Gut 1995; 36: 341-345

Interferon gamma and interleukin 4 secreting cellsin the gastric antrum in Helicobacter pylori positiveand negative gastritis

R Karttunen, T Karttunen, H-P T Ekre, T T MacDonald

AbstractLittle is known of the function of theT cells in the inflammatory infiltrate inHelicobacter pylori associated gastritis.This study thus measured T cell in vivoactivation by enumerating the frequencyof interferon gamma (IFN gamma) andinterleukin 4 (IL 4) secreting cells isolatedfrom the gastric antral mucosa in patientswith or without gastritis and in H pyloripositive and negative gastritis. Fifty foursamples were examined for cytokinesecretion. Four antral biopsy specimensfrom each patient (n=51) were takenduring diagnostic endoscopy. One wasused to estimate histological gastritis andthe presence ofH pylorn, and three of thesamples were used to isolate T cells byenzymatic digestion. IFN gamma and IL 4secreting cells were enumerated byELISPOT. Thirty four samples hadgastritis and 790/0 of those were H pylonipositive. None of the samples from non-inflamed mucosa had H pylorn. Thenumbers ofIFN gamma secreting cells per105 T cells were higher in gastritis than innormal mucosa (145 v 20 IFN gammaspots, p<001), and higher in H pylorinegative than H pylori positive gastritis(371 v 110 IFN gamma spots, p<0.05). Thefrequencies of IL 4 secreting cells did notdiffer between gastritis and non-inflamedmucosa. In conclusion, there is anincrease in IFN gamma secreting cells butnot in IL 4 secreting cells in H pylonipositive and negative gastritis. It is notknown if this TH1 type reaction has apathogenetic or protective role.(Gut 1995; 36: 341-345)

Keywords: Helicobacter pylori, interferon gamma,interleukin 4, gastritis.

Helicobacter pylori causes chronic inflamma-tion, gastritis, which does not generally resolveuntil the bacterium is eradicated by antimicro-bial treatment. Gastric hormone functions areaberrant in this infection, and peptic ulcers,atrophic gastritis, and stomach carcinoma andlymphoma may be longterm consequences ofH pylori infection.'The inflammatory infiltrate in H pylori

associated gastritis consists mainly of mono-nuclear and polymorphonuclear leucocytesbut may include eosinophilic leucocytes,and increased intraepithelial lymphocytes orintestinal metaplasia are sometimes seen.2

Lymphoid follicles often develop. An increasedsecretion of the cytokines tumour necrosisfactor alpha (TNFox), interleukin 6 and 8 hasbeen reported in Hpylori associated gastritis.3 4

There is increasing evidence that the type ofT cell response controls tissue immunopath-ology. Originally based on animal experiments,CD4 positive T cells ('helper' cells) havebeen classified into two main classes: TH 1and TH2, based on differential cytokineresponses,5 for instance TH1 subtype secretesinterleukin 2 (IL 2) and interferon gamma (IFNgamma) whereas IL 4 and IL 5 are TH2products. The biological relevance of thisdistinction has been shown especially in experi-mental animal models for parasitic infectionssuch as schistosomiasis and leishmaniasis whereTH1 cells seem to give protection against thepathogen while TH2 cells exacerbate thedisease.6 The pattern of TH 1 versus TH2activation also explains immunopathology inhuman leprosy as IL 2 and IFN gamma areprominent in tuberculoid leprosy and IL 4, IL5, IL 10 in lepromatous leprosy.7H pylori has been found to stimulate

peripheral blood lymphocytes to proliferateand secrete cytokines IFN gamma and TNFocand the stimulation occurred both in the sensi-tised (=antibody positive) and non-sensitisedpatients. Interestingly, the stimulationresponses were lower in the patients withspecific antibodies.8 9 The results have beenconfirmed in other studies.10- 2We present a hypothesis that the inadequacy

of the immune system to cure H pylori infec-tion might be caused by an incorrect responseelicited, namely an overreaction ofTH2 activa-tion. The finding of a low secretion of IFNgamma in the lymphocyte cultures withH pylori in the antibody positive patients fitsinto this hypothesis.8 It is, however, importantto get information on the actual local T cellactivation process that occurs in the mucosa ingastritis as the in vitro stimulation responses donot always parallel those in vivo. We haveextracted the cells from the biopsy specimensin the patients with and without H pylorigastritis and measured spontaneous cytokinesecretion of the extracted gastric antral cells.By applying a sensitive ELISPOT (enzymelinked immuno spot) method for IFN gammaand IL 4 it was possible to detect cytokinesecretion at a single cell level from a very smallnumber of T cells in biopsy specimens.Choosing IFN gamma and IL 4 as our objectsit was possible to test the hypothesis related toTH1 versus TH2 pattern of the cell mediatedresponse in gastric inflammation.

Department ofPaediatricGastroenterology,St Bartholomew'sHospital, LondonT T MacDonald

Department ofPharmacology 1,Astra-Draco, Lund,SwedenH-P T Ekre

Department ofMedical MicrobiologyR Karttunen

and Pathology,T Karttunen

University of Oulu,Oulu, Finland

Correspondence to:Dr R Karttunen,Department of MedicalMicrobiology, Kajaanintie46E, 90230 Oulu, Finland.

Accepted for publication15 July 1994.

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Methods

PATIENTS AND SAMPLESFifty four samples were collected from 51dyspeptic patients (mean age 56, range 29-83).The patients came to a diagnostic gastroscopyat the Department of Gastroenterology,St Bartholomew's Hospital. The patients withimmunosuppressive therapy or disease, anti-coagulant treatment, antimicrobial treatment,HIV infection or hepatitis, serious heart or lungdisease or earlier stomach resection were

excluded, as well as those who duringendoscopy were found to have candidiasis oftheoesophagus or stomach. Forty nine of thepatients had a single endoscopy while one

patient with normal gastric mucosa had twoendoscopies and specimens taken on both occa-sions. One patient had three endoscopies andtriple therapy against H pylon before the thirdone. The first two samples showed activegastritis and were H pylon positive while thethird sample did not have H pylori and gastritishad become inactive. Four biopsy specimenswere taken with forceps from antrum, fromnormal or inflamed looking mucosa but notfrom erosion site. One specimen was used forhistological examination and three for theextraction of mucosal cells.

HISTOLOGICAL ANALYSISOne antral specimen from each patient wasfixed in formalin and sent to the Department ofPathology, University of Oulu, Finland wherehaematoxylin and eosin and modified Giemsastainings were done. A pathologist (TK)estimated the grade of gastritis (score 0-4)in which score 0 indicated normal (non-inflamed) mucosa, 1 superficial gastritis, 2slight, 3 moderate, and 4 severe atrophicgastritis.13 Score 0 was used to classify thesamples into non-gastritic and scores 1-4 togastritic samples. The gastritic samples were

also classified into inactive (mononuclear cellspresent, polymorphonuclear cells absent) andactive (both cells present). The presence ofH pylori was estimated (positive versusnegative). The histological analysis was donewithout knowing the symptoms of the patients,endoscopy findings or cytokine results.

EXTRACTION OF THE ANTRAL MUCOSAL CELLSThe three samples were first treated withcollagenase (5 mg/ml, Type 1, Sigma) dilutedin RPMI (Flow, Irvine, Scotland) mediumcontaining heparin (5 U/ml), L-glutamine,mercaptoethanol, penicillin, streptomycin,amphotericin B, and gentamycin. The tubeswere incubated at 37°C and vortexedevery 30 minutes. Collagenase was used as theonly enzyme to treat 14 samples and 40 speci-

mens had additional deoxyribonuclease andthermolysin treatment. After four to sixhours, collagenase was washed away anddeoxyribonuclease (2 mg/ml, crude prepara-tion, DN-25, Sigma Chemical, St Louis, MO)and thermolysin (1 mg/ml, T 7902, SigmaChemical, St Louis, MO)14 were added and

the samples were incubated further for 30-60minutes. Incubation was finished when thelarge tissue pieces were dissolved into smallpieces. The overall enzyme treatment took fourto seven hours. Thereafter the cells werewashed three times with RPMI medium. Thecells were suspended in a small volume(250-500 jI) of RPMI medium containing10% fetal calf serum, and the total number ofcells obtained from three specimens werecounted. Two to three cytospin preparationswere made, and kept frozen at -70°C untilimmunostaining.

ENUMERATION OF IFN GAMMA AND IL 4SECRETING CELLSThe mucosal and blood cells were incubatedfor 20 hours in 100 ,l volumes in a humidified5% carbon dioxide atmosphere at 37°C innitrocellulose bottomed microtitre wells(Millipore, Bedford, MA), which had beencoated with a capture antibody. For IFNgamma measurement, two identical wells(about 5X 104 mucosal cells/100 pAL) werecultured without any stimulant (spontaneouscultures) and for some samples, additional twowells with staphylococcal enterotoxin B (SEB)(final concentration 5 pug/ml, Sigma Chemical,St Louis, MO) as a control stimulant.Mononuclear cells from the peripheral bloodsamples of healthy volunteers (n=12) werepurified using Ficoll density gradient centri-fugation and 105 mononuclear cells/well werecultured both with and without SEB stimulantsimultaneously with mucosal cell cultures. ForIL 4 secretion, mucosal cells were culturedin double wells, without any stimulant, tomeasure spontaneous secretion.An earlier described ELISPOT method15

was modified for the mucosal cells as theycontained mucus and cell debris. It was foundnecessary to enhance washing at all phases andnot incubate more than 5-7 X 104 mucosal cellsper well. This made it possible to get ridof artefactual background staining and sodistinguish the true spots. Non-conjugatedmonoclonal antibodies were used as captureand biotin labelled monoclonals to detectsecreted cytokine. IFN gamma antibodies werepurchased from Chromogenix AB, M6lndal,Sweden, and IL 4 antibodies from MabtechAB, Stockholm, Sweden. The wells were firstcoated with a capture antibody (0.7-1Kug/well), diluted in phosphate buffered salineand incubated for three to 24 hours at roomtemperature. The unbound antibody waswashed away with phosphate bufferedsaline. The cells were removed after 20 hourincubation by washing the wells eight timeswith phosphate buffered saline containing0.05% TWEEN 20 (200 gA/well). Biotinlabelled antibody (0 15-0*3 ,ug/well) wasadded and incubated for two to three hoursat room temperature. After six washeswith phosphate buffered saline+TWEEN,streptavidin alkaline phosphatase, in 1:1000dilution (Mabtech AB, Stockholm, Sweden orBio-Rad Laboratories, Hercules, CA, USA),was added and incubated for one to two hours

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Interferon gamma and interleukin 4 in gastritis

Numbers of antral mucosal cells in biopsy specimens (three pieces) in normal and gastritismucosa. Median and range

Normal mucosa (n=20) Gastritis (n=34) p Value

Total cells 382 500 (37 500-680 000) 626 000 (36 000-3 600 000) 0-016T cells 42 220 (2625-126 000) 94 750 (5400-1 044 000) 0-006

at 37°C. The wells were washed six times withbuffer containing TRIS (pH 7.6) and 100 ,ulaliquots ofBCIP/NBT substrate solution (Bio-Rad Laboratories, Hercules, CA, USA) wasadded. The plate was incubated at 37°C untilthe appearance ofdark blue spots (20-40 min).The enzyme reaction was stopped by washingwith tap water. The number of spots/well wereenumerated using a dissection microscope,and a mean of two wells was taken as a result.

ENUMERATION OF T CELLS AMONG THEEXTRACTED ANTRAL MUCOSAL CELLST lymphocyte percentages were estimated oncytospin preparations stained with monoclonalanti- CD3 antibody UCHT-1 and an APAAPmethod. In short, slides were fixed withacetone after which UCHT-1 (a cell linesupematant) was added for 60 minutes atroom temperature. Rabbit antimouseimmunoglobulin (Sigma Chemical, St Louis,MO) in dilution of 1:50 was used as thesecondary antibody and, after removing it bywashing, an APAAP complex (calf alkalinephosphatase coupled with mouse antialkalinephosphatase, Sigma Chemical, St Louis, MO)in dilution of 1:50 was added. The last twosteps were repeated to enhance staining. Afterwashing, BCIP/NBT substrate (Bio-RadLaboratories, Hercules, CA, USA) was added.After a final wash with water, the cells werecounterstained with Mayer's haemalum. Incontrol slides where primary antibody wasreplaced by a TRIS containing buffer, less than1% of cells stained positive. T cell percentageswere used to calculate the absolute numbers ofT cells per well and for three biopsy specimensfrom the known total number of mucosal cells.The numbers of cytokine secreting cells(=spots) were expressed in two ways: per 105T cells (the frequency of secreting cells) andfor three biopsy specimens (total numbers ofsecreting cells).

STATISTICAL ANALYSISThe Mann-Whitney test was used to estimatesignificances of the differences and non-parametric test to estimate correlation betweenthe parameters.

This study was approved by the local ethicscommittee of the Hackney and District Healthauthority.

Results

ENDOSCOPICAL, HISTOLOGICAL, ANDBACTERIAL FINDINGSHistological analysis showed gastritis in 34samples (32 patients, mean age 58, range29-83). These samples had the following

endoscopical findings: normal mucosa (15samples), gastric ulcer (three samples), DU(three samples), duodenal or prepyloricerosion (six samples), stenosis or deformedpylorus (six samples), atrophic gastritis (onesample), and oesophagitis (one sample).Twenty samples had histologically non-inflamed antral mucosa (19 patients, mean age53, range 30-77). Endoscopy was normalexcept for oesophagitis in four samples. A stainfor H pylori gave positive results in 27 of 34samples with gastritis (79%) and in none of thecases with normal mucosa. Inactive gastritis(=increased number of mononuclear cells butno polymorphonuclear cells) was found in 12samples while 22 had active gastritis. Six ofseven samples with H pylori negative gastritisshowed inactive type of gastritis.

NUMBERS OF EXTRACTED ANTRAL CELLS ANDT LYMPHOCYTESTotal cell counts include all cell types, that is,epithelial, mononuclear, and polymorpho-nuclear cells. T cell counts were obtainedusing the total cell counts and T cell percent-ages. More cells and T cells were extracted ingastritis samples compared with normalmucosa (Table).

FREQUENCIES OF IFN GAMMA SECRETING CELLSELISPOT for IFN gamma was performedon 41 samples (1 1 normal, 30 gastritis). Thegastritis samples had a higher frequency ofIFN gamma secreting cells than non-inflamedsamples (145 v 20 spots/105 T cells, p<0-01)(Fig 1). The highest frequency was found inthe patients with H pylori negative gastritis,which differed from that of H pylori positivegastritis (371 v 1 10 spots, p<0 05) (columns3 and 4, Fig 1).The frequencies of IFN gamma secreting

cells tended to be higher in inactive versusactive gastritis (199 v 1 10 spots, p=0.15).The frequencies of mucosal IFN gamma

secreting cells were increased in cultures withSEB versus spontaneous cultures (229 v 110spots, p<0.01; n=31, both normal andgastritis samples included). SEB also stimu-lated peripheral blood mononuclear cells,obtained from the volunteers of the laboratorystaff, to secrete more IFN gamma than theyspontaneously did (101 v 12 spots, p<0.01).

TOTAL NUMBERS OF IFN GAMMA SECRETINGCELLSThe total numbers of IFN gamma secretingcells in biopsy specimens were higher in alltypes of gastritis than the numbers in thenormal mucosa (p<0-01) but there were nodifferences between the different forms ofgastritis (Fig 2).When the gastritis samples were divided into

those with duodenal findings (DU, erosion,pyloric stenosis or deformed pylorus or healedulcer at the region, n= 12) and those without(n= 18), the figures for IFN gamma secretingcells (frequencies and total numbers) did not

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371400 (40-794)

300

0

CL,

E 200 145

E14(0-1110) 110

Z (0-1110)

100

20

(0-140)

0

Normal Gastritis H pylori H pyloripositive negativegastritis gastritis

Figure 1: The frequencies ofIFN gamma secreting cells/O05 T cells isolatedfrom gastricantral biopsy specimens. Samples arefrom patients with normal mucosa (n= 11), gastritis(n=30), H pylori positive gastritis (n=23) or H pylori negative gastritis (n= 7).(Gastritis v normal, p<001; H pylori positive gastritis v normal, p< 0O05; H pylorinegative gastritis v normal, p<0 Ol.) Median and range.

differ between the two groups (data notshown).

IL 4 SECRETING CELLSELISPOT for IL 4 was performed on 26samples (15 normal, 1 1 gastritis). The gastritissamples had a slightly higher frequency of IL 4secreting cells than the normal samples but thedifference was not significant (90 v 40spots/105 T cells, p=0.68). Neither was thereany difference between the total numbers ofIL 4 secreting cells in gastritis versus non-

inflamed samples (32 v12 spots, p=0 25).

DiscussionWe have successfully measured IFN gammaand IL 4 secreting cells in the normal and

400 r

300

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0

0.

CO,

E 200ECOCDzU- 86

(0-1775)100 h

o

79(0-1 775)

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17(0-126)

Normal Gastritis H pyloripositivegastritis

93(54-616)

H pylorinegativegastritis

Figure 2: The total numbers ofIFNgamma secreting cells in cells isolatedfrom gastricantral specimens (three pieces). Samples are the same as in Fig 1. (Gastritis v normal;H pylori positive gastritis v normal; H pylori negative gastritis v normal, p<001.)Median and range.

inflamed gastric antral mucosa. The numbersofIFN gamma secreting cells were increased ingastritis with or without H pylori while thenumbers of IL 4 secreting cells remainedthe same as in the non-inflamed mucosathus pointing to a THi pattern of activation ingastritis. We used the collagenase extractionmethod that has been applied earlier to intesti-nal cells and their cytokine measurements.16The digestion of antral biopsy specimens tookmore time than that of intestinal samples. Theextraction did leave lymphocytes functionallyintact, as IFN gamma secretion was boosted invitro by SEB.ELISPOT method was used to detect

cytokine secreting cells as other methods(ELISA, immunocytochemistry) may not giveas reliable results. There seems to be technicalproblems when immunocytochemistry isapplied to cytokines secreted by T cells, andELISA would also need to work at its lowerdetection level as we are measuring sponta-neous IFN gamma and IL 4 secretion.The frequency ofIFN gamma secreting cells

was increased in gastritis, but the type ofsecreting cell remains unclear. We suggest thatT cells were the secreting cells, as they are themain producers of IFN gamma, but cannotexclude the possibility that some of the cellswere natural killer cells, which can also secreteIFN gamma.17 There are no reports on thefrequency of natural killer cells in the gastriticmucosa.IFN gamma is induced in vivo by antigenic

or superantigenic stimulation and has multipleeffects. It induces HLA class II moleculeexpression on epithelial and endothelial cells.17It decreases epithelial barrier function, but isnot toxic to epithelial cells.'8 19 These twoeffects are especially important with regard togastric mucosa. An enhanced HLA-DRexpression on epithelial cells occurs in Hpyloriassociated gastritis2021 so this is supposinglycaused by local IFN gamma.

Quiding et al14 have reported an averagenumber of 10 IFN gamma spots/105 extractedmucosal mononuclear cells from normal duo-denum, which is comparable with our findingin normal antral mucosa. The higher fre-quency of spontaneous IFN gamma secretingcells in the gastric mucosa than in the blood(145 spots in gastritis; 20 spots in normalmucosa v 12 spots in blood cells) shows thatmucosal lymphocytes tend to be activatedin vivo. Only a few samples from the bloodlymphocytes were measured for spontaneousIL 4 response and the scores were <5/105 cells.It seems from our data that T cells in thestomach mucosa, also with regard to IL 4 (40spots in normal mucosa), are in a higher stateof activation than the blood cells, even if thereis no inflammation. This high figure formucosal cells also shows that extraction wasnot harmful for the capacity of the cells tosecrete IL 4.The frequency ofIFN gamma secreting cells

per 105 T cells was highest in the gastritissamples that were negative for H pylori, eventhough there might actually be H pylori in oneor two cases that were missed by our detection

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Interferon gamma and interleukin 4 in gastritis 345

method, which was based solely on histologicalexamination. On the other hand, six of sevenstaining negative samples had inactive gastritis,which finding correlates with the absence ofH pylori in them. The proportionally highernumber of IFN gamma secreting cells inthese negative samples might be related to theinactive nature of gastritis. On the other hand,the total numbers of secreting cells (in threebiopsy specimens), while being generallyhigher in gastritis than in normal mucosa, weresimilar in different forms of gastritis (H pylornpositive v negative, active v inactive gastritis).The design of our study made it possible toestimate cytokine secretion with regard to thefrequency of secreting cells and also withregard to the total numbers of secretingcells per certain mucosal volume (threebiopsy specimens), which reflects the localaccumulation ofT cells.Our finding of an enhanced 'spontaneous'

IFN gamma secretion in the antrum is seem-ingly in contradiction with the results obtainedwhen blood or mucosal lymphocytes are stimu-lated with H pylori antigen and IFN gammasecretion is measured, which response isdecreased in the patients with Hpylori.8 11 12 Wethink that the results can be explainedon the basis that if the antigen specificlymphocytes are activated in vivo, they maynot respond to further stimulation in vitro.Therefore there is a need to study the immunesystem both by in vivo and in vitro approaches.

In conclusion therefore, we have shownincreased IFN gamma secreting cells in thestomach mucosa in gastritis. Unexpectedly,the highest frequencies were seen in Hl pylorinegative gastritis, thus it is difficult to see whatprecise part H pylori has on the cytokineresponse. In view of the proinflammatoryproperties of IFN gamma, this cytokine maycontribute to the local inflammation in gastri-tis. On the other hand, the presence of IFNgamma in mucosa may have beneficial effects,and our results do not support the hypothesisthat the inability of the immune system toresist H pylori is caused by an overstrong TH2activity.We would like to thank Professor Michael Farthing for permit-ting the samples to be taken at the Department ofGastroenterology, and we express our gratitude to Dr AnnBallinger, Dr Stephen Patchet, Dr Peter Katelaris, Dr FadiMourad, Dr David Gorard, and Dr Charlotte Beaucroft fortheir help and taking the biopsy specimens for the study. Thestudy was supported by Royal Society of Medicine.

1 Blaser MJ. Hypotheses on the pathogenesis and naturalhistory of Helicobacter pylori-induced inflammation.Gastroenterology 1992; 102: 720-7.

2 Heatley RV. Host responses to Helicobacter(Campylobacter) pylori infection. In: MacDonald 'T, ed.Immunology of gastrointestinal disease. London: KluwerAcademic, 1991: 247-62.

3 Crabtree JE, Shallcross TM, Heatley RV, Wyatt JI. Mucosaltumour necrosis factor ot and interleukin-6 in patientswith Helicobacter pylori-associated gastritis. Gut 1991;32: 1473-7.

4 Crabtree JE, Peichl 0, Wyatt JI, Stachl U, Lindley IJD.Gastric interleukin-8 and IgA IL-8 autoantibodies inHelicobacter pylori infection. Scand J Immunol 1993; 37:65-70.

5 Mosmann CNL, Cherwinski H, Bond MW, Giedlin MA,Coffman RL. Two types of murine helper T cell clones.I. Definition according to profiles of lymphokineactivities and secreted proteins. J7 Immunol 1986; 136:2348-57.

6 Sher A, Gazzinelli RT, Oswald IP, Clerici M, Kullberg M,Pearce EJ, et al. Role of T-cell derived cytokines on thedownregulation of immune responses in parasitic andretroviral infection. Immunol Rev 1992; 127: 183-204.

7 Yamamura M, Uyemura K, Deans RJ, Weinberg K, ReaTH, Bloom BR, et al. Defining protective responses topathogens: cytokine profiles in leprosy lesion. Science1991; 254: 277-9.

8 Karttunen R, Andersson G, Poikonen K, Kosunen TU,Karttunen T, Juutinen K, et al. Helicobacter pyloriinduces lymphocyte activation in peripheral blood cul-tures. Clin Exp Immunol 1990; 82: 485-8.

9 Karttunen R. Blood lymphocyte proliferation, cytokinesecretion and appearance ofT cells with activation surfacemarkers in cultures with Helicobacter pylori. Clin ExpImmunol 1991; 83: 396-400.

10 Hatz RA, Brooks WP, Andress H-J, Lange V, Krlmling H-J. Specific suppression of the immune response of periph-eral blood lymphocytes in Helicobacter pylori infection.Digestion 1991; 49: 18-9.

11 Shahi CN, Fou XJ, McDevitt T. O'Connell M, Weir DG,Keeling PW. Defective antigen-specific responses toHelicobacter pylori antigens in patients with gastric Hpylori colonization. Irish J Med Science 1992; 161 (suppl10): 54.

12 Fan SJ, Chua A, Shahi CN, O'Farrelly C, Keeling PWN,Kelleger D. Antigen-specific gastric lymphocyte responsesto patients with gastric H pylori infection and IFNy pro-duction. Gastroenterology 1993; 104: A698.

13 Karttunen T, Niemela S, Lehtola J. Helicobacter pyloriin dyspeptic patients: quantitative association with sever-ity of gastritis, intragastric pH, and serum gastrin concen-tration. Scand J Gastroenterol 1991; 26 (suppl 186):124-34.

14 Quiding M, Nordstr6m I, Kilander A, Andersson G,Hanson iA, Holmgren J, et al. Intestinal immuneresponse in humans. Oral cholera vaccination inducesstrong intestinal antibody responses and interferon-y pro-duction and evokes local immunological memory. J ClinInvest 1991; 88: 143-8.

15 Kabilan L, Andersson G, Lolli F, Ekre H-P, Olsson T,Troye-Blomber M. Detection of intracellular expressionand secretion of interferon-y at the single-cell level afteractivation of human T cells with tetanus toxoid in vitro.EurJl1mmunol 1990; 20: 1085-9.

16 Breese E, Braegger CP, Corrigan CJ, Walker-Smith JA,MacDonald TT. Interleukin-2 and interferon-y-secretingT cells in normal and diseased human intestinal mucosa.Immunology 1993; 78: 127-31.

17 Trinchieri G, Perussia B. Immune interferon: a pleiotropiclymphokine with multiple effects. Immunol Today 1985; 6:131-6.

18 Madara IL, Stafford J. Interferon-y directly affects barrierfunction of cultured intestinal epithelial monolayers.Jf Clin Invest 1989; 83: 724-7.

19 Adams RB, Planchon SM, Roche JK. IFN-y modulationof epithelial barrier function. Time course, reversibility,and site of cytokine binding. J7 Immunol 1993; 150:2356-63.

20 Engstrand L, Scheynius A, Pahlson C, Grimelius L,Schwan A, Gustavsson S. Association of Campylobacterpylori with induced expression of class II transplantationantigens on gastric epithelial cells. Infect Immun 1989; 57:827-32.

21 Valnes K, Huitfeldt HS, Brandtzaeg P. Relation betweenT cell number and epithelial HLA class II expressionquantified by image analysis in normal and inflamedhuman gastric mucosa. Gut 1990; 31: 647-52.

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