THE IMPACT OF INTESTINAL BIOPSY ON GASTROENTEROLOGY

Transcription

1 GAS'fROENTEROLOGY Copyright 1968 by The Williams & Wilkins Cu. Vol. 5-1, No.4, Part 2 of 2 Part., Printed in U.S.A. THE IMPACT OF INTESTINAL BIOPSY ON GASTROENTEROLOGY The development of peroral intestinal biopsy techniques has completely revolutionized our approach to intestinal disease, especially those associated with malabsorption. In addition to providing a greater understanding of disorders such as celiac disease and Whipple's disease, intestinal biopsy has led to the recognition or better characterization of a variety of disorders including intestinal lymphangiectasia, abetalipoproteinemia, tropical sprue, giardiasis, and agammaglobulinemia with malabsorption. A brief discussion of the evolution of our present views of celiac disease and Whipple's disease will serve to illustrate the impact that this technical advance has had on gastroenterology. Celiac Disease Today it is difficult to understand how Thaysen in his monograph on Non-Tropical Sprue written in 1932 could say, "I wish to point out emphatically that in well-preserved material the intestinal epithelium of the intestinal villi shows nowhere any sign whatever of desquamation, degeneration or atrophy... since non-tropical sprue may persist for many years, it must be correct to interpret the intestinal changes described as being of secondary nature." As recently as 1956, the following definition appeared in a prominent review of the pathological physiology, diagnosis, and therapy of nontropical sprue, "Nontropical sprue (idiopathic steatorrhea) may be defined as a disease of unknown etiology without specific anatomico-pathologic changes occurring in adults living in temperate climates in which absorption from and motility of the small bowel are impaired." (Comfort, M. W., and E. E. Wollaeger. Arch. Intern. ~Ied. 98, 1956). What Have Been the Major Factors in the Development of Celiac Disease? First was the demonstration by Dicke in 1950 that it was the cereal protein gluten that induced steatorrhea in childhood celiac disease and that the removal of this protein from the diet of these children led to clinical recovery. Others had suggested that the cause of celiac disease was to be found in dietary factors. Even in 1888, Gee, writing "On the Coeliac Affection," stated that "If the disease is to be cured at all, it is by means of diet." He went on to support his hypothesis by reporting a remission induced by a gluten-free diet (a diet consisting of "a quart of the best Dutch mussels daily") (St. Bartholomew's Hosp. Rep. 24, 1888). Following the lead provided by the Dutch workers, Ruffin et al. and Sleisenger et al. showed that removal of gluten from the diet of patients with adult celiac disease, nontropical sprue, and idiopathic steatorrhea was also associated with clinical remission (New Eng. J. Med. 250, 1954; Trans. Assn. Amer. Physicians 71,1958). The second important development was the demonstration at laparotomy that there is an anatomical lesion in the jejunum of patients with celiac disease (Trans. Roy. Soc. Trop. Med. Hyg. 46, 1952). The third and most important factor was the development of simple and safe peroral intestinal biopsy techniques. If clinicalanatomical correlations could only be made with tissue obtained at laparotomy or autopsy, our progress would have been painfully slow. The exciting opportunities for increasing our understanding of intestinal disease by the use of peroral intestinal biopsy are presented by the early work of Doniach and Shiner.l 797

2 798 IN'l'ES'l'INA.L BIOPSY AND OAS'l'IWEN'J'EROUJOY Vol. 54, No.4, Pari 2 DUODENAL AND JEJUNAL BIOPSIES II. HISTOLOGY 1. DONIACH, M.D., AND MARGOT SHINER, M.R.C.S., D.C.H. From the Departments of Pathology and Medicine, Postgraduate Medical School of London, London, England "As described in the previous paper, duodenal and jejunal biopsies were carried out in a series of 45 patients. We report here the histologic findings. Our object was to find the limits of histologic variation of the normal duodenal and jejunal mucosa and determine the nature of any pathologic processes. In the fundus of the stomach it is comparatively easy to recognize excessive infiltration with inflammatory cells, loss of specialized cells, and metaplasia of glandular epithelium. However, in the mucosa of the small intestine the glands normally are separated by lymphocytes, plasma cells, and eosinophils. There is no process directly comparable with loss of zymogenic and oxyntic cells or their replacement by mucous cells. We hoped, in a mixed series of biopsies, to find a few examples so strikingly different from the rest as to be regarded as pathologic and that we might use the remainder for determining normal limits. DISCUSSION Our findings in the duodenal biopsies give no supporting evidence to the views that duodenitis may (1) be the basis of some dyspepsias, (2) be associated with gastritis, (3) precede duodenal ulcer, or, (4) be associated with peptic ulcer.6-8 None of the 19 patients whose primary symptom was dyspepsia (8 of them with proved gastric or duodenal ulcer) showed a histologically recognizable inflammatory lesion of the duodenum. The only duodenal biopsy with a possible excess of chronic inflammatory cells in the mucosa came from Stanley J., aged 17, with treated iron deficiency anemia; he had no dyspeptic symptoms. The significance of the excess inflammatory cells remains a mystery. There was no atrophy of the duodenal mucosa in the 6 patients with partial or subtotal gastric atrophy not associated with megaloblastic anemia. We have not yet obtained any duodenal biopsies in Addisonian megaloblastic anemia, but the jejunal change we noted in one possible case suggests that the duodenum might show a loss of villi in this disease. The moderate mucosal atrophy and villous irregularity of the duodenum seen in 1 case of duodenal ulcer and 1 case of gastric ulcer might represent regenerated mucosa of a healed erosion or scar. The inflamed duodenal submucosa in patient Winifred W. was an interesting finding. The biopsy was carried out in order to confirm a suggested diagnosis of malignant lymphoma involving the upper intestine. Subsequent laparotomy revealed tuberculous peritonitis. The duodenal biopsy specimen of Ann c., suffering from steatorrhea, was

3 A.pril 1968 INTESTINAL BIOPSY.LYD GASTROENTEROLOGJ' 799 strikingly similar to the jejunal biopsy of Joseph J., who also had steatorrhea and the jejunal biopsy of William B., who suffered from megaloblastic anemia. The impressive finding in all 3 biopsy specimens was the almost total loss of villi. The associated widening of the mucosa and distension of the glands suggests that the mouths of some of the crypts might have been sealed off by adhesions high up between villi. It is tempting to suggest that the malabsorption syndrome of these patients was in part due to the tremendous loss of villous surface epithelium. However, we want only to note the findings at this time; we should like to discuss them when we have seen far more material. This applies also to the 2 borderline cases of jejunal atrophy. The presence of a moderate excess of chronic inflammatory cells in the jejunal villi of 1 case of ulcerative colitis and 1 case of diarrhea, and the absence of this excess in the other cases, suggests that diarrhea might have been the cause rather than the result of the alteration. Our findings in general have proved encouraging from a research point of view. Duodenal biopsy does not seem likely to prove helpful in the elucidation of dyspepsia. But it is clear that jejunal biopsies are of the utmost interest in the studies of malabsorption syndromes of the small intestine. In addition to straight histology, the method is open to bacteriologic and histochemical applications. Biopsies should prove helpful in the differentiation of various types of steatorrhea and might further our understanding of the nature of the mucosal defect." These studies stimulated other clinicopathological studies of celiac disease and amply confirmed Paulley's contention that Thaysen was in error to attribute the pathological changes in the intestine solely to malnutrition or agonal changes. The point was clearly stated by Himes and Adlersberg2 when they said, "The oral technique of obtaining biopsy material represents an important advance in the study of intestinal pathology. The factors of agonal change and post-mortem autolysis are eliminated, thus permitting a more exact interpretation of the pathologic changes in the small intestine." In addition, they observed, "It would appear that the earliest changes occur at the tips of the villi and consist of thickening and swelling (clubbing). Further progression of this process results in fusion of the villus tips, with complete obliteration of the normal pattern. This change results in an almost completely flat surface,yith marked reduction of the area available for absorption from lumen of the small bowel." The early studies raised several questions. (1 ) Was celiac disease in childhood and primary malabsorption in adult life (nontropical sprue or idiopathic steatorrhea) the same disease as suggested by the similar benefit observed when gluten was withdrawn from the diets of these patients? (2) If the same process, was the intestinal lesion congenital or acquired? (3) If intestinal malabsorption was due to loss of absorbing surface area as consequence of the loss of villi, do villi return during clinical remission or in other words, is the intestinal lesion reversible? The first question, the identity of celiac disease and nontropical sprue, was clearly answered in the affirmative by Rubin et aj.3 It is of interest that Gee, writing in 1888, aleo held this view for he clearly states in the opening sentence of his article "On the Coeliac Affection," "There is a kind of chronic indigestion which is met in persons of all ages, yet is especially apt to affect children between one and five years old." In addition to providing strong evidence that celiac disease and nontropical sprue

4 800 INTESTINAL BIOPSY.AND GASTROEN'l'EROLOGY Vol. 54, No.4, Part 2 are identical, Rubin and his co-workers established standards for the preparation and interpretation of intestinal biopsy material that have been invaluable in advancing our understanding of normal and abnormal histology of the small intestinal mucosa. STUDIES OF CELIAC DISEASE I. The Apparent Identical and Specific Nature of the Duodenal and ProxiInal Jejunal Lesion in Celiac Disease and Idiopathic Sprue* CYRUS E. RUBIN, M.D., LLOYD L. BRANDBORG, M.D., PATRICIA C. PHELPS, A.B., AND HAWLEY C. TAYLOR, JR., A.B. From the Division of Gastroenterology, Department of Medicine, University of Washington School of Medicine, Seattie, Washington "In order to investigate systematically the possible relationship between celiac disease and idiopathic sprue, 230 suction biopsies of the duodenum and jejunum were taken from 42 patients with well established diagnoses of celiac disease or sprue and from 73 suitably selected controls. Methods Biopsy In accordance with the principle introduced by Wood et al. 3 and Tomenius" a suction tube was designed" which might be used for duodenal and jejunal biopsy with equal facility in children or adults (fig. 3). In more than 300 small bowel biopsies, 300 gastric biopsies, 100 colonic biopsies, and 25 esophageal biopsies, no complications have occurred. Orientation One cannot place too much emphasis on the necessity for correct orientation of the fresh biopsy by an experienced physician before fixation. Expert serial sectioning of the specimen in a plane exactly perpendicular to the mucosal surface by an adept histology technician is also essential. It is all too easy to make erroneous diagnoses when these small mucosal samples are slightly askew." Gross Evaluation Both scientific information and esthetic pleasure were derived from study of the fresh, unsectioned spec~ens during the process of fixation. The normal small bowel biopsy is strikingly beautiful. Its numerous slender villi, laced through with delicate red capillaries, float in the formalin like the tentacles of a sea anemone (fig. 1). What a marked contrast is offered by the severely abnormal biopsy in sprue or celiac disease, with its barren, knobby surface devoid of normal villi (fig. 2). Diagnostic Specificity It is apparent from table 1 that the controls were chosen to assure a proper age distribution. One-quarter of the controls had normal gastrointestinal function, and the rest had various types of malabsorption or situations which theoretically might produce an intestinal lesion similar to that seen in celiac disease and idiopathic sprue (table 3). An effort was made to examine every conceivable lesion which might offer differential diagnostic difficulty. Eight patients with pancreatogenous steatorrhea had a normal proximal intestinal mucosa. These included 6 patients with chronic recurrent pancreatitis, 1 patient with idiopathic pancreatic atrophy, and 1 patient with cystic fibrosis of the pancreas. The small bowel biopsies in 8 patients with partial gastrectomy with or without accompanying steatorrhea were normal. Several of these patients had complicating illnesses such as pancreatitis and alcoholism which added to their malnutrition. A patient with a total gastrectomy had a normal jejunum 6 years after operation, and hers is the biopsy seen in the gross color illustration

5 .tpl'i11968 INTESTINAL BIOPSY AND GASTROENTEROLOGY 801 as a normal control (fig. 1). These findings conform with those observed by Baird and Dodge18 in 23 of 31 patients who had undergone partial gastrectomy. We feel that the few illustrations of "abnormal" biopsies in the latter paper are really normal specimens which have been cut tangentially. The following patients with various small bowel abnormalities had normal proximal intestinal suction biopsies: 1 patient with extensive duodenal and jejunal diverticulosis with megaloblastic anemia; 1 with intermittent volvulus with steatorrhea; 1, active regional enteritis; 2, ileal resections for regional enteritis; and 1, extensive bowel resection for mechanical obstruction. Histologic Evaluation Even with the best orientation only those serial sections from a central cylindric core of a biopsy of adequate size will have villi which are perpendicular to the mucosal surface and which are free of tangential artifacts (figs. 5 and 6). At least a sliver of muscularis mucosae must be included below this core in order to be certain that the full thickness of the epithelial layer is present. In this study such central areas were used exclusively for histologic interpretation and epithelial surface estimation. In the course of preliminary studies several possible sources of histologic misinterpretation were discovered." As indicated above, tangentially cut normal sections were often confusing; what had not been appreciated was that such sections might be indistinguishable histologically from the milder lesions of sprue and celiac disease. Sections taken from the edge of well oriented, normal biopsy specimens may therefore be wrongly diagnosed as the abnormality of celiac disease or idiopathic sprue. Sections taken further along in the same block from its central core, where the villi are perpendicular to the surface of the bowel, prove its normalcy. Another source of error is the "Brunner artifact," which is not infrequent in normal duodenal biopsies." Brunner's glands may penetrate the muscularis mucosae massively, replacing the crypts of Lieberkiihn and flattening the overlying villi. Normal villi will usually be found somewhere in the serial sections of such biopsies, but they may be missing entirely. The question is then best settled by taking a more distal biopsy, because Brunner's glands are largest and most numerous near the pylorus and diminish practically to the point of disappearance as the duodenojejunal junction is approached: A frequent problem encountered in infants and small children is the "lymphoid artifact." It represents a real source of error to the unsuspecting. Although solitary lymphoid nodules are supposed to be scattered throughout the small bowel, they are largest and most numerous in the ileum: They have never been observed in our biopsies of the duodenum or proximal jejunum in our adult controls, although they are undoubtedly present. Lymphoid nodules, however, were common in duodenal biopsies of normalinfantsandchildren. The villi over a lymphoid nodule were often rudimentary or absent; moreover, the adjacent serial sections might appear abnormal despite the fact that they were beyond the nodule's level. Examination of all of the serial sections of a seemingly abnormal biopsy will avoid the pitfall of the "lymphoid artifact." In our experience the most dependable criterion for differentiating the celiac-sprue lesion from controls was the length of epithelial surface. In the severe lesion the villi were markedly blunted or completely absent, which decreased the number of epithelial cells in comparison to those of the controls and made the diagnosis obvious. On the other hand, broad villi per se were not necessarily abnormal; if they had numerous sawtooth indentations, they might be retracted normal villi which had an adequate area of absorptive surface. Branching or long, bent villi also offered interpretive difficulties which were overcome with increasing experience. Other histologic features which have been emphasized in idiopathic sprue B - 13 and celiac disease" were helpful but far less specific diagnostically; i.e., abnormality of the surface epithelium and infiltration of the lamina propria." These studies firmly established the scientific basis for the diagnosis of a variety of small bowel lesions associated with malabsorption. There were some early observations suggesting that the celiac lesion was irreversible; most evidence, however, indicates that the lesion in celiac disease is acquired as a result of exposure to the intestine of a susceptible individual to gluten and that the lesion is reversible when gluten is withdrawn from the diet. This effect on the histological picture was first shown by Sleisenger, Kowlessar, Law, Pert, and Almy (Trans. Assn. Amer. Physicians 71, 1958) and later confirmed by Rubin (Gastroenterology 43, 1962), Zetterqvist and Hendrix

6 802 INTESTINAL BIOPSY ASD GASTROENTEROLOGr 1'01.54, Xo. 4, Part;! (Bull. Johns Hopkins Hosp. 106, 1960), and Anderson (Arch. Dis. Childh. 35, 1960). The original assumption that malabsorption in celiac disease was due merely to a diminished absorbing surface area and the hope that correlations could be made between intestinal function and histology seemed untenable when it was observed that clinical and functional improvement on a gluten-free diet antedated any restoration of normal mucosal architecture (Gastroenterology 39, 1960). On the other hand, when clinical improvement associated with the institution of a gluten-free diet was compared with the restoration of the absorbing units of the intestine-the surface columnar cells-the correlation was very good (Arch. Dis. Childh. 35: 1960; New Eng. J. Med. 267, 1962). With the general acceptance that celiac disease is associated with a characteristic jejunal mucosal lesion which is reversible in part by the withdrawal of gluten from the diet, inquiry into the pathogenesis of the lesion was undertaken. In the study of Padykula et au the pathogenesis of the celiac lesion was interpreted in the context of the intestinal epithelial cell renewal kinetics established by Leblond (Anat. Rec. 100, 1948) and later confirmed in man by Lipkin et al. 5 A MORPHOLOGIC AND HISTOCHEMICAL ANALYSIS OF THE HUMAN JE JUNAL EPITHELIUM IN NONTROPICAL SPRUE HELEN A. PADYKULA, PH.D.," ELLIOTT W. STRAUSS, M.D.,t AARON J. LADMAN, PH.D., AND FRANK H. GARDNER, M.D. Departments of Anatomy and Medicine, Harvard Medical School, and the R. C. Curtis Hematology Laboratory, Peter Bent Brigham Hospital, Boston, Massachusetts "Recent advances in biopsy methods for the diagnosis of intestinal disordersl -3 have made specimens of the mucosa of the human small intestine available for microscopic study. In this investigation, jejunal mucosal biopsy specimens were analyzed by the methods of histochemistry and electron microscopy. The specimens were obtained from normal adults and from patients with nontropical sprue or with other diagnoses. Nontropical spruet is one variant of the malabsorption or sprue syndrome which is characterized by steatorrhea and weight loss associated with a generalized impaired absorption of food, minerals, and water. The intraluminal biopsy methods have permitted the correlation of clinical findings with characteristic histologic abnormalities of the jejunal mucosa. 4-6, 3 These abnormalities are: short blunt villi, longer dilated crypts, abnormal columnar epithelial cells at the luminal surface, and cellular infiltration of the lamina propria. In this report an attempt was made to define the structural and histochemical properties of the human jejunal epithelium in nontropical sprue and also to delineate the points of deviation from the normal. Evidence is offered for the existence of three distinct epithelial zones in the jejunal mucosa in nontropical sprue. The surface epithelial cells are defective structurally and deficient chemically. The upper part of the apparent crypt has the histochemical characteristics of the villus. Measurements indicate a lengthening of the crypts of Lieberkuhn and increased epithelial cell division in the crypts. The present findings have been interpreted in accordance with the well established biologic facts concerning the origin, migration, and differentiation of the intestinal epithelium, as presented by Leblond and his associates.7-9 Our evidence suggests that the abnormality may be related to the mechanism of epithelial replacement and differentiation." Histologic Zonation of the Human Jejunal Mucosa The normal intestine is divided into two histologically distinct zones, the germinative crypts of Lieberkuhn (zone 1) and the

7 April 1968 INTESTINAL BIOPSY ASD G~\STIWKVTEROJ_OG1 ' 80:3 absorptive villi (zone 2) (diagram 1). The epithelium which lines the crypts and covers the villi is a continuous sheet which is constantly and rapidly being renewed.7 It has long been known that mitosis in the normal epithelium is limited to the crypts. is Isotopic marking of the dividing cells by Leblond and Messier9 has demonstrated conclusively that the cells originate in the crypts, migrate along the villi, and are extruded at their tips. In mammals the turnover time, i.e., the time for complete replacement of the epithelium, is approximately 3 days.is An aspect of this epithelial replacement that has received little attention is the differentiation or maturation which alters the epithelial cells during their escalator-like journey. This progressive modification is illustrated in many histochemical preparations and will be introduced here through a description of cytoplasmic basophilia attributable to ribonucleoprotein (RNP). The germinal cells of the crypts show an over-all, diffuse basophilia throughout the cytoplasm (figs. 1 and 3). As the cells migrate along the side of the villus, they become taller, and there is an apparent decrease in cytoplasmic basophilia as well as an increase in cytoplasmic acidophilia. When the absorptive cells have reached the tip of the villus, they have acquired a predominantly acidophilic cytoplasm, although delicate basophilic strands are still present in the apical cytoplasm (fig. 3). The identity of ribonucleic acid in these locations was established by its removal by ribonuclease and also by the slight metachromasia evidenced through toluidine blue. Although these histochemical preparations do not permit a quantitative statement concerning RNP, the cytologic picture suggests that a rearrangement of substance occurs during the migration of the cells. The Golgi zone is recognized as a supranuclear negative image both in the crypt cells and in the absorptive cells at the bases of the villi (figs. 1 and 6). Paneth cells and goblet cells can be identified after staining with eosin-methylene blue. With appropriate fixation and staining, the acidophilic granules of the Paneth cells are distinct in a basophilic cytoplasm; the mucus of goblet cells is deeply basophilic and metachromatic. 2 --::::: =- --=- - ~--.: ::-.::;- ~ --=-= ~ - - NORMAL NON.TROPICAl SPRUE ~ - 2 DIAGRAM 1. Epithelial zonation in the human jejunum. The zonation of the normal and abnormal intestinal epitheliums is derived from histochemical characteristics. Zone 1 comprises the crypts, and this germinal region is more extensive in nontropical sprue. Zone 2 represents the villous epithelium; in sprue this area is considerably smaller. Zones 1 and 2 in sprue are identical histochemically to zones 1 and 2, respectively, of the normal intestine. Zone 3 is the location of the peculiar surface epithelium of sprue, and it shows striking deviations from the normal absorptive epithelium. The precise observations supporting this zonation are presented in the text and photographs. The jejunal epithelium of nontropical sprue has three distinct zones (diagram 1). Long, distended "crypts" open onto a flattened surface. The apparent crypt can be differentiated by several methods into two zones: a basal region (zone 1) of smaller cells which have a higher mitotic rate than normal (tables 1 and 2), and an upper part (zone 2) which has taller cells possessing the properties of villous cells. With respect to RNP, the basal cells of the crypt resemble the normal crypt cells by having a rich diffuse basophilia throughout their cytoplasm. The taller cells of zone 2 (fig. 6) appear identical with those along the side of the normal villus by an apparent diminution of cytoplasmic basophilia, by the prominence of the brush border and Golgi zones, by the regular arrangement, and by enzymatic properties to be described later. The surface cells of zone 3 differ sharply from the typical normal simple columnar epithelium and have a pseudostratified appearance. A unique feature of these cells is

8 804 IXTESTIi\dL BIOPSY.IND G.ISTROENTEROLOGY Vol. 54, No.4, Part 2 the prollounced cytoplasmic basophilia of their apical cytoplasm (figs. 2, 4, and 5). Most of this basophilia can be eliminated by ribonuclease, although some of it is resistant to digestion. A metachromatic reaction with toluidine blue occurs in this cytologic location, which again suggests the presence of RNA.19 If these surface cells originate from those of zone 2, an abrupt increase in the concentration of RNP must occur as the epithelial cells appear at the luminal surface. In contrast to this apparent abundance of RNP, later descriptions will show deficiencies in various enzymatic activities of this zone of the jejunal epithelium. Further investigations in sprue should he directed toward defining the morpholog.0 and histochemical effects of the gluten and hormonal therapy and determining the origin of the surface epithelial cells. Identification of the factors which control the process of epithelial replacement in the normal intestine would provide information essential to an understanding of the abnormal manifestations in sprue." The preceding selected examples served to illustrate how strikingly our views of celiac disease have been modified by studies based on peroral intestinal biopsy. Past accomplishments, although impressive, have not solved the fascinating puzzle that is celiac disease. It seems unlikely, although not conclusively proved, that there is any defect or abnormality in intraluminal digestion of gluten. The identification or even the existence of a defect in the intracellular disposition of the products of gluten digestion has not been established. Finally, it remains to be determined whether local immunological factors are primary in the pathogenesis of the celiac lesion or whether they are merely secondary to damaged intestinal epithelium. Whipple's Disease More cases of Whipple's disease have been described and more interest has been generated in the pathogenesis of this disorder in the decade since the introduction of peroral intestinal biopsy techniques than in the half-century following Whipple's original description. The occasional case reports that followed Whipple's article tended to concentrate attention on the lipid accumulations in the bowel and mesenteric glands and to attribute etiological implications to these findings. With the demonstration that the characteristic macrophages found in Whipple's disease reacted with periodic acid-schiff stain (Proc. Soc. Exp. BioI. Med. 72, 1949), a number of hypotheses were advanced suggesting that Whipple's disease was metabolic, storage, or hypersensitivity disorder (Amer. J. Med. 19, 1955; J. Path. Bact. 68, 1954). Two lines of evidence have recently developed which have materially altered our concepts of Whipple's disease: (1) the long term beneficial defects of antibiotics upon the course of the disease,6 and (2) the identification of bacteria in the lamina propria and macrophages of the intestine. It is of interest to note that it has taken almost 60 years to appreciate the importance of the observations presented by Whipple in his initial case report (Bull. Johns Hopkins Hosp. 18, 1907). "Gland tissue treated by the Levaditi's method shows great numbers of a peculiar rod-shaped organism most numerous in the vacuoles and in the neighborhood of the peculiar 'foamy' cells which often include these structures... It is not claimed that this is the etiological factor in this disease but its distribution is very suggestive." These observations were not repeated until peroral intestinal biopsy tissue became available. When these organisms so clearly described by Whipple were "rediscovered" in electron micrographs they were considered to be protein-carbohydrate complexes which were extended from the cytoplasm of the "foamy" macrophages.7 Yardley and Hendrix, studying similar intestinal biopsy material, pointed out that these particles described by Haubrich et al. had all of the characteristics oi bacteria and confirmed Whipple's original observations (Bull. Johns Hopkins Hosp. 109, 1961). These workers suggested that the

9 April 1968 IN'J'BS'J'INAL BIOPSY AND GASTIWENTEIWLOGY 80;) characteristic periodic acid-schiff (PAS) positive foamy macrophages were the result of phagocytosis of bacteria by these cells. Following destruction of the bacteria an undigestible PAS-positive residue of bacterial capsular material was left in the macrophages. Meanwhile, clinical studies gave added support to Whipple's original observation suggesting the etiological role of bacteria in Whipple's disease. England et ai.6 and Davis et ai.9 reported regular recovery of patients.1ith Whipple's disease treated with antibiotics, whereas other forms of therapy ultimately failed. Trier et a1.1 demonstrated in an elegant fashion that the bacteria seen in the intestinal biopsies and the clinical response to antibiotics could be correlated. It wab shown by serial intestinal biopsies that when patients with Whipple's disease were treated with antibiotics they became asymptomatic, the bacteria disappeared from the mucosa, and the PAS-positive macrophages decreased. They were also able to show that clinical relapses occurring when antibiotics were discontinued always were preceded by the reappearance of bacteria in the mucosa. Others have shown that the intestinal biopsy may return completely to normal with long term antibiotic treatment.9 In spite of all of these indications that bacteria are involved in the pathogenesis of Whipple's disease, there has been little success in culturing an organism that could be clearly implicated. The most convincing report to date is that of Charache et ai. (Trans. Assn. Amer. Physicians 79, 1966). An atypical group D enterococcus was repeatedly isolated from their patient's blood and peripheral lymph nodes. The patient had a high titer against the isolated organism, but not to other organisms. The patient's clinical response to antibiotic therapy paralleled the in vitro sensitivities of the organism. It remains to be proven that the organism seen in Whipple's disease is always an atypical enterococcus and that the organism isolated is the same as seen in the biopsy tissue. It should not be assumed that Whipple's disease is merely an infection with a peculiar organism. There is every indication that in Whipple's disease the host's response to the infection also is very peculiar. No technique since the introduction of radiography has had as great an impact on gastroenterology as peroral intestinal biopsy has had. Not only has peroral intestinal biopsy made the diagnosis of intestinal disease easier and more precise, but it has made it possible to follow the histological consequences of therapy. Probably it is of greater importance that intestinal biopsy has made it possible for the clinical investigator to apply techniques and concepts formerly found only in the physiology or biochemistry laboratories to the study of his patients with intestinal disease. REFERENCES 1. Doniach, I., and M. Shiner Duodenal and jejunal biopsies. II. Histology. Gastroenterology 33: Himes, H. H., and D. Adlersberg Pathologic changes in the small bowel in idiopathic sprue; biopsy and autopsy findings. Gastroenterology 35: Rubin, C. E., L. 1,. Brandborg, P. C. Phelps, and H. C. Taylor, Jr Studies of celiac disease. I. The apparent identical and specific nature of the duodenal and proximal jejunal lesion in celiac disease and idiopathic sprue. Gastroenterology 38: Padykula, H. A., E. W. Strauss, A. J. Ladman, and F. H. Gardner A morphologic and histochemical analysis of the human jejunal epithilium in nontropical sprue. Gastroenterology 40: Lipkin, M., P. Sherlock, and B. Bell Cell pi'oliferation kinetics in the gastrointestinal tract of man. Cell renewal in stomach, ileum, colon, and rectum. Gastroenterology 45: England, M. T., J. M. French, and A. B. Rawson Antibiotic control of diarrhea in Whipple's disease. A six-year follow-up of a patient diagnosed by jejunal biopsy. Gastroenterology 39: Haubrich, W. S., J. H. L. Watson, and J. C. Sierscki Unique morphologic features on Whipple's disease. A study by light and electron microscopy. Gastroenterology 39:

10 Chears, W. C., Jr., and C. T. Ashworth Electron microscopic study of the intestinal mucosa in Whipple's disease. Demonstration of encapsulated bicilliform bodies in the lesion. Gastroenterology 41: Davis, T. D., Jr., J. W. McBee, J. L. Borland, Jr., S. M. Kurtz, and J. M. Ruffin The effects of antibiotic and steroid therapy in Whipple's disease. Gastroenterology 44: Trier, J. S., P. C. Phelps, S. Eidelman, and C. E. Rubin Whipple's disease: light and electron microscopic correlation of jejunal mucosal histology with antibiotic treatment and clinical status. Gastroenterology 48: