ihc chemotaclicresponsiveness of mononuclear phagocytes is defective inpatients with malignancies [6,7] or chronic inflammatory respir-atory disease [8]. It was demonstrated that LMWF (<25 kD)isolated from tumours and serum were responsible for this

98 R. A. Scheeren et al.

Table 3. Frozen tissue reactivity of MoAbs directed against pi 5E withchronic inflamed human ti.ssue specimens

Tissue

Inflamed mucosa of nasal polypsChronic inflamed mucosa of ihe middle earChronic inflamed bronchial mucosa in purulent

bronchitisCholesteatoma of the middle earChronic dermatitisUlcerative colitisMorbus Crohn

Positive/tested

23/23'4/4*

3/3t0/10/20/20/2

* Positive staining in basai cell layer of respiratory epithelium. Morethan 75% of the cells showed a staining intensity from + to + + .

t Positive slaining in respiratory epithelium of the bronchus. Morethan 75% of ihe cells showed a staining intensity from + to + + .

LMWF LMWF+19F8 LMWF+

control MoAb

Fig. 3. Inhibition ofthe fMLP-induced polarization of healthy donormonocytes by serum factors < 25 kD (low mol. wt factors, LMWF) ofhealthy controls and patients wilh carcinoma ofthe cervix uteri (Cervixca.J; morbus Crohn; nasal polyposis (PPN): chronic purulent rhinosi-nu.sitis (CPR); primary ciliary dyskinesia (CPR); or carcinoma of thehead and neck region (H/Nca.) (mean ±s,d.). To validate the pl5E-likecharacter of the inhibition, the polarization assay was also performedwith serum factors after adsorption with anti-pl5E MoAb or controlMoAb. Inhibition of the polarization was significant (P<0001) withsera of PPN. CPR. PCD and H/N ca.

suppression of chemotactic responses [6]. This inhibitory effectcould be abolished by blocking these factors with antibodiesdirected against pl5E. the transmembrane envelope protein ofrelroviral murine leukaemia virus [6,7]. Transmembrane enve-lope proteins related to pl5E are assumed to play a role insuppression ofthe immune system, as seen in retroviral infection[6,16,20-22],

Here we have shown that the pl5E-like proteins are onlyfound in the sera of patients with upper airway diseases (Fig. 3)and that they can be demonstrated exclusively in normal andpathologic epithelium of the upper respiratory tract. Since envgenes that share homology with retroviral pl5E have been

found in human DNA and the presence of mRNA derived fromthese genes has been detected in most normal tissues and tumourcell lines [23], an endogenous origin ofthe pl5E-likc proteins islikely. Moreover., endogenous retroviral sequences reside in thegenome of all the vertebrate species examined so far. includinghumans [24]. Most of these sequences are structurally defectiveand are not correlated with a manifest retroviral infection.However, activation of these sequences, subsequently followedby production of proteins as pl5E. might initiate cell dysfunc-tion.

We favour the view that chronic stimulation of the upperrespiratory tract epithelium, seen in inflammatory and malig-nant processes, triggers the production of pl5E-like proteins.The quenching effect of these proteins on the cellular immuneresponse results in a lower inflammatory reaction. This wouldresult in a reduced eradication of noxious stimuli and thereforean ongoing stimulation, which may in turn result in an increasedproduction of pl5E-like proteins.

Stimulation of the respiratory tract epithelium will alsooccur in healthy persons, hence the presence of pl5E-likcproteins in normal respiratory Iraet epithelium. However, theexpression of p 15E-like proteins in normal epithelium is low andthese proteins could not be detected in serum of healthy persons.Thus, stimulation of respiratory tract epithelium in healthypersons is not sufficient to produce a detectable serum level ofpI5E.

Monitoring pI5E serum levels ean be of diagnostic valuesince decreased levels of pi 5E have heen found after appropriatetherapy, including radical excision of tumour [7] and functionalendoscopic nasal sinus surgery (unpublished data). Further-more, measurement of pi 5E serum levels can be a too! in earlydetection of local recurrence of neopiastic or inflammatorydisease ofthe upper airway traet. In addition, measurement ofp 15E serum levels can also be used in evaluating immunopoten-tiation therapies, as shown by Tas et al. [25]. In this studypatients with chronic, recurrent upper airway infections, refrac-tory to the current therapies, were treated with a thymus extract(TP-l, Serono. Italy) or a placebo; during the TP-1 treatmentperiod ofthe patients, the subjective improvement was accom-panied by a decrease of inhibitory activity of p 15E-like proteinsin their serum.

We have shown that the presence of pl5E-like proteins islimited to the epithelia of the upper respiratory tract. Theproduction of these proteins is triggered by malignant andinflammatory processes and may contribute to immunosuppres-sion. Monitoring p 15E serum levels can be of value in the followup of patients with neopiastic or chronic inflammatory diseaseofthe upper airway tract.

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