Vol. 30, No. 8JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 2013-20180095-1137/92/082013-06$02.00/0Copyright X 1992, American Society for Microbiology

Immunoglobulin A (IgA) and IgG Serum Antibodies toMycobacterial Antigens in Crohn's Disease

Patients and Their RelativesLAWRENCE G. WAYNE,'* DANIEL HOLLANDER,2 BEATRIZ ANDERSON,1 HILDA A. SRAMEK,1

CONSTANCE M. VADHEIM,3 AND JEROME I. ROTrER3Tuberculosis Research Laboratory, Department of Veterans Affairs Medical Center, Long Beach, California908221; Gastroenterology Division, Department ofMedicine, California College of Medicine, University of

California, Irvine, California 927172; and Division ofMedical Genetics, Departments ofMedicine andPediatrics, Cedars Sinai Medical Center, University of California, Los Angeles, California 900483

Received 4 February 1992/Accepted 18 May 1992

Sera from patients with Crohn's disease, their relatives, their spouses, and unrelated healthy controls wereassayed by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) and IgA antibodies toMycobacterium tuberculosis, M. avium, and M. gordonae. The patients had significantly higher IgA responsesto mycobacterial antigens than did either their relatives or the controls. On the other hand, both the patientsand their relatives had significantly higher IgG responses against these antigens than did the controls. Theelevated IgA response was more pronounced against isopentanol-extracted whole bacterial cells than it wasagainst soluble protein extracts, and it appeared to be directed against fixed surface antigens that lie under theloosely bound peptidoglycolipid or glycolipid antigens of mycobacteria.

An etiologic role for mycobacteria in Crohn's disease haslong been the subject of study and speculation. Interest inthis possibility was stimulated by recent reports of theisolation of mycobactin-dependent mycobacteria from theintestinal tissues of patients with Crohn's disease (2, 3).These organisms have been shown by nucleic acid analysesto be indistinguishable from Mycobacterium paratuberculo-sis (15, 33), the agent of Johne's disease, a granulomatousenteritis of ruminants. However, these mycobactin-depen-dent strains have been isolated from only a small proportionof human bowel specimens examined, and evidence for thepresence of a variety of other mycobacterial species hasbeen found in the tissues of patients with Crohn's disease (6,8, 34). This calls into question the specificity of the possibleetiologic role of these bacteria and suggests that such a role,if it does exist, may belong to environmental mycobacteria,in general, rather than to a single species.With this in mind, we undertook a serologic survey of

patients with Crohn's disease, as well as their blood rela-tives, their spouses, and unrelated control subjects. The serawere examined for antibodies of both the immunoglobulin A(IgA) and IgG types to whole cells of three species ofmycobacteria from which the loose outer envelope of sero-var-specific antigen (1) was removed and to crude proteinextracts of sonically disrupted mycobacteria. The mycobac-teria selected for study were the pathogen M. tuberculosis,which is not found free in the environment; M. avium, whichis an environmental opportunistic pathogen; and M. gordo-nae, which is a common environmental organism that israrely, if ever, the cause of disease (25, 28).

MATERIALS AND METHODS

Antigens used. Most of the studies were done with whole-cell antigens prepared from M. tuberculosis H37Rv, M.avium SJB-2, M. intracellulare TMC 1403, and M. gordonae

* Corresponding author.

TMC 1324, all of which are maintained in the Long Beachlaboratory collection. Limited additional studies were alsodone with M. paratuberculosis Linda, which was providedby Roderic Chiodini, and strains of M. avium and M.intracellulare representing various serovars, which wereprovided by Anna Tsang. The mycobacteria were grown inDubos broth base (Difco Laboratories, Detroit, Mich.) con-taining 1% (wt/vol) glycerol and were enriched with Dubosmedium albumin (Difco) for M. tuberculosis and M. gordo-nae or with Dubos oleic albumin complex (Difco) for M.avium and M. intracellulare. For growth of M. paratuber-culosis, the medium was also supplemented with 2 ,ug ofmycobactin J (Allied Laboratories, Ames, Iowa) per ml. Themethods used to grow and harvest the mycobacteria havebeen described previously (27).Two types of antigens were used, isopentanol-extracted

formalin-fixed whole cells and crude, protein-enriched sonicextracts of the bacilli. Salcedo et al. (21) reported thatisopentanol can extract polar lipids such as nonionic deter-gents from protein solutions without denaturing or otherwisedamaging the proteins. We used this solvent to remove theloosely bound serovar-specific polar peptidoglycolipid cellwall sheaths (1) and to expose the fixed cell surfaces of themycobacteria for use as whole-cell antigens. Samples of 200,ul of 10% (vol/vol) aqueous suspensions of formalin-fixedmycobacterial cells were mixed vigorously with 10 ml ofisopentanol and centrifuged (900 x g, 30 min). The supema-tant solvent was discarded, and the isopentanol-treated cellswere washed once in 5 ml of aqueous 0.02% Tween 80 andresuspended in water to a concentration of approximately 10mg of moist cells per ml. The method for preparation ofcrude protein-enriched sonicates has been described previ-ously (27).One study was carried out with baker's yeast (Saccharo-

myces cerevesiae ATCC 7754), which was provided by RuthRussell. The yeasts were grown and the whole cells werefixed for use as an antigen as described by Main et al. (14).Human sera. Sera from the following categories of human

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subjects were examined (the average + standard deviationage in years is given for each group): patients with Crohn'sdisease (43.5 + 16.5), blood relatives of patients withCrohn's disease (45.9 + 18.0), spouses of patients withCrohn's disease (48.0 + 13.8), and normal Red Cross blooddonors and other unrelated control subjects (42.6 + 16.1).The criteria for inclusion of patients with Crohn's diseaseand their relatives were the same as those described for priorstudies (10, 12). Sera and clinical information were collectedunder the guidelines of the human studies committees of ourrespective institutions, and the privacy of the informationwas protected.

Rabbit sera. Antisera raised against unextracted cells ofM. avium complex agglutination serovars 1 to 9, 12, 14, 15,18 to 20, 23, and 25 were provided by Anna Tsang. The seraagainst unextracted cells of M. paratuberculosis Linda wereraised in the Long Beach laboratory by methods describedby Good and Beam (7).ELISAs. For enzyme-linked immunosorbent assays

(ELISAs) with isopentanol-treated whole-cell antigens, thesuspensions were treated briefly in a low-energy Bransonic 3cleaning bath (Heat Systems-Ultrasonics, Farmingdale,N.Y.) to disrupt clumps without breaking the cells. Forty-microliter volumes of the cell suspensions were dispensed towells of flat-bottom NUNC II ELISA plates (Vangard Inter-national, Neptune, N.J.) and were incubated overnight (un-covered) at 37°C to dry. On the following day, 50 ,ul ofphosphate-buffered saline, (pH 7.5) containing 100 ,ug ofthimerosal per ml (PBSM) and 50 Ru of 0.05 M bicarbonatebuffer (pH 9.4) were added to all wells and the plates wereincubated at room temperature for 30 min. The buffer wasthen aspirated, and the wells were blocked for 2 h at 37°Cwith 2% normal equine serum in PBSM. The plates werewashed with a solution of 200 ,ug of bovine serum albuminper ml in PBSM and could be used immediately or after drystorage at 5°C for 2 weeks or more. For tests with crudeprotein antigens, the wells of the plates were primed over-night with 50 ,ul of a solution containing 40 ,ug of the desiredprotein per ml in PBSM and 50 ,Il of 0.05 M bicarbonatebuffer (pH 9.4).

For human antibody assays, 100-,ul samples of the testsubjects' sera, diluted 1:100 (for IgG) or 1:20 (for IgA) inPBSM, were dispensed to wells coated with the selectedantigens and to a blocked well without antigen. After 2 h ofincubation at room temperature, the serum was aspiratedand the wells were washed three times with 0.05% Tween 20in normal saline and once in saline without Tween 20. Onehundred microliters of second antibody, peroxidase-conju-gated immunoaffinity-purified goat anti-human IgG or IgA(Sigma Chemical Co., St. Louis, Mo.) diluted 1:400 in 10%equine serum in PBSM, was dispensed to each of the testwells, and the wells were incubated at room temperature for2 h. After aspiration of the second antibody, the wells werewashed four times with Tween 20-saline and once withPBSM. After the addition of 200 ,ul of a freshly preparedsolution of 0.33 mM 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) and 0.34 mM H202 in 0.1 M acetatebuffer (pH 4.6) to each well, the plates were incubated in thedark for 2 h at room temperature. The reaction was stoppedwith 10 ,ul of 1 M oxalic acid, and the optical A405 wasmeasured on a model EL307 ELISA reader (Bio-Tek Instru-ments, Burlington, Vt.).For assay of rabbit antibodies, the procedure was per-

formed as described above, except that the dilutions ofantibody to the different serovars were based on their knownagglutinating titers, and the second antibody was peroxi-

dase-conjugated goat anti-rabbit IgG (ICN Immunobiologi-cals, Lisle, Ill.).The readings for each specimen were corrected for non-

specific background by subtracting the A405 of the corre-sponding antigen-free well from those of the antigen-primedwells. When theA405 of a sample was off scale on the reader,i.e., greater than 2.0, half of the sample in the well wastransferred to an empty well; the sum of the A405 of theresidual fluid in the original well and that of the transferredaliquot represented the ELISA score for that sample.The commercial goat anti-human and anti-rabbit globulins

that were used as peroxidase conjugates for second antibod-ies were produced with Freund's adjuvant, which containskilled mycobacteria. To ensure that false-positive reactionswere not produced in our tests by antimycobacterial anti-bodies in these conjugates, control wells in the ELISA plateswere primed with the mycobacterial antigens and challengedwith the second antibody without exposure to human orrabbit serum. No reactions with A405 values higher thanthose of the substrate blanks were seen between the antigensand the second antibodies with the reagent concentrationsused for these tests.

Statistical tests. Statistical significances of the differencesbetween distributions of raw scores for serum among thehuman subjects were determined by the nonparametricrank-order Mann-Whitney U test (22). Correlation coeffi-cients were calculated with the Sigmaplot version 3.10program (Jandel Scientific, Corte Madera, Calif.).

RESULTS

In a preliminary experiment (data not shown), suspensionsof unextracted cells of a strain each of M. avium complexagglutination serovars 1 to 9, 12, 14, 15, 18 to 20, 23, and 25and of M. paratuberculosis Linda were dotted onto nitrocel-lulose membranes and tested against homologous and heter-ologous rabbit antisera by using peroxidase-conjugated goatanti-rabbit IgG and hydrogen peroxide-diaminobenzidinereagent (24) to detect antibody binding. None of the antiserato the M. avium complex serovars gave positive reactions tothe M. paratuberculosis cells, but antisera to M. paratuber-culosis appeared to react as strongly to the cells of the otherselected strains as they did to their homologous strain. Thissuggested that the dominant antibody in the anti-M. paratu-berculosis serum is to an underlying nonpeptidoglycolipidsurface antigen that is common to the entire complex. Totest this hypothesis, unextracted and isopentanol-extractedwhole cells of M. intracellulare TMC 1403 serovar 14 wereassayed by ELISA against homologous and heterologousserovar rabbit antisera by using peroxidase-conjugated anti-rabbit IgG as the second antibody. Isopentanol extractioncaused an average of 72% reduction in the ELISA score ofhomologous serovar reactions against whole cells, whereasextraction had little or no effect on most of the heterologousreactions (Table 1). When rabbit antisera raised againstunextracted cells of M. paratuberculosis Linda were testedagainst isopentanol-extracted and unextracted cells of thehomologous strain, the isopentanol-treated cells gaveslightly higher responses than did the extracted cells (ratio ofthe A405 of isopentanol-treated to A405 of untreated cells =1.16). These results demonstrated that isopentanol extrac-tion removes the loosely bound serovar-specific peptidogly-colipid surface antigen and exposes a common surfaceantigen on cells of the M. avium complex, and that even theserovar-specific antisera have some antibody to that under-lying antigen. Our antibody that was raised against the

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TABLE 1. ELISA of unextracted and isopentanol-extracted cells of M. intracellulare serovar 14 against selected rabbit antisera tohomologous and heterologous serovars

Rabbit serum A405 against serovar 14 cellsRelationship Batch Serovar or Dilution (A) (B) Ratio (B/A)

no. strain Dlto nxrce A xrce B

Homologous 567 14 1:1,280 2.750 0.953 0.35756 14 1:320 2.781 0.833 0.30806 14 1:640 1.074 0.269 0.251294 14 1:1,280 2.406 0.513 0.21

Mean ± SD 0.28 ± 0.06

Heterologous 692 6 1:640 1.006 0.487 0.48500 7 1:640 1.841 1.753 0.951094 7 1:640 0.339 0.348 1.031240 8 1:1,280 1.118 1.135 1.02159 Linda 1:400 3.707 3.562 0.96160 Linda 1:200 3.766 3.577 0.95

Mean ± SD 0.90 + 0.21

unextracted whole cells of M. paratuberculosis appears tobe directed to that common antigen. M. paratuberculosis hasbeen shown by nucleic acid homology to belong in thespecies M. avium (15, 33). The demonstration of the com-mon surface antigen reactivity led us to conduct our humanserum survey with isopentanol-treated cells of M. aviuminstead of M. paratuberculosis, which is difficult to grow inquantity.When isopentanol-treated whole mycobacterial cells were

used as antigens and the human sera were tested for anti-body of the IgG class, the reactions of sera from patients andtheir relatives and spouses were not significantly differentfrom one another, but they were significantly higher thanthose of the unrelated control group (Fig. 1A, C, and E) forall mycobacterial antigens used.On the other hand, when the sera were tested for antibody

of the IgA class, the reactions of sera from patients withCrohn's disease against the same three whole-cell antigens(Fig. 1B, D, and F) were significantly elevated over those ofsera from all other subject groups, including the relatives ofpatients with Crohn's disease. This experiment was repeatedwith the same M. avium isopentanol-treated whole-cellantigen and was also done with a comparable antigen pre-pared from M. intracellulare TMC 1403 (data not shown);again, the distributions of IgA scores of the patients withCrohn's disease were significantly higher (P < 0.01) thanthose of their relatives.

Species-versus-species regression analyses of the IgAELISA scores with the whole-cell antigens were run for theM. avium, M. tuberculosis, and M. gordonae antigens(Table 2). Among the two most reactive groups of humansubjects, i.e., the patients with Crohn's disease and theirrelatives, the mean IgA ELISA scores were greatest againstM. avium and lowest against M. gordonae. Among these twosubject populations, the correlation coefficients betweenpaired antigens ranged from 0.61 to 0.90; the highest corre-lations were seen between the responses to the M. aviumand M. gordonae antigens with both the patients withCrohn's disease (r = 0.89) and their relatives (r = 0.90).When soluble protein extracts of either M. avium or M.

tuberculosis were used as antigens, nonsignificant (P > 0.05)

or borderline significant (P > 0.03) differences in the IgGresponses were seen among all groups of subjects. The M.avium-soluble antigen yielded IgA scores (data not shown)with sera from patients with Crohn's disease that were low(median A405, 0.104) but significantly higher than those oftheir relatives (median A405, 0.054; P = 0.007) or the normalcontrols (median A405, 0.031; P = 0.0003); no significantdifference was seen between the relatives and the normalcontrols. With M. tuberculosis soluble antigen, a very small,but significant (P = 0.012) elevation in the median IgA scorewas seen in patients with Crohn's disease over the normalcontrols, but no difference was seen between these patientsand their relatives.When whole baker's yeast cells were used as the antigen,

the sera from patients with Crohn's disease exhibited signif-icant elevations of both IgG (P = 0.011) and IgA (P = 0.0025)ELISA scores over those seen with serum from their rela-tives (Fig. 2), as well as those seen with sera from the 10other control subjects, which comprised the patients'spouses and other healthy individuals (P < 0.025) (data notshown in Fig. 2); the healthy relatives' responses were notsignificantly different from those of the other controls. Whenthe IgG reactions to yeast cells of serum from patients withCrohn's disease were compared with their IgA reactionsagainst that antigen, a high correlation (r = 0.84) was seen.On the other hand, a similar comparison between IgG andIgA reactions with the M. avium antigen yielded a muchlower correlation (r = 0.46).

DISCUSSION

The data from the present study indicate that sera bothfrom patients with Crohn's disease and from their relativesexhibit higher IgG ELISA scores to fixed mycobacterialsurface antigens than do those of normal control subjects.This could be due to increased environmental exposure tothe bacilli or increased serological response to a commonenvironmental exposure. An environmental factor must beconsidered in view of the significant elevations in antimyco-bacterial IgG seen in sera from the small number of patients'spouses in this series. However, this factor will have to be

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reexamined in a new study with larger numbers of bloodrelative and spousal controls. No significant differences wereseen in the IgG ELISA between the patients and theirrelatives, regardless of the type of mycobacterial antigentested.

In contrast to the results seen with IgG, the IgA responsesto whole-cell mycobacterial antigens were significantly ele-vated in sera from patients with Crohn's disease in compar-ison with those of their relatives. The elevated IgA responseto mycobacterial antigen in patients with Crohn's disease isespecially noteworthy in light of the reported selectivereduction in total IgA production by isolated intestinalmononuclear cells of patients with Crohn's disease (32).The ubiquity of human antibody responses to environmen-

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FIG. 1. Serologic response of serum from four groups of humansubjects to whole-cell antigens of three species of mycobacteria.The top shows the distribution of individual ELISA scores (A40)with median bars, and the bottom shows the significance (P) of thedifferences in score distributions among the groups as determined bythe nonparametric Mann-Whitney rank order test. N, not signifi-cant, i.e., P > 0.05. (A and B) M. tuberculosis whole-cell antigen; (Cand D) M. avium whole-cell antigen; (E and F) M. gordonaewhole-cell antigen. (A, C, and E) Sera tested at a 1:100 dilution forIgG; (B, D, and F) sera tested at a 1:20 dilution for IgA. CRO,patients with Crohn's disease; REL, blood relatives of patients withCrohn's disease; SPO, spouses of patients with Crohn's disease;NOR, unrelated normal control subjects.

(26) make it unlikely that the mere presence of an antibody toa mycobacterial antigen will prove diagnostic of a mycobac-terial role in Crohn's disease. Quantitative differences inantibody responses to such antigens can, however, serve assupporting evidence for an association of mycobacteria withthis disease. Thayer and colleagues (23) tested a formalin-fixed M. paratuberculosis whole-cell antigen, using a mix-ture of anti-human IgA, IgE, IgG, and IgM as secondantibody, and found significantly higher reactions with serafrom patients with Crohn's disease than with sera fromeither normal subjects or patients with ulcerative colitis;both of the last two groups gave similar, low responses. Choet al. (4) were unable to demonstrate significant elevations inIgG or IgA antibodies in the sera of patients with Crohn'sdisease when either a sonic extract or purified glycolipid ofM. paratuberculosis was used as an antigen. The failure todetect a glycolipid response is compatible with our observa-tions that isopentanol extraction of cells of M. paratubercu-losis did not diminish their reaction with rabbit antiserumthat was raised against unextracted whole cells of thisspecies. Grange et al. (9) have reported a very small, butsignificant elevation in the mean antimycobacterial IgAscores of patients with Crohn's disease when a sonic extractofM. bovis BCG was used as the antigen. We also recordedelevations in IgA against sonic extracts of mycobacteria insera from patients with Crohn's disease, but they were not as

striking as those seen with whole cells. Kobayashi andcolleagues (13) have reported that they did not detect any

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TABLE 2. Mean IgA ELISA scores of serum from threecategories of human subjects to isopentanol-extracted whole cellsof M. tuberculosis, M. avium, and M. gordonae and correlationcoefficients between scores in each category to paired antigens

Subject category Whole-cell antigens of: r

Crohn's disease M. tuberculosis (0.365) versus 0.82M. avium (0.406)

Crohn's disease M. tuberculosis (0.365) versus 0.80M. gordonae (0.315)

Crohn's disease M. avium (0.406) versus 0.89M. gordonae (0.315)

Relatives M. tuberculosis (0.270) versus 0.61M. avium (0.291)

Relatives M. tuberculosis (0.270) versus 0.71M. gordonae (0.215)

Relatives M. avium (0.291) versus 0.90M. gordonae (0.215)

Normal M. tuberculosis (0.184) versus 0.60M. avium (0.160)

Normal M. tuberculosis (0.184) versus 0.06M. gordonae (0.114)

Normal M. avium (0.160) versus 0.69M. gordonae (0.114)

a The numbers in parentheses are the mean IgA scores (A45) for theindicated category of subjects tested against the indicated antigen.

significant elevation in either IgA or IgG scores in sera frompatients with Crohn's disease tested against two mycobac-terial antigens. One of them, M. leprae lipoarabinomannan,was a 70% ethanol-soluble cell wall antigen that was purifiedby ion-exchange and gel filtration chromatographies, that iscommon to all mycobacteria, and that would be expected toyield results similar to those obtained with our isopentanol-treated whole cells; the other was a partially purified mixtureof proteins from an extract of M. paratuberculosis Linda.However, the logarithmically transformed datum plots pub-lished by Kobayashi et al. (13) do actually show elevated IgAresponses to the lipoarabinomannan antigen in patients withCrohn's disease, and the decision of those investigators toconsider that elevation insignificant may be based on theirselection of an unusually rigorous criterion for significance(P < 0.003). Furthermore, their IgA analyses were based on1:100 dilutions of sera; we found serum dilutions of 1:20 tobe optimal for the detection of IgA in our study subjects.None of the four reports (4, 9, 13, 23) on antibody tomycobacteria in patients with Crohn's disease cited herespecifically included relatives of the patients as a studygroup.Main and colleagues (14) have reported comparably ele-

vated reactions of both IgG and IgA in the sera of patientswith Crohn's disease when the sera were tested againstwhole yeast cells. In contrast to this similarity in responsesof the two classes of immunoglobulins to yeasts, we reporthere evidence of selective elevations of IgA responses inpatients with Crohn's disease when mycobacterial antigensare used. This difference in responses to the two types ofantigens is not due to differences in technique or patientselection, since we have confirmed the published results foryeast cells by our ELISA technique (Fig. 2) using the samepatients' sera that we studied for the mycobacterial antibod-ies. The IgG and IgA results with yeast cells are highlycorrelated with one another in serum from patients withCrohn's disease, but the results with mycobacterial cells are

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FIG. 2. Serologic response of serum from patients with Crohn'sdisease and their blood relatives to whole cells of baker's yeast.When the A4,5 of a sample was off scale on the reader, i.e., greaterthan 2.0, half of the sample in the well was transferred to an emptywell, and theA405 of the residual fluid in the original well and that ofthe transferred aliquot were added together to yield the ELISAscore for that specimen. See the legend to fig. 1 for definitions of theabbreviations and other details.

not. The apparent broad immunologic response of patientswith Crohn's disease to yeast antigens that are ingested inrelatively large quantities in bakery products may be aconsequence of the increased intestinal permeability re-ported in patients with Crohn's disease (10, 12), since it isnot seen in patients with ulcerative colitis (14, 16).The selective IgA response of patients with Crohn's

disease to whole cells of mycobacteria may be related to alimited localized topical exposure to ingested intact environ-mental mycobacteria, which are common in the stool (19).Mycobacterial cell walls are rich in lipids (18) and interactwith detergents (5), so when they are ingested they probablypartition into bile salt micelles. They could then be selec-tively deposited at the site of active absorption of bile saltsnear the terminal ileum and stimulate a local immune re-sponse. While monomeric IgA or IgA immune complexeshave little, if any, ability to trigger inflammatory responses,IgA aggregates can mediate them (30). Phagocytosis that wasmediated by suboptimal concentrations of IgG has also beenreported to be enhanced by IgA (17).The elevated antimycobacterial IgA reported here may

play no causal role in Crohn's disease, but it may simply bea reflection of a secondary response to the disease. How-ever, the observations reported here are compatible with theetiologic hypothesis of Roediger (20), which incriminates thelocalization of an amalgam of lipids and bacterial fragments,some of which have strong inherent adjuvant activities, inregions of the bowel that are predilective sites of Crohn'sdisease. We found the elevations in IgA scores to be morestriking when fixed whole cells of the mycobacteria wereused as antigen than when sonic extracts were used, sug-gesting a dominant reaction to the bacterial cell surface. Theinterpretation is also compatible with a report of a strikingremission in Crohn's disease of 18 years' duration in a

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patient who subsequently developed AIDS (11); patientswith AIDS and with disseminated M. avium infections donot produce antibody to these organisms (29, 31). Membersof other lipid-rich, mycolate-producing environmental gen-era, such as Corynebacterium, Nocardia, and Rhodococcus,could also be deposited at bile acid-active absorption sites,leading to the concentration of a variety of particulateantigens with adjuvant properties at the terminal ileum. Thishypothesis would not require invasive action by the bacteria,nor would it require that they even be viable.

ACKNOWLEDGMENTS

This study was supported by the Research Service of the Depart-ment of Veterans Affairs and by the Goldsmith Family Foundation.

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4. Cho, S.-N., P. J. Brennan, H. H. Yoshimura, B. I. Korelitz, andD. Y. Graham. 1986. Mycobacterial aetiology of Crohn's dis-ease: serologic study using common mycobacterial antigens anda species-specific glycolipid antigen from Mycobacteriumparatuberculosis. Gut 27:1353-1356.

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6. Gitnick, G., J. Collins, B. Beaman, D. Brooks, M. Arthur, T.Imaeda, and M. Palieschesky. 1989. Preliminary report on isola-tion of mycobacteria from patients with Crohn's disease. Dig.Dis. Sci. 34:925-932.

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