Immunohistochemical Changes in MorphologicallyInvolved and Uninvolved ColonicMucosa of Patients with Idiopathic Proctitis

KIRON M. DAS, WILLIAM F. ERBER, and ARYERUBINSTEIN

From the Division of Gastrointestinal and Liver Disease, Department of Medicine, Cell Biology,and Pediatrics, Albert Einstein College of Medicine, Bronx, New York 10461

A B S T RA C T Alterations in secretory component,IgA, IgG, and IgM were studied by immunofluorescenttechniques in mucosal biopsy specimens obtainedat colonoscopy from inflamed and grossly uninvolvedcolonic mucosa from 12 patients with idiopathic proc-titis. Parotid-salivary secretory component and IgAand serum immunoglobulins were also investigated.Decreased secretory IgA was observed in the epi-thelium of all grossly involved rectal mucosa and in40% of proximal normal mucosa. Salivary secretoryIgA was not diminished. These observations suggestthat a local immune defect may be pathogeneticallyrelated to idiopathic proctitis.

INTRODUCTION

Systemic and local immunologic abnormalities havebeen described in ulcerative colitis and Crohn'sdisease (1-10); however, data regarding the localimmunologic factors are conflicting. Immunologicabnormalities have also been reported in idiopathicproctitis (11), a localized form of inflammatory boweldisease (12). Although abnormalities in IgA in colonicmucosa occur (5-8), the status of colonic mucosalsecretory IgA, which is the predominant local anti-body (13, 14), has not been established in idiopathicproctitis. The present study examines secretory IgAin histologically involved as well as in uninvolvedmucosa in an attempt to clarify the possible patho-genetic role of this system in indiopathic proctitis

A preliminary report of this material was presented, inpart, at the Meeting of the Eastern Gut Club, New York,in March 1976 and in abstract form in 1975. Gastroenterology.68: 880. (Abstr.)

Dr. Das is the recipient of a Clinical Investigator Awardfrom the National Institutes of Health, IK08 AM00237.

Received for publication 16 July 1976 and in revisedform 16 November 1976.

which is considered to be a forerunner of ulcerativecolitis (12, 15, 16). For comparison between colonicand extraintestinal S-IgA system, salivary S-IgA in thesame group of patients was also investigated.

METHODSPatients. The untreated patients with idiopathic proctitis

(aged 20-55 yr, mean 32 yr; 11 female, 1 male) verifiedby sigmoidoscopy had a normal mucosa on barium enemaexaminations. All the patients had mild to moderate diseaseon the basis of number of bowel movements, amount ofblood and mucus in the stool, and sigmoidoscopic appear-ance. The duration of the disease ranged from 3 wk toseveral mo before the present episode. After initial in-vestigations, all patients were colonoscoped while theywere symptomatic to determine the extent of the lesion andto obtain multiple mucosal biopsy specimens. Endoscopicmucosal disease was localized to the rectum (up to 15-20 cm from the anus) (12, 15). In no case did the diseaseextend endoscopically beyond the rectum. All patientswere subsequently treated with hydrocortisone enemas(Cortenema, Rowell Laboratories, Inc., Baudette, Minn.)with or without salicylazosulfapyridine (SAS-500, RowellLaboratories, Inc.; Azulfidine, Pharmnacia Fine Chemicals,Inc., Piscataway, N. J.). Nine additional patients in whomcolonoscopy was required for a workup of diarrheal prob-lems unrelated to idiopathic proctitis or any other inflamma-tory bowel disease served as controls. In addition, biopsyspecimens were obtained at colonoscopy in one patient withclindamycin-associated colitis and another patient withgonococcal proctitis. Informed consent was obtained from allpatients before colonscopy.

Biopsy specimens. Colonscopic mucosal biopsies wereobtained from rectum, sigmoid, or descending colon andtransverse colon in each patient and included macroscopi-cally involved and uninvolved mucosa. Similar biopsy speci-mens were obtained from the control subjects. Specimens forimmunofluorescent studies were immediately placed in iso-pentane, then in liquid nitrogen and stored at -70°C for amaximum period of 6-8 wk. The biopsy specimens weremounted on a block, embedded in OCTcompound (AmesCo., Div. of Miles Lab., Inc., Elkhart, Ind.) and seriallysectioned at 4 iLm with a cryostat at -20°C. Parallelspecimens were fixed in formalin for light microscopy.

The Journal of Clinical Investigation Volume 59 March 1977 379-385 379

Collection of parotid saliva. Parotid saliva was col-lected for 30 min from 7 of the 12 patients with idiopathicproctitis and from 10 control subjects by uitilizing specialcups to fit duct openings while stimulating secretion withcitric acid. Saliva specimens were dialyzed and concen-trated 25-fold by freeze drying. Presence of secretory IgA,free and bound secretory component, was verified bydouble diffusion in agar gel with anti-IgA and anti-free andanti-bound secretory component sera with control salivasand colostrum IgA as standards (17). Quantitative deter-minations of salivary secretory IgA was determined byradial immunodiffusion and compared to 10 controls (IgA:range 0.56-2.2 mg/100 ml; meean valuie 1.4 mg/100 ml).Electrophoretic mobility of the patienit's salivary proteinswas determined by microzone electrophoresis (BeckmanInstruments, Inc., Fullerton, Calif) on cellulose acetate mem-brane (18). Salivary proteins of the patients were comparedwith three nonrmal control saliva specimens, secretory IgA,secretory component, and lactoferrin p1urified by uis fromhuman colostrum (19) and myeloma IgA. Although this isan insensitive technique to (quantitate salivary proteins, itwas found adequate to compare their electrophoretic mobilityto normal controls.

Immunofluorescent methods

Antisera. Rabbit antihumilcani IgA, IgG, IgM, f,3C, andsecretory component unconjuigated and (or) con-jugated withfluorescein isothiocyanate were obtaiined from Behring Diag-nostics, American Hoechst Corp., Somerville, N. J. Rhoda-mine-conjugatedl IgG goat antirabbit gamimiia globtilin wasobtained froIn Cappel Laboratory, Downingtown, Pa. Xlono-specificity of antisera was screened by imnillltiioelectro-phoresis before use. Two types of antisera to secretory com-ponent were used, one that reacted only with free secre-tory component (Behring Diagniosties) and one that reactedin addition with colostral IgA. Each batch of antiserumiii wastested in douible diffuisioni in agar againist SC anid S-IgAisolated by us from human colostrumil (19). The ptirity ofSC and S-IgA pturified by uis was compared to referencesamples (kindly stupplied by Doctors D. Goodenberger andW. Strober, National Cancer Institute, National Instittutes ofHealth, Bethesda, Md.), by uising SDS acrylamide gels,double diffusion in agar, and i mmunoelectrophoresis setups.An antifree SC antiserum was defined as that reacting tofree SC and not to S-IgA. The antibound SC reacted withboth S-IgA and free SC. Anti-IgA was tested in immuino-electrophoresis and double diffusion in agar not to crossreact to SC or other salivary protein except for IgA.

Direct techriique. Cryostat sections were fixed in 95%ethanol, washed with phosphate-buffered saline (0.1 M, pH7.2) for 5 min, anid stained with fluoresceini-con-jugated anti-sera to IgG, IgM, IgA, andl ,1C. To remove any aggregatedimmunoglobulin which may be presenit in the antisera andthat canl react with Fe receptors every fluorescent anti-serumLi was ultracentrifuiged iminediately before its use.After 1 h of incubation in a Imloist chamber, slides were rinsedwith buffer and washed twice in buffer for 10 min. Sec-tions were dried and sealed using elvanol and cover glass.Specificity of the staining of each conjugate in the directtechni(lue was confirmed by incubating parallel sectionsfirst with unconjugated antisera to IgA, IgG, IgM, washingin buffered saline and then adding the corresponding con-jugated anti-immunoglobulin for an additional 20 min.

Indirect techni(que. Sections were processed as in thedirect technique and were incubated vith unconjugated anti-sera to human free secretory component followed by a vash

380 K. M. Das, IV. F. Erber, antd A. Ruibitnsteint

in phosphate-buffered saline. Sections were then stained withrhodamine-conjugated IgG goat antirabbit gamma globulinand incubated for 1 h in a moist chamber. Slides wererinsed, washed with buffer, and mounted as above.

Unstained sections mouinted in buffered glycerin weretused to estimate autofluorescence. A parallel section wasincubated with conjugated antisera as control for non-specific fluoreseence. The specificity of the antifree SCwas tested by preincubationi and precipitation of the anti-free SC with free SC followed by the use of the stupernateof the antiserum for immuiiinofluiorescent staining. No immuno-fluiorescence for SC was obtained by this experimenital setuip. Serial sections were also staine(d with hemiiatoxylinand eosin.

Fluiorescentt inicroscopyj. A Zeiss microseope (Carl Zeiss,Inc., New York) equipped with vertical illuminator HBO200 WMerecury lamiip, fluioreseeini isothiocyanate and KP-600exciter filters, FL-500 anid 580 reflectors, 53- anid 44-barrier filters were uised for fluorescein isothioeyanate andrhodamine stainings, respectively. Photographs weretaken with Kodak Ektachrome film ASA 160 (Eastman KodakCo., Rochester, N.Y.).

Sections were examined witlh 400 x 900 x magnifica-tion. 30 sections from each biopsy specimen were stainedwith different antisera. Immunofluorescence of mucosalglandular epithelia was quantitated by utilizing a 0 to+++ scale: 0, no fluoreseence; +, minimal fluiorescencedetected only after close observation; + +, clear-ecut fluiores-cent pattern of epithelial suibeellular struietures; + + +, diffuiseband of fluoreseeinee of epithelia. The scale of 0 or + wasconsidered as diminished and ++ to +++ was consideredto be normal. The slides were examinied douible blind bythree of uis anid individually scored. The suiperficial colono-scopic muiticosal biopsy specimiienis uisedi did niot enable uisto (Iquantitate the iillmmunocytes in the lamiiina propriai.

RESULTS

Lighlt indicroscopiJ. Biops specim ens from sites ofobvious lesionis seeni by colonoscopy showved at leastthree or miiore of the following histologic abnormiialities:incireased inflamiimatorv cells (ineluidiing neuitropllils)in the lamina propria, glanidtular necrosis, tulceratioins,crypt abscesses, depletion of goblet cells, and miiucosalatrophy. Mucosal atrophyv was judged by the lengthof crypts and the density of glands as compared withthat seen in niormiial controls. Biopsy specimiens fromproximal colonic mucosa of normiial appearaince werehiistologically normiial in 7 out of 12 patienits. In theremiiainiing five, the iut'cosa of the sigmoid colon re-vealed atrophy, depletion of goblet cells, excesschron-ic inflaimmatory cells, and, in one patieint, cryptabscesses. Two of the five patienits had these mor-plhological abnormalities in the biopsy specimiienis takenfrom the transverse colon. The detailed morphologyis being reported elsewlhere (20).

Fluiorescentt microscopil. In all sections fromi thenine control subjects, secretory component appearedas a dense bland at the apices (+++) and in smallquantity (+ +) at the basal membrane and intercellutlarareas of glanduilar epithelia (Figs. 1A and 2A). Secre-tory componenit was not seen in the lamina propria.

The patients with clindamycin-associated colitis andgonococcal proctitis showed immunohistochemicalpatterns similar to those of control subjects.

The immunohistochemical findings at various levelsof colon in the 12 patients with idiopathic proctitisare shown in Table I. All specimens obtained fromgrossly involved areas showed a significant decreaseof secretory component (O or +). Apical epithelialfluorescence of secretory component appeared as avery irregular line compared with the homogenousband of staining observed in control specimens. 6of the 12 patients showed decreased (+) epithelialsecretory component in grossly uninvolved areas ofthe transverse colon (Figs. 1B and 2B). Two of these

|4

FIGURE 1 Immunohistochemical localization of secretorycomponent (SC) in glandular epithelium of comparablefields from sections of the alcohol-fixed biopsy specimenstaken from the mucosa of the transverse colon: (A) from acontrol subject. There is intense staining for SC in the luminalside (L) of the glandular columner epithelium. Note thatSC is present also along the cell membranes includingthe basement membrane areas (B) from a patient with idio-pathic proctitis. Note the marked diminution of staining ofSC at the luminal aspect (L) of the glandular columnerepithelium and the staining is practically nil along the cellmembranes including the basal areas (x400).

The distribution of IgA was similar to that of secre-tory component and was markedly dense (+ + +)(Fig. 3A). Plasma cells in the lamina propria con-tained primarily IgA. No epithelial fluorescence wasseen on staining for IgG and ,31C. There was minimalepithelial fluorescence when staining for IgM (Fig. 4).

FIGURE 2 Immunohistochemical localization of SC. Highermagnification of the same specimens as in Fig. 1: (A)from a control subject and (B) from a patient with idiopathicproctitis (x900). L indicates the lumen of the gland.

Mucosal S-IgA System in Idiopathic Proctitis 381

FIGURE 3 Imtmunohistochemical localization of IgA in theglandular epithelium in comparable parts of the biopsyspecimens taken from the mucosa of the transverse colon:(A) from a control subject, the distribution and intensityof staining is more or less the same as for secretory com-ponent; (B) from a patient with idiopathic proctitis, notethe marked diminution of staining for IgA both at the luiminalaspect (L) and along the cell membranes (x400).

six patients had histological abnormalities in thesesame grossly normal areas.

When stained with anti-IgA, sections from areasshowing gross and histological proctitis revealed sig-nificant diminution of epithelial fluorescence (0 to +)in 8 of the 12 patients (Table I). Epithelial fluores-cence for IgA in the proximal uninvolved colonicmucosa was also significantly diminished (0 to +) in5 of the 12 patients (Fig. 3B and Table I).

Anti-IgM sera did not give any staining above +in the biopsy specimens of nine patients or in any of

the control subjects. In three patients, the IgM staini-ing was, however, increased. IgG staininig in grosslyinvolved mucosal specimens appeared as spicules inthe lamnina propria, and distinct localization withinlplasma cells was lacking. In no case was IgG foMindwithin muciosal epithelial cells. Three of six patienitsstudied demonstrated faint epithelial staininlg for81C whiclh was absent in controls.

Sermin and salivary immunoglobuilins a ud secretorycomponent (Table II). Serum IgA deviatedl f'rom thenormal controls only in three patients: 350, 380, anid75 mg/100 ml (normal range = 77-257 mng/10() ml).Serumn IgG and IgM were essentially normiial.

Salivary IgA was slightly elevated in two patielitscompared to adult controls. Patient's parotid salivarytotal secretory component gave precipitationi linlessimilar to normals by the double immulnodifhlisiointest. Saliva was serially diluted to the end pointwhere no precipitation line could be detectedl andclthen compared to the controls. The mobility on imiero-zone electrophoresis of patients' salivary IgA and( secre-tory component was comparable to that of controlsaliva, purified colostral IgA, and purified colostralsecretory component.

DISCUSSION

The pathogenesis of inflammatory bowel disease isstill enigmatic. Several immunologic abnorm-alitieshave been described during the course of inflaimmatorybowel disease, none of which could be clearly de-

FIGURE 4 Immunohistochemical localization of IgM in the glandular epithelium of colonicbiopsy specimen from a control subject. Minimal staining was noticed mainly along theluminal aspect (L) of the epithelial cells.

382 K. M. Das, W. F. Erber, and A. Rubinstein

q

TABLE IIninn111tnlObistocl1einical Changes at Different Levels of

Colotnic Mftcosal Epitheli mfrom Patientswcith Idiopathic Proctitis

SC IgA 1gM 8C,

Nunm}ber of patienits 12 12 12 6Colonic level of biopsy

fromn anal verge<20 cim (rectumn)* 4 12 4 8 t 3 T 3>20 < 40 cm11 (sigm-noid

colonl)* 4 8 46 T 3 T 2>40 cmil (descending

and transverse colon)* 4 6 1 5 N N

The figuires indicate the number of the patients with abnor-malities as indicated by arrows. The findings in the rest ofthe patients wvere within normal limits. 1, increased; 1, de-creased; N, normiial in all.

fined as primiiary. In this sttudy, we attempted to de-fine the role of the secretory IgA system in idiopathicproctitis.

Local muvcosal immlutne abnormalities are fre(quentlyassociated wvith clhronic inflmmation and auitoimmuine(lisease (13, 14, 21, 22). Suich abnormalities especiallythose of the secretory IgA system may occuir seconidarilyto actute or chronic infections (23). Previouis reportsregardinig the colonic IgA system in inflmmnatory boweldisease were coniflicting (3, 5-8, 10); in uilcerativecolitis the cleinsity of IgA positive lymphocytes is de-creased in the rectal mutcosa (3). In some instanceswhere IgA immulllnocytes were normal or even in-creased (5-8), a paradoxical depletion of glaniduilarIgA occuirred (7). Stuch a depletion of epithelial secre-tory IgA wvas reported to be restricted only to histo-logically involved areas obtained at colectomny frompatients with Crohn's disease (10). These observedvariations inl distribtution of colonic IgA raise the(Iliestion as to whether they are primary or secondaryto epithelial (lestruietion. 'We approached this quiestionby uitilizing histologically normal and abnormal colonicmutcosa obtained during colonoscopy from the samepatient. Since previouis sttudies have revealed histologi-cal abnormalities in proximal macroscopically normalcolon (20), "uiininvolved areas" were strictly definedas those whiclh were not only macroscopically butalso histologically normal. Any abnormality in thesecretory IgA system in uninvolved areas could bemore likely a primary disorder. To further minimizeartifactual immulllnofluorescent abnormalities presentin advanced disease, only uintreated patients in theinitial phase of idiopathic proctitis, an inflammatorydisease of predominantly restricted extension, wereincluded. The sttudy showed that in patients withidiopathic proctitis, secretory IgA and free secretory

component were markedly diminished, not only inareas of histologic involvement btut also in histo-logically uninvolved areas in about onie-third of thepatients.

The meaning of this local S-IgA deficiency is specuila-tive at this stage. A certain concentration of free secre-tory component is important in maintaining the carriertransport milieui for IgA (24). Its lack (i.e., a localimmutne deficiency) couild be implicated as a cauisativefactor and may precede proximal spread of the diseaseleading to generalized colitis in the patients withidiopathic proctitis. This finding was also comparedwith other extraintestinal sites of the secretory IgAsystem and with sertum IgA levels. Seruim and salivaryIgA were, however, not significantly diminished inany of the stuldied patients (Table II). Stuch a dissocia-tion between the abnormal colonic IgA and thenormal sertum and salivary IgA system is uintustual. Aselective global secretory piece deficiency (22) aswell as absent parotid and normal gtut IgA (25) de-ficient senim IgA with normal guit IgA (26) havebeen reported. In our patients, however, the reversesittuation was observed, namely a depletion of colonicsecretory IgA with normal salivary and serum IgA.This cannot be explained on the basis of the phylo-genetic and ontogenetic developmenit of the IgAsystem and thuis raises the alternative possibility thata local exogenouis toxic process may have depletedthe epithelial secretory IgA even before any histologicchanges were noted. In support of this concept arerecent observations that grantulomas can be elicited infootpads of mice by injection of homogenates fromdiseased bowel tissuie with Crohn's disease as well asfrom histologically normal resection margins (27).Moreover, in Crohn's disease, increased gluicosamninesynthetase activity was noticed in apparently normalrectal biopsy specimens (28). The latter data indicate

TABLE IISalivary and Serum IgA in Patienits wvith

Idiopathic Proctitis

Parotid saliva SeniuIgA IgA

ilng/loo10 11 ng/loo nl

Idiopathic proctitis 1.2 350(n = 7) 1.3 75

0.09 3801.8 1581.3 1583.2 2052.02 165

Normal range(n = 10) 0.56-2.2 77-257

Mucosal S-IgA System in Idiopathic Proctitis 383

that a "toxic agent" or a pathological process may bepresent in tissues long before morphological ab-normalities are detectable.

Abnormal systemic and local cellular immunity mayalso contribute to failure of the secretory IgA system.Preliminary studies in our patients failed to reveal amajor deficiency in systemic cellular immunity (uin-published data). Secretory component may be presentin two distinct immunochemical and physicochemicalforms in rats (29). Our initial studies by microzoneelectrophoresis could not, however, detect an ab-normal electrophoretic mobility of secretory IgA orsecretory piece from patients' saliva. Further analysisof secretory IgA isolated from mucosal secretions isnecessary to examine the role of breakdown of hostdefenses in the pathogenesis of inflammatory boweldisease. Abnormalities in glycosylation of secretorycomponent or in polymerization, assembly, or bindingof IgA to secretory component may result in a bio-logically deficient secretory IgA which escapes detec-tion by the immunofluorescence techniques employed.

ACKNOWLEDGMENTS

The authors wish to acknowledge the invaluable help thatBoyce Bennet, Professor of Pathology, and Rachel Morecki,Associate Professor of Pathology, Albert Einstein College ofMedicine, gave in critical interpretation of immunohisto-chemical and morphological findings. We are also gratefulto Irwin M. Arias, Professor of Medicine, for helpful criticismsin the preparation of this manuscript.

This investigation was supported by grants Al 10702 andAM05384 from the National Institutes of Health and by agrant from the Rowell Laboratories, Inc.

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Mucosal S-IgA System in Idiopathic Proctitis 385