Immune responses and the gut

Vol. 56, No. 5

GASTROENTEROLOGY

Copyright

© 1969 b y The Willia ms

Printed in U.S.A.

& Wilkins Co.

PROGRESS IN GASTROENTEROLOGY IMMUNE RESPONSES AND THE GUT DAVID W. WATSON, M .D.

Department of Medicine, University of Michigan Medical Center, Ann Arbor, Michigan

The ever widening interests of the immunologist and the continued efforts of the gastroenterologist to understand presently obscure conditions have combined to focus increasing attention on the immune mechanisms of the gut. Most studies concerned with immune processes and the gastrointestinal tract have sought to implicate such events directly or indirectly in the pathogenesis of various disorders not heretofore assigned a specific cause. In some instances the accumulated evidence is moderately persuasive. In no case is it conclusive. The inability to convincingly relate various cellular and serological findings to gastrointestinal disease stems in part from our relatively meager knowledge of immune reactions generally, especially as they may be involved in the genesis of pathophysiological states. An equally important factor is the frequent failure to consider the immunological capabilities of the gut itself. Unlike most other organs such as the heart or kidney, the gastrointestinal tract is replete with immunologically competent tissue. It is capable not only of experiencing but also of mounting an immune response. In this respect it maintains functional identity with other peripheral lymphoid tissues such as the lymph nodes and spleen. Little is known, however, regarding either the qualitative or quantitative contributions of the gut to the body's immune responses. Of further importance is the realization that all of the presently recognized, soAddress requests for reprints to: Dr. David W. Watson, Department of Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48104.

called central lymphoid tissues which determine the immunological capabilities of the whole organism are lymphoepithelial derivatives of the gut. The gut, therefore, assumes a position of considerable importance to the immunologist as well as to those who seek an understanding of its more classic functions. Any attempt to relate abnormalities of the gastrointestinal tract to concurrently observed immunological changes must proceed from an awareness of the gut as both a central and peripheral lymphoid organ and as an immunological target.

The Gut As Central Lymphoid Tissue The central lymphoid organs are lymphoepithelial tissues which do not participate directly in immune responses but exert a profound influence over the immune capabilities of the peripheral lymphoid tissues especially during neonatal life. They are all gut derived and include the thymus, the avian bursa of Fabricius, and probably the lymphoid tissues of the appendix and small intestine of mammals, including man. Some perspective concerning the relationship between the gut and the lymphoid system can be obtained by examining the latter's phylogenetic development, a subject recently reviewed by Good et al. l The lymphoid system and its subserved immunological functions are late occurrences in both phylogeny and ontogeny, and in mature mammals, including man, the lymphoid system is composed of a complex group of cells and tissues which includes the thymus, tonsils, Peyer's patches, lymph nodes, spleen, bone mar944

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row, and widely distributed nonaggrega'ted lymphoid cells of many tissues. The intestinalcassociated lymphoid tissues, lymph nodes, and germinal center structures represent the most. recent additions to the body's lymphoid system and their appearance at a time when life was adapting to a nonaquatic environment suggests some phylogenetic advantage to this segment of the immune system much of which is intimately related to body surfaces. This phylogenetic parallelism between the intestinal lymphoid tissues, lymph nodes, and germinal centers forms one basis for the currently popular . thesis that certain of the intestinal-associated lymphoid tissues constitute a central lymphoid organ controlling some aspects of immunoglobulin production in mammals including man. While the phylogenetic approach provided a morphological and developmental basis for the existence of a controlling dichotomy within the immune system, it remained for ontogenetic studies to establish the fact of separate central and peripheral lymphoid tissues in functional terms. They are most clearly defined in avian species where the thymus and bursa of Fabricius exercise control over two functionally distinct segments of the peripheral lymphoid tissues. The thymus is derived from the third and fourth pharyngeal pouches and is composed of mesenchymal, epithelial, and lymphoid elements. Although some antigens penetrate the thymus, there is little evidence of a local immune response.i-6 The principal function of the thymus is to confer immunological competence upon a portion of the peripheral lymphoid tissues. Thymectomy at birth or shortly thereafter significantly reduces immunological responsiveness in the mouse, rat, hamster, guinea pig, and rabbit; in the mouse, rat, hamster, and rabbit the thymus is essential for the re-establishment of immunological competence after ablation of the immune system. The immunological consequences of thymectomy have been most clearly

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defined in the chicken where the development of the cell-mediated immunities (delayed hypersensitivity, homograft immunity, and graft-versus-host reactions) are thymus dependent and humoral immunity (antibody mediated) is not. 7 The latter, at least in avian species, is primarily · dependent upon another gutassociated lymphoepithelial structure, the bursa of Fabricius, which occurs.' as an outpouching of the cloaca. The thymus-dependent immunological functions are carried out by a ' small lymphocyte population found in blood, ' the deep cortical areas of lymph nodes, and the perifollicular regions of the spleen. Thymectomy experiments demonstrate the thymus to be essential for the initial development, maintenance, and , repopulation of these sites in peripheral ' lymphoid tissues. 8 - 11 Although the thymus, with the help of the bone marrow, functions to establish and maintain ' this peripheral cell population, the manner in which it imparts to it the aforementioned immunological capabilities remains uncertain. A humoral mechanism appears likely.12 - 13 The most profound and clear cut effects are obtained when thymectomy is carried out neonatally. There is evidence, however, which suggests a continuing function of . the thymus in adult life. 14 , 15 Even though the principal function of the thymus appears to be the regulation of cell-mediated immune responses ' and immunoglobulin synthesis is not greatly decreased by thymectomy, there is nonetheless a variable defect in the ability to express humoral immunity following neonatal removal of this organ, some antigens eliciting a response and others not. 16 - 19 The thymus also appears to play some part in the development and maintenance of tolerance to specific antigens. The hypothesis has been put forward that antigens present in or gaining access to the thymus are dealt with as self. Experimental data involving both proteins and cellular alloantigens lend support to this concept. 20 - 22 The case for similar thymic functions

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in man rests primarily upon a singular experiment of nature generally referred to as DiGeorge's syndrome. 23 Affected children manifest congenital aplasia of the thymus and parathyroids secondary to a developmental failure involving the third and fourth pharyngeal pouches. Such infants have normal immunoglobulin levels and produce antibodies but exhibit marked defects in delayed hyperand sensitivity, allograft rejection, phytohemagglutinin responsiveness of lymphocytes. Humoral immunity and antibody synthesis, on the other hand, are associated with germinal centers, lymphocytes in the cortical regions of lymph nodes, plasma cells in the medullary cords of lymph nodes, spleen, and the intestinal lamina propria as well as scattered lymphocytes and plasma cells in vario\ls connective tissues. The central lymphoid tissue controlling humoral immunity has been clearly defined only in the chicken where this function can be ascribed to The bursa the bursa of Fabricius. 24 develops at the opposite end of the gut from the thymus as an epithelial invagination from the dorsal cloaca to which it retains luminal connection. It, too, is a lymphoepithelial structure. As in the case of the thymus, its development is independent of antigen exposure and uninfluenced by the germ-free state. It was a chance event that bursectomized birds were challenged with antigens in a classroom demonstration,25 but this coincidence provided one of the most basic observations in all of immunology. Neonatal bursectomy abolishes or greatly retards plasma cell and germinal center development, immunoglobulin production, and antibody responses. 11 , 26 - 29 As in the case of the thymus, evidence has been presented that the bursa may influence its dependent tissues via a humoral mechanism, 30, 31 The counterpart of this bursal system in mammals, especially in man, is presently the object of considerable speculation, centering around three gut-asso-

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ciated lymphoepithelial tissues; the tonsils, the aggregated lymphoid tissues of the intestine represented by the appendix and Peyer's patches, and the nonaggregated lymphocytes of the intestinal epithelium and lamina propria. The pattern of development and involution of the tonsillar tissues and their intimate lymphoepithelial relationship present obvious similarities to the avian bursa. Direct extirpative experiments referred to by Good et al.I have, however, failed to establish a bursal function. Further studies are required before these structures can be assigned a position among either the central or peripheral lymphoid tissues. Studies of the appendix and Peyer's patches have been carried out only recently. The rabbit has been the object of these experiments because of the well developed lymphoid tissue in the appendix and closely associated sacculus rotundus. These structures exhibit somewhat of a lymphoepithelial relationship and morphological features at least superficially resembling bursal tissue. In the case of the Peyer's patches, growth and regression curves parallel those of the bursa, and their development has been reported to coincide with the appearance of immunoglobulin-producing cells elsewhere. In irradiated mice in which Peyer's patches were shielded, regeneration of spleen and lymph nodes but not bone marrow has been demonstrated. 32 These studies would be more convincing if thymectomy had also been performed. Neonatal extirpation of the appendix and sacculus rotundus has been shown to interfere with some but not all aspects of humoral immunity.16 - 18, 33,34 These defects are measurably enhanced by the addition of thymectomy or splenectomy. Of greater interest are two studies reported by Cooper et al. 35 and Perey et al. 36 In the first instance, removal of the appendix, sacculus rotundus, and all of the Peyer's patches in young adult rabbits together with heavy total body irradiation produced a lasting and selective suppression

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of humoral immunity while leaving given intravenously did not migrate to cellular immune mechanisms intact. In this area suggesting a closed population. the second study, removal of the same Most recently Fichtelius et al. 44 have tissues followed by irradiation and re- reported data in essential aggreement constitution with fetal liver cells at age with these investigators. He further that epithelial lymphocytes states 4 weeks abolished or markedly reduced differ ultrastructhe antibody response to brucella immu- (theliolymphocytes) nization. Appendectomy alone delayed turally from those in the lamina propria comparative but did not suppress the antibody re- (propriolymphocytes) . In sponse while thymectomy or splenectomy studies involving the mouse and pig the and removal of the mesenteric lymph earlier appearance of theliolymphocytes nodes had no effect. Certain features of in the latter correlates with the earlier the Peyer's patches, however, point to appearance of humoral immunity and desome dissimilarities in respect to the velopmental and involutional features of bursa. Evidence has been presented that these theliolymphocytes and the bursa are following irradiation the Peyer's patches similar. In a separate study involving are repopulated largely by cells of numerous assumptions the same authors lymph node origin rather than bone propose that theliolymphocytes migrate marrow37 and in this respect resemble to the tips of villi in close association with peripheral rather than central lymphoid epithelial cells and there enter the lamina tissue. Also, in germ-free animal experi- propria to be' subsequently seeded to the ments full Peyer's patch development peripheral lymphoid tissues. 45 These studies taken together show only seems dependent upon the presence of bacteria,38 again resembling peripheral that some lymphocytes maintain a lympholymphoid tissues. In ungulates39 and epithelial relationship but provide no man,40 however, Peyer's patches are information regarding their function. well developed at birth. Thus, there may Nature has been only partially helpful be considerable species variation in this in defining the bursal equivalent in man. relationship and Peyer's patches may in The counterpart of DiGeorge's syndrome fact be composed of both types of tissue. is the congenital sex-linked agammaglobuIn the case of the nonaggregated linemia of Brutton. While this defect lymphoid tissue of the intestine the evi- further emphasizes the duality of the dence linking it to a bursal function is lymphoid system, it provides no clue to highly circumstantial. The studies of the identity of the defective central lymphAndrew41 originally suggested that the oid organ responsible. epithelial lymphocytes assumed an intraClearly much work is needed to establish cellular location and were extruded into which if any of the intestinal associated the lumen of the gut with the epithelial lymphoid tissues constitutes a mammalian cells. A recent study by Meader and bursal equivalent. Landers 42 of the intestinal epithelium of The Gut as Peripheral the rat, mouse, hamster, and man indiLymphoid Tissue cates that these lymphocytes occur intercellularly within deep niches in the It seems rather evident that, if properly epithelial cell surface, are located basal stimulated, the lymphoid cells of the gut to the epithelial cell nuclei, and are not would be capable of forming both serum extruded. They also did not appear to and coproantibodies. The evidence for transform into other cell types and mi- local antibody production is, however, grated between the epithelium and the largely indirect and surprisingly incomlamina propria. Darlington and Rogers43 plete. Kinetic data derived from studies have shown that approximately 10% of of cholera immunization have often been the cells in the epithelium of mice are cited in support of in situ antibody prolymphocytes, and labeled lymphocytes duction. 46 . 47 Following oral immunization

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the appearance, peak ~iter, and decline of coproantibody precedes the corresponding serum values and the relatively low levels of serum antibody produced appear insufficient to account for the amount of coproantibody found. Nonetheless, without knowledge of the manner in which antibodies are transported by the intestinal epithelium such information is inferential. The only recent studies of immunoglobulin handling by the gut are those of Pierce and Smith. 48, 49 Using everted sacs from newborn pigs they found the absorption of colostral antibodies to be concentration dependent and variable between different segments of the gut. It was enhanced by prior feeding of colostrum and inhibited by human serum albumin and certain L-amino acids suggesting an active transport mechanism. More direct evidence for local antibody production has been provided by Farr and co-workers.50, 51 Following oral administration of bovine serum albumin to rabbits in amounts insufficient to evoke humoral antibody formation , coproantibodies were detected. A recent study by Cooper and Turner52 is also of interest. Mter injection of Peyer's patches with antigen, the immune response was confined to the Peyer's patches and regional lymph nodes as judged by the presence of antibody-forming cells. This, however, does not preclude the possibility that all such cells were derived from the regional nodes. In fact, Cooper et al. 53 have shown that antigens injected into isolated intestinal loops are absorbed and distributed to the spleen and other lymphoid tissues with antibody forming cells being found first in the spleen and later in the Peyer's patches. To what extent such events occur in the undisturbed intestine is not known. The gut also imposes the additional variables of antigen degradation and absorption. Two recent studies emphasize the divergent effects possible with orally administered antigens. In the first, evidence is presented that prior sensitization may increase antigen absorption,54 whereas in the second prolonged minimal antigenic stimulation by the oral route appeared to produce a degree of toler-

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ance. 55 Since antigen exposure via the gastrointestinal tract is highly variable, it is apparent that many factors must be taken into consideration when attempting to study an antibody response by the gut. Apart from the question of local antibody production, considerable interest has developed in the immunoglobulins of the gastrointestinal tract and it is now well established that IgA is the predominant immunoglobulin in the lymphoid cells and secretions of the gut.56 - 59 Considerably lesser amounts of IgM and IgG are present with the ratio of IgA-containing cells to IgM - and IgG-containing cells being approximately 20 : 1. IgD has recently been shown to occur in a few lymphoid cells of the rectal mucosa but has not been found as yet in any of the external secretions. A fifth immunoglobulin, IgE, has been demonstrated only in serum. The human infant is born totally lacking IgA in serum and external secretions, and secretory IgA rises sooner and reaches adult levels more quickly than serum IgA. The gut and other tissues contributing to the external secretions have been regarded by some as potentially important sources of both secretory and serum IgA. 60 - 62 Tomasi59 has recently reviewed the characteristics of secretory IgA which differs in several important respects from serum IgA. Serum IgA is a 7S immunoglobulin with a molecular weight of 180,000, while secretory IgA has a sedimentation coefficient of 11S and a molecular weight of 390,000. The two also differ in amino acid composition and antigenicity. The physicochemical and antigenic differences are due to the presence in secretory IgA of a nonimmunoglobulin glycoprotein with a molecular weight of 60,000 referred to as secretory piece or T (transport) component. It seems likely that the lIS secretory IgA is a dimer of 7S serum IgA plus the T component. No doubt the secretory piece which appears to be synthesized in epithelial cells63 confers some biological advantage on IgA in the external secretions. How and where it combines with IgA is not known but

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Gelzayd et al. 64 have demonstrated IgA within the apical cytoplasm of the epithelial cells of the small bowel, appendix, and colon. The role played by lIS IgA in external secretions remains unsettled. It has been shown to function as an isohemagglutinin and to possess antibacterial65 and virusneutralizing properties66 and probably constitutes an important defense against certain microorganisms and perhaps other potentially harmful substances. 60, 67

Gastrointestinal Disorders with Immunological Manifestations As previously emphasized, the normal gastrointestinal tract contains significant numbers of immunologically competent cells which are invariably increased in numbers or abnormal in distribution in those disorders suspected of having an immune origin. Furthermore, the gastrointestinal tract may serve as an immunological target by participating in most, if not all, of the known reactions involving antibodies or antigen-antibody complexes including the Arthus and Auer phenomena, Prausnitz-Kustner reactions, localized and systemic anaphylaxis, and, possibly, atopic sensitivity.68 - 73 The ability of the small and large intestine to also develop delayed hypersensitivity reactions, although known for some time, has recently been reinvestigated by Bicks and coworkers. 74, 75 Since potential antigens abound in the tissues and luminal contents of the gut, considerable latitude is afforded those favoring an immune etiology for various gastrointestinal disorders and several continue to be the object of such investigations. Pernicious anemia and gastritis. Discussion here is confined to material appearing in the literature subsequent to the reviews by Taylor 76 and Taylor and Fisher77 except in some instances where certain points require emphasis and further comment. With the loss of parietal cell mass associated with atrophic gastritis, a significant decrease in intrinsic factor (IF) production would be expected. Whether

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the gastritis is immunologically mediated or sufficient to cause pernicious anemia without the additional insult of immune mechanisms directed toward IF is, of course, the point at issue. It is perhaps pertinent that all methods utilized to detect IF depend upon vitamin B12 binding and, as used in most studies, do not permit distinction between absent or defective IF and IF inactivated by antibody. IgG and IgA immunoglobulins exhibiting specificity for an insoluble microsomal component of parietal cells are demonstrable by complement fixation or fluorescent antibody techniques in the serum of at least 80% of patients with pernicious anemia 78, 79 and in a significant number of patients with diabetes mellitus, especially if insulin dependent,80 iron deficiency anemia,81 idiopathic Addison's disease in females, 82 and patients with atrophic gastritis without pernicious anemia. 83 They are also present in many relatives of patients with pernicious anemia, thyroid disorders,84 and iron deficiency anemia. 85 It is increasingly clear that the common denominator for parietal cell antibodies is atrophic gastritis and not vitamin B12 malabsorption. Previous studies reporting the presence of parietal cell antibodies in some normal individuals assumed the absence of gastritis or relied on blind gastric biopsy. In patients with thyroid disease, in whom the incidence of such antibodies is appreciable, their presence tends to be associated with defective acid production and, therefore, most likely with gastritis. 78 Although not clearly established, the same is probably true for other disorders having a significant incidence of parietal cell antibodies. Recent studies by Fisher and co-workers,83 Fixa et al.,86 and Samloff et a1. 87 have further emphasized the close relationship of parietal cell antibodies to gastritis and the lack of correlation with IF antibodies or B12 malabsorption. This latter finding is of interest in view of the correlation between IF secretion and acid output following stimulation with histamine or pentagastrin.88 Why antibodies are not found in all patients with gastritis is not clear. The low

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incidence of parietal cell antibodies in patients with postgastrectomy gastritis83 or gastric carcinoma with atrophic gastritis89 suggests that elements in addition to histological damage are operative in their formation. The familial occurrence of pernicious anemia and the presence of antibodies in many relatives without pernicious anemia point to genetic influences as one probable factor. The significance of circulating parietal cell antibodies in patients with atrophic gastritis with or without pernicious anemia remains to be determined. The finding of diminished B12 absorption in some of the latter has led to the unfortunate term "latent pernicious anemia." The problem in part, therefore, is one of definition. In any slowly evolving disorder the time course is bound to be variable and studies at a fixed point in time are likely to be discrepant. The possibility that locally produced parietal cell antibodies might damage the gastric mucosa has received relatively little attention. Jeffries and Sleisenger90 have demonstrated the presence of IgGand IgM-containing lymphoid cells in the mucosa of patients with pernicious anemia but without apparent antibody activity. Other investigators91 . 92 have reported an increase in the small amounts of IgG and particularly IgA present in the lymphoid cells of normal mucosa, but mucosal immunoglobulin content could not be correlated with either circulating IF or parIetal cell antibody titers or IF output, and no local parietal cell antibody activity was detected. Parietal cell antibodies have been demonstrated in the gastric juice of some subjects who also had serum antibodies but their local production has not been established. 90 . 93 Two studies confirming earlier work have reported the production of gastric atrophy in dogs following immunization with autologous mucosa or gastric juice and complete Freund's adjuvant. 94 . 95 Although the gastric lesion was accompanied by delayed cutaneous reactions and precipitating antibodies to gastric juice, no information regarding IF activity or long term effects

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was reported. Curiously, infiltration with mononuclear cells was not a conspicuous feature as it is in human gastritis. To date no studies involving delayed hypersensitivity in pernicious anemia have been published. Bicks et al. 96 have reported the production of a 2, 4-dinitrochlorobenzene-induced "contact gastritis" in miniature swine which correlated with circulating parietal cell antibody titers. Although not disproving the potential importance of parietal cell antibodif's, this finding nevertheless emphasizes their possible secondary nature. Numerous studies have confirmed the presence of IgG immunoglobulins with two types of specificity for IF in the serum of some patients with pernicious anemia. 97 • 98 Blocking antibody (type I, combining site antibody) inhibits the combination of Bl2 with IF while binding antibody (type II, complex antibody, precipitating antibody) combines with the B12-IF complex or IF alone but does not prevent IF binding of B 12 . This suggests two single but separate and antigenically distinct sites for antibody attachment. Ashworth et al. 99 employed a new method for demonstrating IF antibodies based upon the ability of guinea pig small intestinal homogenates to take up BwIF complexes but not B12 or binding antibodies. They have published data suggesting no single site of attachment for either antibody since the antigen-antibody' reactions observed were characteristic of combinations in multiple proportions similar to the classic pattern of increasing antibody reacting with a constant amount of antigen in a nonprecipitating system. Sera from patients with pernicious anemia may contain only blocking antibody, both blocking and binding antibody, or neither. Rarely, if ever, only binding antibody has been demonstrated. The dose and quality of . the antigen and the immunological capacity of the host are among the factors which may determine the different patterns of antibody response observed. Data has been presented which suggests a stronger immunogenicity for the blocking antibody site87 as well as the BwIF complex. loo It is

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evident that IF antibodies might arise either from the emergence of an abnormally reactive clone of antibody-forming cells or a change in the antigenicity and availability to the immune system of IF. The former would be expected to produce a monoclonal response, assuming the development of a single population of such cells. Evidence has been presented, however, that IF antibodies are heterogeneous,lol similar to antibodies reacting with thyroglobulin or nuclear antigens. 102 Both K and A light chains have been found associated with binding and blocking antibody activity and in 1 patient studied their ratios were different in the two antibody types. The significance of serum antibodies to IF remains controversial. Despite their absence in some 30 % of patients with pernicious anemia, they correlate more closely with BI2 malabsorption and pernicious anemia than parietal cell antibodies, being rarely if ever present in other conditions or normal individuals. Fisher et al. B3 have demonstrated a close correlation between diminished BI2 absorption and the presence of circulating IF antibodies implying biological significance. Other studies, however, tend to cast doubt on their importance to BI 2 malabsorption. Transplacental transfer of both parietal cell (lgG) and IF antibodies has been demonstrated but the results of assays for infant IF in the recipients have been at variance. 103 - 105 In some, IF activity was absent while in others it was undiminished. Although Ungar et al. 106 found their incidence to increase with the duration of the disease, Samloff and co-workers B7 could demonstrate no relationship of IF or parietal cell antibodies to sex, duration of disease, or age at diagnosis. Yates and Cooperl07 were further unable to find any difference in the amount of normal gastric juice required to enhance BI2 absorption in patients with or without IF antibodies in serum, and feeding an excess of gastric juice prior to a Schilling test failed to increase BI2 absorption, implying that endogenous secretory antibody was not a factor. The latter conclusion. however,

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would require knowledge of the rate of formation and secretion of both antibody and IF. Another study based on 22 patients with gastric carcinoma demonstrated a significant decrease in IF activity and acid output without circulating IF antibodies. loB This may only indicate that more than one mechanism can be responsible for IF deficiency. Patients with pernicious anemia have also been shown to respond to prednisone therapy with an increase in BI2 absorption as measured by the Schilling test, increased IF activity, and regeneration of gastric glands but without any change in serum antibody titers to IF or parietal cells. l09 Since prednisone may interfere with antigen-antibody reactions without appreciably affecting serum titers, this finding cannot be used to impugn the potential relevance of such antibodies. Most recently, 9 patients with hypogammaglobulinemia and pernicious anemia have been reported in whom no IF or parietal cell antibodies could be detected in serum despite the presence of atrophic gastritis with heavy mononuclear cell infiltration. 110 Equally bothersome are those patients with atrophic gastritis, little or no detectable IF, and preserved BI2 absorption. This might indicate that, while IF bound to antibody is undetectable by current assay techniques, it still functions in some instances to promote BI2 absorption. An early study by Kaplan et al. 111 demonstrated resistance to hog IF after oral but not parenteral administration despite the presence of circulating IF antibodies in each case. This suggested a local origin for antibodies to IF and indeed both types of antibody have been demonstrated in gastric juice, but here too accompanied by discordant findings .112, 113 Previous studies have found the IF antibodies in gastric juice to belong to the IgG class of immunoglobulins, suggesting an origin from serum. 112, 11 4 In saliva IgA antibody has been detected although it was not shown to be of the secretory type. 11 4 A more recent study has demonstrated binding antibody activity in gastric

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JUIce associated with secretory IgA but not with IgG or IgM immunoglobulins. ll5 One point in the sequence of events involved in B12 absorption at which antibodies might interfere has only recently received attention. MacKenzie and coworkers l16 have reported that antibodies to distal but not proximal intestinal microvillous membranes inhibit IF -mediated Bl2 attachment, an effect which could be overcome by an excess of BI2-IF complex. Crahn's disease. Crohn's disease has continued to elude the interest and imagination of investigators, a situation both curious and unfortunate. Surprisingly little evidence has accumulated to suggest an immune etiology for this disorder but, for reasons not entirely clear, it has come to be recognized as having immunological features. It had been previously reported that patients with Crohn's disease often developed anergy to antigens normally eliciting a delayed cutaneous response. In what appears to be a much better controlled study, Fletcher and Hinton 117 could find no evidence for such anergy, at least in the case of tuberculin sensitivity. Employing graded doses of tuberculin, cutaneous responses were found to be independent of the presence or absence of granulomas as well as the age of the patients. There is evidence that in some situations granulomas constitute morphological evidence of an immune response. The granulomatous reaction to tubercle bacilli and many fungi has long been understood as a manifestation of delayed hypersensitivity; more recently evidence has been presented that the tissue response in schistosomiasis can be similarly regarded. Neonatal thymectomy in mice prevents the granulomatous response to schistosome eggs 11 8 as does the administration of antilymphocyte serum prior to experimental infection.ll9 The granulomatous response to foreign bodies is, however, relatively unaffected. Warren and colleagues l2o have shown that granulomas developing after the intravenous administration of eggs are larger in animals previously

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sensitized by the intraperitoneal injection of eggs or sensitized spleen cells. Although obviously tangential, these observations may have potential relevance to many granulomatous disorders including Crohn's disease. The recent demonstration that lymphocytes from patients with Crohn's disease are cytotoxic for human colon epithelial cells but not ileal cells would seem to be a finding without apparent significance. It is conceivable, however, that these lymphocytes are reacting to nontissue antigens, resulting in a nonspecific cytotoxic effect on surrounding tissue cells. This possibility is discussed more fully in relationship to ulcerative colitis. Although an infectious origin for Crohn's disease has often been considered, no recent investigation of this possibility has been undertaken. Goldie 121 had drawn an interesting analogy between Crohn's disease and the tuberculoid form of leprosy. In the latter, leprosy organisms are rarely demonstrated histologically and have only recently been cultured with difficulty on highly specialized media. The organism is not sensitive to the usual antibiotics or antituberculous drugs and its epidemiology is highly complex and unlike most infectious diseases. Furthermore, Koch's postulates have never been fullfilled. While no conclusions can be drawn from such a comparison, it should serve to encourage further search for an infectious agent in Crohn's disease. Recent developments relative to sarcoidosis are of interest in this regard. 122 While patients with tuberculosis frequently have antibodies to mycobacteriophages, they are generally absent in patients with sarcoidosis. These phages convert microorganisms to a lysogenous state wherein they can be neither seen nor cultured but may retain their antigenicity. Utilizing serum-containing antibodies to mycobacteriophages, anonymous mycobacteria have been cultured from some patients with otherwise classical sarcoidosis. The implications for Crohn's disease although perhaps a little strained are nonetheless obvious. As in the case of tuberculosis, both

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infectious and immune processes may be involved and further work in both directions should be profitable. Nontropical sprue. Gluten sensitivity has been regarded as due either to a mucosal enzyme defect permitting accumulation of noxious peptides or an allergic reaction to certain cereal proteins. Circulating antibodies to gluten fractions and other food substances, especially milk proteins, are frequent in both childhood and adult celiac disease particularly prior to gluten elimination. 123 - 127 Variations in the antigens studied and the antibody assay techniques employed preclude meaningful comparisons, however. Whether these antibodies are developmentally related to the intestinal lesion remains unknown. They may result from increased permeability of the gut allowing for greater antigenic stimulation but it seems paradoxical that in malabsorption permeability to antigens should be increased. It is perhaps pertinent that, in tropical sprue where the structural abnormality closely resembles that of celiac disease, antibodies to gluten fractions are seldom found .128 - 130 Immunoglobulin levels in patients with nontropical sprue have been variable. Earlier studies in small groups of patients reported an increase in IgA and IgG concentrations. More recently a modest decrease in IgM levels was demonstrated in untreated patients followed by a rise after gluten elimination. No relationship between immunoglobulin concentrations and specific antibody activity has been demonstrated. The possibility that locally produced antibodies might be of importance has received little attention. Rubin et al. 131 have demonstrated the usual predominance of IgA-containing mononuclear cells in the mucosa of patients with celiac disease but no gliadin binding by any of the immunoglobulins was detected. Conversely, epithelial cells were often found to bind gliadin but no antibodies could be localized to these sites. Another study has reported increased numbers of IgG-con-

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taining mononuclear cells in the mucosa associated with free interstitial IgG.132 These changes were unaffected by either gluten elimination or gluten challenge and similar findings were present in patients with various viral illnesses, further attesting to their nonspecificity. Cutaneous reactions to gliadin or its fractions have not been elicited l33 and, in treated patients at least, these antigens do not cause lymphocyte stimulation. 134 Gastrointestinal symptoms and malabsorption can be produced in patients with nontropical sprue by feeding peptides whose molecular weights are less than 1500, 135 well below that of synthetic amino acid polymers shown to be significantly immunogenic. 136. 137 Such peptides by themselves are not likely to initiate appreciable antibody titers although presumably would be capable of reacting with antibodies once the latter were formed. Gluten is an antigenically complex material 138 and it is doubtful that critical studies of antibody formation can be carried out until its antigens are purified and better characterized. Molecular recombinations within the gut lumen might also result in the formation of antigenic determinants bearing little or no relationship to those naturally present in the ingested material. The lymphoreticular dysfunction that has been associated with nontropical sprue (intestinal reticulosis and lymphoma, hypogammaglobulinemia, defective lymphocyte transformation, and splenic atrophy) suggests some underlying abnormality of the lymphoid system.139 - 144 The possibility that sprue results from a local immunological deficiency rather than a hyperimmune response deserves consideration. The patients with steatorrhea and IgA deficiency reported by Crabbe and Heremans, I 4 5 although of uncertain relationship to what is generally understood as nontropical sprue, support this view. If immune responses are involved in this disease, it is difficult to understand the 30% of patients with otherwise identical lesions who do not respond to gluten

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elimination,146 as well as patients with a variety of nongluten sensitive disorders who exhibit similar histological changes. As in the case of pernicious anemia, a problem of definition exists. How do we know, for example, that those patients with one or another immunological abnormality do not in fact comprise a homogeneous group in which abnormal immune responses are responsible? The answer must await studies utilizing purified and well characterized antigens and uniform and sensitive assay techniques in clearly defined patients subjected to controlled antigenic challenge. Intestinal lymphangiectasia. Proteinlosing enteropathy associated with dilated intestinal lymphatics may be accompanied by both hypogammaglobulinemia and lymphopenia with a loss or diminution of related immunological functions. A study of 18 patients with this disorder by Strober et al. 147 is worth discussion. Serum concentrations and miscible pools of IgG, IgM, and IgA were diminished and the daily fractions lost into the gut increased. Synthetic rates were generally normal, emphasizing again that immunoglobulin depletion does not constitute a stimulus for increased production. Antibody responses to some but not all antigens were diminished. Lymphocytopenia was often present, being associated with anergy to cutaneous delayed hypersensitivity and 4 of the patients were able to retain skin homografts for at least 12 months. Other disorders sometimes associated with dilated intestinal lymphatics (Whipple's disease, regional enteritis, and' constrictive pericarditis) may also exhibit both hypoand lymphocyte gammaglobulinemia depletion, whereas protein-losing states unassociated with lymphangiectasia develop only a modest decrease in immunoglobulins. Measures which correct the underlying disorder generally reverse the immunological hyporesponsiveness. This study serves to emphasize that in some situations even widespread immunological defects may be secondary to a gastrointestinal abnormality. Milk sensitivity. Among food antigens

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the proteins of cow's milk are in a class by themselves. Hardly a disorder with immunological implications has escaped association with antibodies to these substances. Ulcerative colitis has been the most frequent victim of this approach and the relationship of milk protein antibodies to this disease has been reviewed .148 Suffice it to say here that such antibodies are the rule in normal infants ingesting cow's milk and are not infrequently present in adults. They may occur in somewhat higher titers in a number of gastrointestinal disorders such as ulcerative colitis, nontropical sprue, and many instances of infectious gastroenteritis. They have yet to be understood in the majority of instances as anything but secondary events occurring in the course of intestinal disorders having in common the undistinguishing feature of an abnormal mucosa. There is one probable, currently ill defined exception to this generalization. Gryboski149 has described a group of 21 patients, mostly infants, with diarrhea and bleeding which appeared to bear a close relationship to milk ingestion. In 7 infants, milk administration was followed by cardiovascular collapse and in 2 by gastrointestinal hemorrhage . In 8 patients, 7 of whom had no evidence of lactose intolerance, sigmoidoscopic findings were compatible with ulcerative colitis. Histologically, there was infiltration with lymphocytes and plasma cells, epithelial destruction and crypt abscesses. Such findings probably reflect only the nonspecific character of the histological changes in ulcerative colitis rather than any relationship of milk sensitivity to that disease per se. Studies in 4 children with similar symptoms of diarrhea and bleeding related to milk intake have also been reported by Katz et al. 150 Precipitins to whole milk were present in the stools of all 4, but absent from serum and disappeared within 48 hr of milk elimination. One patient reported separately by the same group was found to have immunoglobulin-containing cells in the rectal mucosa capable of binding a-Iactalbumin. 151 The possibility

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that coproantibodies may be involved in some cases of milk or other food sensitivity must be further explored. Waldmann and co-workers I52 have recently described 6 children with what they have termed allergic gastroenteropathy. Although seemingly milk sensitive, they differ from the aforementioned patients in several respects. Characteristic features included diarrhea, edema, growth retardation, extreme hypoalbuminemia, hypogammaglobulinemia, anemia, eosinophilia, and various allergic manifestations such as eczema and allergic rhinitis. The hypoproteinemia, including the hypogammaglobulinemia, was related to increased gastrointestinal protein loss and responded in some cases to either milk elimination or steroid therapy. A single adult with similar manifestations has been reported, however, in whom milk elimination and corticosteroids were not effective. 153 Although the abnormalities exhibited by these different patients very likely represent a spectrum of adverse responses to milk proteins further study is required to confirm their allergic origin. Nonetheless some conditions commonly accepted as allergic such as eczema and many cases of asthma rest on credentials no more impressive. Ulcerative colitis. A more complete discussion of immune mechanisms in ulcerative colitis may be found in a recent review. 148 Discussion here is limited to current publications and certain features which merit emphasis. Investigation has been hampered in part by an inability to produce a comparable lesion in experimental animals. In the continuing search for an investigative model of ulcerative colitis, Singer et al. 154 studied the colonic manifestations of runt disease, a form of graft-versushost reaction. Newborn rats injected with homologous spleen cells frequently exhibited diarrhea and dilation of the colon in addition to the other features of runting. Atrophy and disruption of the epithelium . and invasion with mononuclear cells were observed but ulcerations and crypt abscesses characteristic of ulcerative

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colitis were not found. Perhaps we expect too much of our animal friends. There is really no reason to believe that they can be made to develop a lesion that is identical in all respects to one which occurs spontaneously only in man. The colonic flora continue to be the object of sporadic investigation but no systematic study of their potential relevance to immunological alterations in ulcerative colitis has been undertaken. It is difficult to imagine that this prodigious and intimate companion to the colon can be blameless in the development of this disorder. In a somewhat tangential study, human group B erythrocytes and Escherichia coli 086 organisms have been shown to contain antigenically similar lipopolysaccharides with the erythrocyte appearing to possess surface receptors capable of binding these bacterial antigens. 155 That the same might be true for colon epithelial cells is an important possibility. In fact human, germ-free rat, and rabbit colon appear to contain one or more antigens in common with the lipopolysaccharide of E. coli 014.156 Any studies attempting to assess the immune response to bacterial antigens, however, must take into account the variables inherent in them. Although the common enterobacterial antigen (Kunin) is widely distributed among Enterobacteriaceae, only the antigen in E. coli 014 engenders antibodies in high titer. I57 When separated from the lipopolysaccharide 0 antigen (endotoxin), the Kunin antigen is highly immunogenic, whereas, combined with 0 antigen (except in E. coli 014), it is not. Additional confusion is provided by the observation that, although antibodies react with all bacteria containing Kunin antigen by passive hemagglutination, they do not cause agglutination of the bacteria or precipitate the isolated antigen. Another group has reported the presence of circulating endotoxin in patients with ulcerative colitis. I58 Since endotoxin can function as an adjuvant, it too may have important contributions to make to immune reactions in patients with this disease.

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Most studies have been concerned with some aspect of humoral antibody formation. The sera of patients with ulcerative colitis have been shown by a variety of techniques to contain antibodies to numerous exogenous and endogenous antigens. These antibodies in general bear no relationship to the severity, duration, or extent of disease; are neither tissue, disease, nor species specific; occur also in some normal individuals; are not readily demonstrable in tissue lesions; and do not appear in vitro to be cytotoxic. It appears increasingly correct to view them, whatever their orientation, as secondary events and to consider their relevance as pathogenetic factors in ulcerative colitis as doubtful. Several recent studies further support this view. Kraft et al.,1 59 utilizing both direct and indirect immunofluorescent techniques in autologous, homologous, and heterologous systems, failed to confirm earlier reports of immunospecific in vitro binding of sera from patients with ulcerative colitis by colonic mucosal glands. Their finding of specific fluorescence in the cytoplasm of lymphocytes and plasma cells suggesting antigen uptake deserves further investigation. McGiven and co_workersI 60. 161 in two separate studies further demonstrated the nonspecificity of fluorescent staining by sera from patients with ulcerative colitis. Normal human sera frequently stained colonic mucosal glands and hemagglutinating antibodies to rat feces which shares a common antigen with human colon were no more frequent in patients with ulcerative colitis than in normal control subjects. Antibodies reacting with antigens from normal adult and fetal colon as well as colonic carcinomas have also been found in the sera of patients with colon cancer, further emphasizing their nonspecificity.1 62 Of additional interest is the finding that anticolon antibodies occur with comparable frequency in patients with ulcerative colitis, granulomatous colitis, and regional enteritis. 163. 164 Again coproantibodies have been largely neglected. Gelzayd et al. 165 demonstrated the usual preponderance of IgA-containing

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cells in the rectal mucosa of patients with ulcerative colitis but found both IgAand IgG-containing cells decreased in absolute numbers with IgA often being found interstitially in an extracellular location. The significance of these changes is not yet apparent. Recent studies have produced evidence tending to implicate lymphocytes more directly in the tissue-destructive processes characteristic of ulcerative colitis. This evidence consists of the recognized capability of these immunologically competent cells to produce tissue destruction and cell death in various in vivo and in vitro systems as well as their demonstrated cytotoxicity for the epithelial cells of the colon. 148 . 166 - 169 Certain features appear to characterize this cytotoxicity. (1) It is demonstrable in virtually all patients with ulcerative colitis and Crohn's disease regardless of anatomic distribution. (2) Lymphocytes from patients with a variety of disorders including other inflammatory and neoplastic lesions of the gastrointestinal tract do not share this property. (3) Only colonic epithelial cells are affected whereas cells derived from liver, kidney, small intestine, and stomach are not. (4) The effect in vitro is rapid in onset, occurring within 2 hr and may be complement dependent, at least when intact lymphocytes are employed. (5) Cell-free extracts of appropriate lymphocytes are also cytotoxic. (6) Cytotoxicity disappears soon after removal of diseased tissue although persisting during clinical remission and can be inhibited in vitro by antilymphocyte serum. The nature of this effect and the factors which mediate it remain unknown. An obvious temptation is to consider it to be an expression of delayed hypersensitivity since the lymphocyte is known to play a fundamental role in this type of immune reaction. The rapidity of the in vitro effect and the disappearance soon after colectomy do not favor such an interpretation. Lymphocyte transformation, an in vitro correlate of delayed hypersensitivity, has been investigated using antigens derived from germ-free rat feces, several strains of E . coli. and normal

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human colon. 170, 171 Although the results of these studies have b&en negative, there is no assurance that the proper antigens have been examined. In a separate study, Stefani and Fink l72 observed reduced blastoid transformation of lymphocytes from patients with active as compared to quiescent disease upon exposure to a pathogenic strain of E. coli, 0119: B14. This finding is reminiscent of the situation in tuberculosis and indicates further study of this and other organisms. An attempt has been made to apply the inhibition of cell migration technique to a study of this problem. The in vitro migration of peripheral leukocytes from patients with ulcerative colitis was inhibited by homogenates of sterile fetal colon and jejunoileal mucosa, being most marked during active disease while leukocytes from patients with Crohn's disease were not affected. l73 As an in vitro correlate of delayed hypersensitivity, this technique has been firmly established only with macrophages. The suitability of substituting peripheral leukocytes requires further study with known delayed hypersensitivity reactions prior to interpretation of these results. Some thought must be given to the possibility that we are not dealing with an exclusively immune reaction. Moeller et al. l74 have shown that nonspecific aggregation of unsensitized allogeneic lymphoid cells to target cells results in a cytotoxic effect. Holm and Perlmann 175 further demonstrated that nonspecific and nonaggregating stimuli also caused allogeneic lymphoid cells to become cytotoxic. More recently Ruddle and Waksman 176 observed a cytotoxic effect on fibroblasts ("innocent bystanders") in both syngeneic and allogeneic systems associated with the interaction of sensitized lymph node cells and specific antigen. It appears likely, therefore, that, although specific immune mechanisms may be operative at some point in this process, the cytotoxic effect per se is nonspecific. Thus substances bearing no relationship to tissue antigens, by interacting with sensitized cells may bring about the destruction of surrounding tissue. This

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could provide an explanation for the seemingly contradictory finding that lymphocytes from patients with either Crohn's disease or ulcerative colitis are cytotoxic for colonic but not ileal cells.

Immunological Disorders with Gastrointestinal Manifestations Patients with either hypogammaglobulinemia (reduction of all immunoglobulins) or dysgammaglobulinemia (selective decrease or increase in one or more immunoglobulins) may develop diarrhea and malabsorption which appear to be related to the immunological defect rather than to malfunction of the gut per se. No attempt here will be made to classify these disorders since hopeless confusion presently surrounds such efforts. It is convenient for this discussion to consider patients with immunoglobulin abnormalities and gastrointestinal symptoms in four groups: (1) hypogammaglobulinemia, (2) nodular lymphoid hyperplasia of the small intestine, (3) isolated IgA deficiency, and (4) thymoma with hypogammaglobulinemia. Most patients with hypogammaglobulinemia who have diarrhea and malabsorption have an aquired rather than a congenital defect. Small intestinal histology is usually normal but villous atrophy indistinguishable from nontropical sprue may be present. The relationship between the immunoglobulin deficiency and the gastrointestinal symptoms remains obscure since attempts to demonstrate a specific infection have generally failed. Patients with nodular lymphoid hyperplasia of the small intestine comprise an interesting but increasingly heterogeneous group. 177, 178 Germinal centers in the gut are hyperplastic and plasma cells are sparse, but the mucosa is otherwise normal. Immunoglobulin defects range from absent IgA and IgM to frank hypogammaglobulinemia. Diarrhea and malabsorption are frequently present and in many patients Giardia lamblia can be recovered. Following Crabbe's original description several patients have been reported with isolated deficiencies of IgA, diarrhea, and

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malabsorption. 179 -181 In some a striking decrease in IgA-containing cells in the gut has also been demonstrated. One patient has been reported with extreme deficiencies of serum IgA and IgM, diarrhea, steatorrhea, and small intestinal changes resembling nontropical sprue in whom normal numbers of immunoglobulin containing cells were present in the intestine. 182 Some patients with a decreased or absent IgA have been reported to be gluten sensitive and to exhibit subtotal villous atrophy. Since most patients with the usual type of gluten sensitive sprue do not have immunoglobulin abnormalities, these features are of uncertain significance. On the other hand isolated IgA deficiency occurs with an incidence of about 1 in 500 indicating that not all patients with IgA deficiency have gastrointestinal symptoms. 183 A few patients, mostly over the age of 50, with hypogammaglobulinemia and diarrhea also have a thymoma but they do not appear to differ substantially from other patients with hypogammaglobuand gastrointestinal symplinemia toms.184, 185 The relationship between immunoglobulin defects and gastrointestinal symptoms is uncertain in view of the inconstant . histological findings and the unpredictable response to treatment; some patients responding spontaneously, others to ,),-globulin, antibiotics, steroids, gluten-free diets, or not at all. An interesting hypothesis has been advanced by Hermans. 177 Evidence has been presented that intestinal lymphoid cells may be derived from the primitive gut epithelium. If this were true an abnormal anlage could be expressed as concurrent but independent abnormalities of the lymphoid cells (immunoglobulin production) and epithelium (absorption). Nodular lymphoid hyperplasia might then represent the gut equivalent of a thymoma, namely hyperplasia of the bursa-related central lymphoid tissue resulting in or from inadequacy of its dependent immunoglobulin-producing system. It seems likely that some abnormality of this type

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affecting the gut-associated lymphoid tissue governing humoral immunity must inevitably occur; with this bit of conjecture we have come full circle. REFERENCES 1. Good, R. A., J. Finstad, H. Gewurz, M. D. Cooper, and B. Pollara. 1967, The development of immunological capacity in phylogenetic perspective. Amer. J. Dis. Child. 114: 477-497. 2. Metcalf, D., and M. Brumby. 1966. The role of the thymus in the ontogeny of the immune system. J. Cell. Physiol. 67: 149-163. 3. Knight, S., J. Bradley, J. J. Openheim, and N. R. Ling. 1968. The in vitro transformation of thymocytes and lymphocytes from humans, rabbits and guinea pigs and from thymomas. Clin. Exp. Immunol. 3: 323-341. 4. Davies, A. J. S., E. Leuchars, V. Wallis, R. Marchant, and E. V. Elliott. 1967. The failure of thymus-derived cells to produce antibody. Transplantation 5: 222-231. 5. Davies, A. J. S., E. Leuchars, V. Wallis, and P. C. Koller. 1966. The mitotic response of thymus-derived cells to antigenic stimulus. Transplantation 4: 438-451. 6. Leuchars, E., A. J. S. Davies, V. Wallis, and P. C. Koller. 1966. Further studies upon the mitotic response of thymus-derived cells to antigenic stimulus. Ann. N. Y. Acad. Sci. 129: 274-282. 7. Cooper, M. D., R. D. A. Peterson, M. A. South, and R. A. Good. 1966. Functions of thymus system and bursa system in chickens. J. Exp. Med. 123: 75-102. 8. Miller, J. F. A. P. 1961. Immunological function of thymus. Lancet 2: 748-749. 9. Good, R. A., A. P. Dalmasso, C. Martinez, O. K. Archer, J. C. Pierce, and B. W. Papermaster. 1962. Role of the thymus in development of immunologic capacity in rabbits and mice. J. Exp. Med. 116: 773-795. 10. Jankovic, B. D., B. H. Waksman, and B. G. Amason. 1962. Role of thymus in immune reactions in rats. I. Immunologic response to bovine serum albumin (antibody formation, Arthus reactivity and delayed hypersensitivity) in rats thymectomized or splenectomized at various times after birth. J. Exp. Med. 116: 159-175. 11. Warner, N. L., A. Szenburg, and F. M. Burnet. 1962. Immunological role of different lymphoid organs in chicken. Aust. J . Exp. Bioi.

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12. Law, L. W., and H . D. Agnew. 1968. Effect of thymic extracts on restoration of immuno-

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logic competence in thymectomized mice. Proc . Soc. Exp. Bioi. Med. 127: 953-956. 13. Levey, R. H., N. Trainin, and L. W. Law. 1963. Evidence for function of thymic tissue in diffusion chambers inplanted in neonatally thymectomized mice. J. Nat. Cancer Inst. 31: 199- 217. 14. Metcalf, D. 1965. Delayed effect of thymectomy in adult life on immunological competence. Nature (London) 208: 1336. 15. Miller, J . F. A. P. 1965. Effect of thymectomy in adult mice on immunological responsiveness. Nature (London) 208: 1337. 16. Sutherland, D. E. R., O. K. Archer, and R. A. Good. 1964. Role of the appendix in development of immunologic capacity. Proc. Soc. Exp. Bioi. Med. 115: 673-676. 17. Archer, O. K., J . C. Pierce, B. W. Papermaster, and R. A. Good. 1962. Reduced antibody response in thymectomized rabbits. Nature (London) 195: 191-193. 18. Konda, S., and T . N. Harris. 1966. Effect of appendectomy and of thymectomy with irradiation on the production of antibodies to two protein antigens in young rabbits. J . Immunol. 97: 805-814. 19. Mitchell, G. F., and J . F. A. P. Miller. 1968. Immunological activity of thymus and thoracic duct lymphocytes. Proc. Nat. Acad. Sci. 59: 296-303. 20. Staples, J. P ., I. Gery, and B. H . Waksman. 1966. Role of the thymus in tolerance. III. Tolerance to bovine gamma globulin after direct injection of antigen into shielded thymus of irradiated rats. J. Exp. Med. 124: 127-139. 21. Horiuchi, A., and B. H. Waksman. 1968. Role of the thymus in tolerance. IV. Tolerance to bovine gamma globulin in rats given a low dose of irradiation and injection of nonaggregated or aggregated antigen into the shielded thymus. J. Immunol. 100: 974-986. 22. Vojti§kova, M. , and A. Lengerova. 1968. Thymus mediated tolerance to cellular alloantigens. Transplantation 6: 13-24. 23. Kretschmer, R., B. Say, D . Brown, and F. S. Rosen. 1968. Congenital aplasia of the thymus gland (DiGeorge's syndrome). New Eng. J. Med. 279: 1295-1301. 24. Cooper, M. D., A. E. Gabrielsen, and R. A. Good. 1967. Role of the thymus and other central lymphoid tissues in immunologic disease. Ann. Rev. Med. 18: 113-134. 25. Glick, B., T. S. Chang, and R. C. Jaap. 1956. The bursa of Fabricius and antibody production. Poultry Sci . 35: 224-225. 26. Glick, B. 1964. The bursa of Fabricius and the

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development of immunologic competence. In R. A. Good and A. E. Gabrielsen [eds.]: The thymus in immunobiology, p. 343-351. Hoeber and Harper, New York. 27. Warner, N. L., and A. Szenburg. 1964. Immunologic studies on normally bursectomized and surgically thymectomized chickens: dissociation of immunologic responsiveness. In R. A. Good and A. E. Gabrielsen [eds.), The thymus in immunobiology, p. 395-411. Hoeber and Harper, New York. 28. Cooper, M . D., R. D. A. Peterson, and R. A. Good. 1965. Delineation of the thymic and bursal lymphoid system in the chicken. Nature (London) 205: 143-146. 29. Amason, B. G., and B. D. Jankovic. 1967. Immunoglobulins after surgical bursectomy in chickens. J. Immunol. 99: 917-924. 30. Cooper M. D., M. L. Schwartz, and R. A. Good. 1966. Restoration of gamma globulin production in agammaglobulinemic chickens. Science 151: 471-473. 31. St. Pierre, R. L. , and G. A. Ackerman. 1965. Bursa of Fabricius in chickens: possible humoral factor. Science 147: 1307-1308. 32. Jacobson, L. 0. , E. K. Marks, D. L. Simmons, and E. O. Gaston. 1961. Immune response in irradiated mice with Peyer's· patch shielding. Proc. Soc. Exp . Bioi. Med . 108: 487-493 . 33. Clawson, C. C. , M . D. Cooper, and R. A. Good. 1967. Lymphocyte fine structure in the bursa of Fabricius, the thymus, and the germinal centers. Lab. Invest. 16: 407-421. 34. Konda, S. 1967. Immune response and changes in gamma globulins in thymectomized and/or appendectomized rabbits. Acta Haem. Jap. 30: 51-67. 35. Cooper, M . D., D .Y. Perey, M . F. McKneally, A. E. Gabrielsen, D. E. R. Sutherland, and R. A. Good. 1966. A mammalian equivalent of the avian bursa of Fabricius. Lancet 1: 1388-1391. 36. Perey, D. Y. E., M. D. Cooper, and R. A. Good. 1968. Lymphoepithelial tissues of the intestine and differentiation of antibody production. Science 161: 265-266. 37. Evans, E. P ., D. A. Ogden, C. E. Ford, and H. S. Micklem. 1967. Repopulation of Peyer's patches in mice. Nature (London) 216: 36-38. 38. Miyakawa, M. 1959. The lymphatic system of germ-free guinea pigs. Ann. N. Y. Acad. Sci. 78: 221- 236. 39. Carlens, O. 1928. Studies uber das lymphatische, gewebe das darmkenels beienigen haustieren, mit benonder berucksichtigung der embryonolen entwicklung, der mengenverroltnisse und der altersinovoilution dieses

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gewebes im dunndaum des rindes. Z . Anat. Entwicklungsgesch. 86: 393-493. 40. Cornes, J. S. 1965. Number, size and distribution of Peyer's patches in the human small intestine. I. The development of Peyer's patches. Gut 6: 225-229. 41. Andrew, W. 1965. Lymphocyte transformation in epithelium. J. Nat. Cancer Inst. 35: 113137. 42. Meader, R. D., and D. F . Landers. 1967. Electron and light microscopic observations on relationships between lymphocytes and intestinal epithelium. Amer. J. Anat. 121 : 763-773. 43. Darlington, D., and A. W. Rogers. 1966. Epithelial lymphocytes in the small intestine of the mouse. J. Anat. 100: 813-830. 44. Fichtelius, K. E., E. J . Junis, and R. A. Good. 1968. Occurrence of lymphocytes within the gut epithelium of normal and neonatally thymectomized mice. Proc . Soc . Exp. Bioi. Med. 128: 185-188. 45. Fichtelius, K. E. 1968. The gut epitheliuma first level lymphoid organ? Exp . Cell Res. 49: 87-104. 46. Freter, R. 1964. Comparison of immune mechanisms in various experimental models of cholera. Bull. W. H . 0.31 : 825-834 . 47. Freter, R. 1962. Detection of coproantibody and its formation after parenteral and oral immunization of human volunteers. J. Infect . Dis. 111: 37-48. 48. Pierce, A. E., and M. W. Smith. 1966. The in vitro transfer of bovine immune lactoglobulin across small intestines of newborn pigs. J. Physiol. (London) 186: 85-86P. 49. Pierce, A. E .,and M . W. Smith. 1967. The in vitro transfer of bovine immune lactoglobulin across the intestine of newborn pigs. J. Physiol . (London) 190: 19-34. 50. Farr, R. S., W. Dickinson, and K. Smith. 1960. Quantitative aspects of oral immunization against bovine serum albumin. Fed. Proc . 19: 199. 51. Farr, R. S ., and W. Dickinson. 1961. The transfer of antibody producing capacity from intestinal to other lymphatic tissue. Fed. Proc. 20: 25. 52. Cooper, G. N ., and K. Turner. 1967. Immunological response in rats following antigenic stimulation of Peyer's patches. I. Characteristics of the primary response. Aust. J . Exp. Bioi. Med . Sci. 45: 363-378. 53. Cooper, G. N ., W. J. Halliday, and J. C. Thonard. 1967. Immunological reactivity associated with antigens in the intestinal tract of rats. J. Path. Bact. 93: 223-233.

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54. Bockman, D., and W. B. Winborn. 1966. Light and electron microscopy of intestinal ferritin absorption. Observations in sensitized and non-sensitized hamsters (Mesocricetus auratus). Anat. Rec. 155: 603-609. 55. Korenblatt, P. E ., and R. Rothberg. 1968. Immune response of human adults after oral and parenteral exposure to bovine serum albumin. J. Allerg. 41: 226-235. 56. Jeffries, G. H. 1965. Immunofluorescent studies in adult celiac disease. J. Clin. Invest . 44: 475-485. 57. Crabbe, P. A., and J . F. Heremans. 1966. The distribution of immunoglobulin-containing cells along the human gastrointestinal tract. Gastroenterology 51: 305-316. 58. Gelzayd, E. A., S. C. Kraft, and J. B. Kirsner. 1968. Distribution of immunoglobulins in human rectal mucosa. I. Normal control subjects. Gastroenterology 54: 334-340. 59. Tomasi, T. B., Jr. 1968. Human immunoglobulin A. New Eng. J. Med. 279: 1327-1330. 60. South, M . A., M .D. Cooper, F . A. Wollheim, and R. A. Good. 1968. The IgA system. II. The clinical significance of IgA deficiency: studies in patients with agammaglobulinemia and ataxia telangiectasia. Amer. J . Med . 44: 168-178. 61. Eidelman, S. , S. D. Davis, D. Lagunoff, and C. E. Rubin. 1966. The relationship between intestinal plasma cells and immunoglobulin A (lgA) in man. J . Clin. Invest. 45: 10031004. 62. Eidelman, S., and S. D. Davis. 1968. immunoglobulin content of intestinal mucosal plasma cells in ataxia telangiectasia . Lancet 1: 884-886. 63. Tomasi, T. B. Jr., E. M. Tan, A. Solomon, and R. A. Prendergast. 1965. Characteristics of an immune system common certain external secretions. J. Exp. Med . 121 : 101-124. 64. Gelzayd, E. A., S. C. Kraft, and F . W. Fitch. 1967. IgA: localization in rectal mucosal epithelial cells. Science 157: 930-931. 65. Adinolfi, M., A. A. Glynn, M . Lindsay, and C. M. Milne. 1966. Serological properties of gamma A antibodies to E. coli present in human colostrum. J . Immunol . 10: 517- 526. 66. Tokumaru, T . 1966. A possible role of gamma A immunoglobulin in herpes simplex virus infection in man. J. Immunol. 97: 248-259. 67. Stobo, J. D., and T . B. Tomasi, Jr. 1967. A low molecular weight immunoglobulin antigenically related to 19S IgM . J. Clin. Invest. 46: 1329-1337. 68. Goldgraber, M. D., and J . B. Kirsner. 1959.

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