Both Celiac Disease (Nontropical Sprue) and Tropical Sprue Are Char- Acterized by Diarrhea, Weight Loss, Steatorrhea and Malabsorption of Various Foodstuffs

MORPHOLOGIC CHARACTERISTICS OF JEJUNAL BIOPSY IN CELIAC DISEASE AND TROPICAL SPRUE

ERIc A. SCHENK, M.D.; I. MICHAEL SAMLOFF, M.D., AND FREDERICK A. KLIPSTEIN, M.D. From the Departments of Pathology and Medicine, University of Rochester School of Medicine and Dentistry, Rochester, N.Y., and College of Physicians and Surgeons of Columbia University, New York, N.Y. Both celiac disease (nontropical sprue) and tropical sprue are char- acterized by diarrhea, weight loss, steatorrhea and malabsorption of various foodstuffs. Malabsorption of folic acid and vitamin B12 commonly results in a megaloblastic anemia. Celiac disease may be dis- tinguished clinically from tropical sprue by its hereditary nature, its occurrence in temperate as well as tropical climes, and its relationship to dietary gluten. Tropical sprue is found exclusively in tropical and subtropical climes, and occurs in endemic, and occasionally epidemic form. Tropical sprue is not influenced by a gluten free diet, but therapy with folic acid, vitamin B12 or oral antibiotic agents commonly result in a clinical remission. Histologically definable abnormalities of the jejunal mucosa in celiac disease and in tropical sprue have thus far not allowed differentiation of these conditions. Peroral biopsy specimens in both show partial or complete absence of villi, an abnormal surface epithelium and an inflammatory cell infiltrate in the lamina propria. It has been proposed that this pattern of change in the jejunal mucosa is an entirely non- specific reaction to injury regardless of the inciting cause.' Since the epidemiologic features and response to therapy indicate that celiac disease and tropical sprue are distinct and separate disease processes, the present study was undertaken to determine whatever morphologic similarities and differences might be definable in these conditions by the use of more elaborate cytochemical and histochemical techniques than have heretofore been utilized.

This work was supported in part by grants HE o6089 and CA-02332 from the United States Public Health Service, an Advanced Research Fellowship from the American Heart Association (Dr. Schenk), and an Advanced Clinical Fellowship from the American Cancer Society (Dr. Samloff). Presented in part at the Annual Meeting of the International Academy of Pathology, Philadelphia, Pa., March, x965. Accepted for publication, June 22, I965. 765 766 SCHENK, SAMLOFF AND KLIPSTEIN Vol. 47, No. 5

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MATERIAL AND METHODS Peroral jejunal biopsy tissues were obtained from 7 patients with untreated celiac disease and from 4 patients with untreated tropical sprue. Pertinent clinical features and laboratory data are outlined in Table I. Of the 7 patients with celiac disease, 4 (Cases 2, 4, S and 7) had had recurrent episodes of diarrhea and weight loss since childhood, and 3 (Cases I, 3 and 6) first developed these symptoms in adult life. Two of the patients (Cases I and 2) were relatively asymptomatic at the time of biopsy. Of the patients with tropical sprue, symptoms began, in case i,S months before admission while she was still a resident of Puerto Rico. Case 2, a native of Puerto Rico but a resident of New York City for 14 years, had symptoms for 4 months. Case 3 developed symptoms I year after leaving Puerto Rico and was ad- mitted i month later. Case 4 had symptoms for 6 years while a resident in the Domin- ican Republic and subsequently for i year as a resident of New York City. All biopsies were made under fluoroscopic control in the region of the ligament of Treitz in the morning after a 12- to 14-hour fast. Usually 2, and at times 3 specimens were obtained during each intubation without moving the tube by turning the patients into supine, prone and lateral positions. Each biopsy specimen was placed on a no. i Onksponge (Histomed, Inc., Paterson, N. J.) and oriented so that its submucosal surface rested on the sponge. One of the specimens from each case was placed on a cryostat chuck, quick-frozen in dry ice, and sectioned at io p with a Lipshaw cryostat at-28' C. Serial sections were taken up on slides and immediately stored at-85o C in a cascade type deep freeze unit. The following histochemical stains were performed on the frozen sections: acid phosphatase by the method of Burstone2 at pH 5.2, using naphthol AS-TR as substrate; alkaline phosphatase by the method of Burstone2 at pH 8.2, using naphthol AS-MX as substrate; succinic dehydrogenase by the method of Nachlas and asso- ciates,3 using tetranitroblue tetrazolium, but no phenazine methosulfate; lactic dehydrogenase by the method of Hess, Scarpelli and Pearse,4 using tetranitroblue tetrazolium and DPN but no polyvinyl pyrrolidone; adenosinetriphosphatase (ATPase) by the lead nitrate method of Wachstein and Meisel,5 and also by the calcium-cobalt method of Padykula and Herman;6 cytochrome oxidase by the method of Burstone 7; and diphosphopyridine nucleotide diaphorase (DPN) and triphosphopyridine nucleotide diaphorase (TPN) by the nitroblue tetrazolium method of Scarpelli, Hess and Pearse.8 Appropriate controls for these histochemical procedures consisted of (a) the use of specific inhibitors, as discussed by the authors of the various methods given above; (b) the incubation of duplicate slides in media with and without substrate; (c) the use of jejunal biopsy tissues obtained from control patients in each incubation media; and (d) the simultaneous incubation of specimens obtained at different times and from different patients during this study. Fresh frozen sections, fixed in io per cent neutral buffered formalin, were stained forlipid with oil red 0. The other specimens were fixed in io per cent neutral buffered formalin, embedded in paraffin, sectioned at 6 g and stained with hematoxylin and easin and with the Gomoritrichrome (for connective tissue), the periodic acid-Schiff (PAS), Alcian blue and mucicarmine (for mucins and mucopolysaccharides) methods. Control patients comprised 13 native inhabitants of the continental United States, 6 males and 7 females, ranging in age from 27 to 74. There were also 2 normal

control Puerto Ricans. None of these patients had celiac disease or tropical sprue.

Two of the controls were normal male volunteers.Jejunal tissues were obtained and the specimens were processed as described above. 768 SCIIENK, SAMLOFF AND KLIPSTEIN Vol. 47, No. 5

RESULTS Histologic Features of Jejunal Mucosa Normal. With the dissecting microscope the normal villi appeared as tall, delicate, round, finger-like projections. Occasional tongue or leaf shaped forms were present. The average height of the mucosa, measured from the base of the crypts to the tips of the villi, was o.85 mm with a range of 0.78 to 0.9I mm. Microscopic sections showed tall and slender villi. The surface epithelium consisted of regular columnar cells. De- generating cells were frequently seen at the tips of villi. Occasionally, sloughing of single or clumps of the degenerating cells from the tips of the villi was apparent. The lamina propria contained a sparse infiltrate of lymphocytes, plasma cells, histiocytes, and rarely, eosinophils. In- flammatory cells were extremely uncommon between surface or crypt epithelial cells. Endothelial lined vascular and lymphatic channels were always distinct and adjacent to a delicate basement membrane which separated the surface epithelium from the lamina propria (Fig. io). Celiac Disease. Examination of tissue from 5 of the 7 patients with the dissecting microscope showed a flat mucosa with an occasional mosaic pattern. Histologically, the jejunal mucosa was flat because of an absence of villi in all patients. The average mucosal height was 0.72 mm, with a range from o.6i to o.88 mm. The superficial epithelial cells were pseudostratified and non-columnar, with considerable variation in both size and shape. There were nuclear pyknosis and focal cytoplasmic vacuolization. An inflammatory cell infiltrate composed of lymphocytes and plasma cells was present within the surface epithelium and occasionally within that of the crypts. The basement membrane separating the surface eipthelium from the lamina propria was thickened in many areas and stained as collagen. An increase in the amount of connective tissue staining as collagen was present in the lamina propria which contained a dense inflammatory cell infiltrate consisting of lymphocytes, plasma cells, histiocytes and eosinophils. Lymphatic and vascular channels here, especially in the region beneath the basement membrane, were difficult to define and either absent or obscured by the dense inflammatory cell infiltrate (Fig. ii). Alcian blue and PAS stains showed a decreased number of goblet cells in the surface epithelium and an increased number in the crypt epithelium. The local- ization and staining reaction of the acid mucopolysaccharides of goblet cells stained with Alcian blue, and the neutral mucopolysaccharides of the brush border stained with PAS, were similar to those seen in control biopsy tissues (Figs. I3 and 14). Tropical Sprue. There was considerable variation in the degree of Nov., z965f JEJUNAL BIOPSY 769 mucosal abnormality. Examination with the dissecting microscope re- vealed a normal villous pattern in case 2, prominent villous ridging in cases i and 4, and a flat convoluted pattern in case 3. The histologic changes corresponded to the dissecting microscopic appearance. In case 3 there was a completely flat mucosa with complete loss of villi (Fig. 3). Cases i and 4 showed blunting and broadening of the villi, and case 2 had a normal villous pattern (Fig. 6). The height of the mucosa in cases i, 2, 3 and 4 was o.63 mm, 0.84 mm, 0.52 mm and 0.55 mm respectively. Although the mucosal height was usually uniform throughout a section, the degree of villous alteration was often non- uniform. In the same section, markedly broad villi and areas of villous fusion with a flat surface were often found adjacent to slightly broad and non-fused villi. Changes in the surface epithelium varied from normal (Case 2) to markedly atypical (Case 3). The atypical surface epithelial cells were low columnar to cuboidal and showed slight pseudo- stratification and variation in cell size and shape. Nuclear pyknosis, abnormal cytoplasmic staining and vacuolization were less marked than in celiac disease. A dense inflammatory cell infiltrate consisting of lymphocytes, plasma cells and eosinophils was present in the surface epithelium (Fig. I2). The basement membrane was thickened and stained as collagen. This was most prominent below the surface epithelium and less so adjacent to the crypt epithelium. The amount of thickening was quite variable within the same section. There was no correla- tion between the degree of basement thickening and the loss of villous pattern or mucosal atrophy. Increased connective tissue was present in the lamina propria, where in some areas it clearly surrounded vascular and lymphatic channels (Fig. I2). The inflammatory cell infiltrate in the lamina propria consisted of lymphocytes, plasma cells, histiocytes and eosinophils. Eosinophils were occasionally present in exceptionally large numbers, especially at the bases of crypts and in the subjacent submucosa. Goblet cells were decreased in number in the surface epithelium and increased in the crypt epithelium in the 3 specimens showing villous alteration. The staining reaction of acid mucopolysaccharides in goblet cells and neutral mucopolysaccharides in the brush border was similar to that seen in the controls (Fig. I5). Lipid Distribution Normal and Control Subjects. All patients were fasting for I2 to 14 hours prior to biopsy. Lipid droplets were present in the lamina propria lacteals. A fine haze of lipid was frequently found in the cytoplasm of the surface epithelial cells, but distinct lipid droplets were unusual in this location, and not seen at all in the 2 normal volunteers (Fig. i). 770 SCHENK, SAMLOFF AND KLIPSTEIN Vol. 47, No. 5 Lipid droplets were occasionally present in degenerated cells being sloughed at the tips of villi. Celiac Disease. There was an accumulation of lipid droplets ranging in size from approximately 0.5 to 4 u in the superficial epithelial cells, predominantly in the supranuclear region. Droplets were also occasionally present in the upper lamina propria beneath the basement membrane. In this location they were more uniform in size, ranging from 0.5 to I P; for the most part they lay within histiocytes but were also apparently extracellular. Lipid was not found in the mid or lower lamina propria except within occasional histiocytes. This distribution of lipid was a constant feature in all specimens from patients with celiac disease. The amount of stainable lipid in the same section, however, was variable in that no lipid was found in some areas (Figs. 2 and 4). Tropical Sprue. Lipid droplets measuring approximately o.s to I ,u were present within the thickened basement membrane (Figs. 3, and 5 to 9). This was a constant feature in all of the tissues obtained from patients with tropical sprue and the only evidence of abnormality in case 2, in whom the mucosal pattern was normal (Fig. 6). The amount of stainable lipid in the basement membrane in the same section was variable, and there was no apparent relationship between the degree of basement membrane thickening and the presence of lipid within it. In case i only, fine lipid droplets measuring o.s to I u in size, also appeared in the surface epithelial cells with both an infra- and supranuclear location (Fig. 7). Lamina propria lipid was absent from lymphatic channels and present only within histiocytes (Fig. 8). Enzyme Distribution and Activity Normal and Control Subjects. Alkaline phosphatase activity was present in the brush border of villous columnar epithelium. Acid phosphatase activity was located immediately beneath the brush border in the supranuclear region of the cells. Histiocytes in the lamina propria also contained this enzyme. Lactic and succinic dehydrogenase, cytochrome oxidase and DPN and TPN diaphorase appeared mainly in the apical and supranuclear portions of the cytoplasm and were less prominent lateral to the nucleus and at the base of the cell. ATPase activity was prominent in the apical cytoplasm and along the cell membranes. Both the Wachstein and Meisel lead method and Padykula calcium method for ATPase gave similar results but the Padykula technique appeared to stain the cell membranes more distinctly. ATPase activity was also present in the walls of blood vessels in the lamina propria and submucosa by both techniques (Fig. I6). Untreated Celiac Disease. ATPase activity was markedly decreased Nov., tg65 JEJUNAL BIOPSY 77I1 to absent in the surface epithelium. This loss was most severe along the free border of the cells, where the most prominent activity was observed in the controls (Fig. I7). Intracytoplasmic ATPase activity was occasionally present but diminished in cells in which brush border activity was lacking. The activity of alkaline phosphatase was the same as that in control subjects. A patchy decrease in acid phosphatase activity in the surface epithelium was present in most sections. There was also a marked increase in the number of acid phosphatase containing histiocytes in the lamina propria. A variable decrease in activity was also evident for lactic and succinic dehydrogenase, cytochrome oxidase and DPN and TPN diaphorase. This decrease in activity was more marked for succinic dehydrogenase and cytochrome oxidase than for lactic dehydrogenase and DPN and TPN diaphorase. Tropical Sprue. Alkaline phosphatase activity was normal in all specimens. Acid phosphatase, lactic dehydrogenase and DPN and TPN diaphorase were slightly decreased, while ATPase, succinic dehydrogenase and cytochrome oxidase were moderately to markedly decreased. In contrast to celiac disease, ATPase activity, although decreased in terms of intensity of staining, was present in the surface epithelium (Fig. i8). The degree of enzyme activity loss was again related to the degree of abnormality of the mucosal villous pattern. Case 2 had both a normal villous pattern and a normal spectrum of enzyme activity, while case 3 whose mucosa was flat, showed the most prominent decrease in enzyme activity. DISCUSSION Our findings indicate distinct and characteristic morphologic changes in jejunal biopsy tissues from patients with untreated celiac disease and tropical sprue. These changes relate to (a) the overall mucosal height and villous pattern, (a) the distribution of lipid and (c) the activity of certain epithelial enzymes. Mucosal Pattern. All of the specimens from patients with celiac disease examined with the dissecting microscope showed a flat surface with or without a mosaic pattern, and complete absence of villi on microscopic section. In contrast, all of the abnormal tropical sprue specimens exhibited a ridged or convoluted pattern with the dissecting microscope, and a variable loss of villous pattern histologically. The marked varia- bility of mucosal changes in even the small number of patients with tropical sprue examined is of interest. One patient in our group with clinical and histochemical evidence of tropical sprue indeed had an entirely normal villous pattern. This finding, together with the observa- 772 SCHENK, SAMLOFF AND KLIPSTEIN VoI. 47, No. 5 tions of Baker and colleagues9 that the extent of villous loss in tropical sprue may be slight, may well explain the confusing statements in the early literature summarized by Thaysen 10 and Manson-Bahr," about the absence or presence of intestinal mucosal lesions in this condition. The terms "subtotal villous atrophy" and "partial villous atrophy" were introduced by Doniach and Shiner12 to describe the structural changes seen in the jejunum in patients with celiac disease. Subtotal villous atrophy referred to a mucosa which was flat and devoid of normal villi. Partial villous atrophy was used to describe a mucosa with shortened, broadened and thickened villi. The use of the terms subtotal and partial villous atrophy, although widely accepted, has been criticized since the mucosal changes observed may not represent an atrophic process,18 especially if atrophy is defined classically as an acquired decrease in size of a tissue or organ resulting from a reduction in cell size or a decrease in the total number of cells. In support of the view that atrophic changes are not present in celiac disease, Reid and Brunser'4 have shown that the total thickness of the jejunal mucosa is not significantly different from that in normal individuals. Our observations in 7 adult patients with celiac disease confirm this. The mucosal height in all of these was not markedly different from that seen in normal controls. In contrast, tissues from 3 of 4 patients with tropical sprue, and from a number of additional patients with tropical sprue which we have recently examined,15 consistently showed a 30 to 6o per cent decrease in mucosal height. A possible explanation for this would be intestinal dilatation. Radiographic examination, however, has not impressed us that significantly more small bowel dilatation is present in individuals with tropical sprue than in those with celiac disease. Although the mechanisms responsible for the loss of villous pattern and decrease in mucosal height are not entirely clear,"' our observations suggest that an atrophic process is a more reasonable explanation for the alterations encountered in tropical sprue than for those in celiac disease. In addition to the variations in mucosal height, there are a number of less clearly definable and subtler morphologic differences between celiac disease and tropical sprue. In general, the surface epithelium in tropical sprue shows less atypism. The surface epithelial cells in celiac disease are pseudostratified, cuboidal or irregularly shaped, and exhibit cytoplasmic vacuolization and nuclear pyknosis. In tropical sprue the epithelium is frequently columnar with regular basally or midcellular located nuclei, and the major abnormality seen is a dense inflammatory cell infiltration between epithelial cells. The severity of histologic changes in the surface epithelium in tropical sprue is directly related to the loss of villous pattern. Nov., z965 JEJUNAL BIOPSY 773 Morphologic features which appear to be similar in both conditions are an inflammatory cell infiltrate in the lamina propria, variable thickening of the basement membrane separating the surface epithelium from the lamina propria and varying degrees of fibrosis within the lamina propria. Eosinophils and plasma cells are usually abundant in both conditions. The number of goblet cells in the surface epithelium is in- variably decreased in both, while the number of goblet cells in the crypts is usually increased. Both acid and neutral mucopolysaccharides in goblet cells and in the brush border of epithelial cells give a normal staining reaction. We have not observed any abnormalities in Paneth cells in either condition. Lipid Distribution. The use of frozen sections stained with oil red O provides a simple procedure for the demonstration of lipid distribution. Our findings show that there are characteristic patterns of distribution in celiac disease and in tropical sprue which allow differentiation between these conditions and from normal controls. In celiac disease, lipid droplets of varying size appear principally in the surface epithelium; in tropical sprue, uniform small droplets are present in the basement membrane subjacent to the surface epithelium. In both conditions, lipid is usually absent from the lymphatic channels of the lamina propria but is present in histiocytes. In control tissues droplets are found principally within lymphatic channels in the lower one-half of the lamina propria, occasionally within histiocytes, and not in the surface epithelium. In a prior communication we have reported that lipid droplets are present in the upper lamina propria in the region of the basement membrane in celiac disease.17 In this location, lipid was found both extra- cellularly and within histiocytes. Subsequent examination in patients with tropical sprue has clearly demonstrated lipid accumulation not only in the region of the basement membrane but distinctly within a thickened basement membrane. None of our biopsy specimens from patients with celiac disease showed lipid accumulation within the basement membrane, although in some cases the degree of membrane thickening was equal to that seen in tropical sprue. The finding of lipid in the basement membrane exclusively in tissues from clinically documented tropical sprue, even in those with a normal or near normal villous pattern, suggests that this finding may be pathognomonic. Enzyme Distribution. A spectrum of histochemically definable enzyme changes in the jejunal mucosa of celiac disease has been demonstrated by Padykula, Strauss, Ladman and Gardner,18 Spiro and asso- ciates19 and Samloff, Davis and Schenk.17 Of all of the enzyme systems 774 SCHENK, SAMLOFF AND KLIPSTEIN Vol. 47, No. 5 evaluated, only alkaline phosphatase supposedly has a normal staining reaction, although we have seen 3 patients with a probable variant of celiac disease who had decreased to absent activity of this enzyme as well.20 Histochemical analysis of a number of enzymes in two patients with tropical sprue and partial villous atrophy of the jejunum by Spiro and colleagues19 showed a slight general decrease of activity. The alteration of enzyme activity in the surface epithelium in our patients with tropical sprue differs from that in celiac disease in two major respects: (a) the degree of decreased activity in tropical sprue is related to the severity of villous alteration, although patients with tropical sprue and a characteristic jejunal mucosal lipid distribution (vide infra) may have a normal villous pattern and normal epithelial enzyme activity. This situation has not been seen by us or reported previously by others in celiac disease. (b) Even in a "flat" mucosa in a patient with tropical sprue, ATPase activity is demonstrable, although with decreased intensity, in the surface epithelium. This enzyme is markedly decreased to absent in all cases of celiac disease which we have seen. Unfortunately, the major limitation of the histochemical techniques we and others 18"91 have employed lies in the interpretation of any quan- titative differences observed. While quantitation of demonstrable enzyme activity may be possible on a rough scale ranging from normal to decreased to absent, the significance of any degree of reduction except that of absent activity is impossible to interpret. The actual per- cent decrease of any enzyme in a given cell which gives a particular intensity of staining, and its significance in terms of altered cellular metabolism, are unknown. There is, furthermore, no evidence to indicate whether the observed decreased enzyme activity represents a primary direct inhibitory effect of a toxin (gluten) or an infectious agent on the enzyme system, or whether these changes are merely a secondary expression of epithelial atypism. Relationship of the Morphologic Alterations to Lipid Malabsorption. Malabsorption of lipid as well as of other foodstuffs is a feature of both celiac disease and tropical sprue. In our discussion attention is focused principally on the mechanism of lipid malabsorption because of the ease with which lipid may be identified in tissue sections. Normally, fatty acids and monoglycerides enter the epithelial cell along the brush border by a non-energy requiring process. The absorbed fatty acids are converted into triglycerides and phospholipids and incorporated into chylomicrons. In addition to these two lipids, chylomicrons contain protein which has also been synthesized by the epithelial cells. The chylomicrons probably find egress from the lateral border of the epi- Nov., I965 JEJUNAL BIOPSY 775 thelial cells at approximately the level of the nucleus and pass into the extracellular space through the basement membrane into lymphatic channels in the lamina propria. Short chain fatty acids may be trans- ported through the epithelium and into the lamina propria capillaries without being metabolically altered.2" An impairment of lipid absorption may be postulated as a conse- quence to a decreased absorptive area or to a defect in any of the processes responsible for the transfer of lipid from the intestinal lumen to the circulation. There is little doubt that the absorptive area of the small intestine is probably decreased in cases of celiac disease and in most cases of tropical sprue. Creamer16 has demonstrated that with increasing abnormality of the villous shape, from ridged to convoluted to flat, the number of adult epithelial cells covering the villi is progres- sively reduced to approximately 25 per cent of normal. While it is not possible to evaluate the relative role of a decreased absorptive area, our findings of the distribution of lipid in the surface epithelial cells in celiac disease, and in the basement membrane in tropical sprue, indicate that more basic mechanisms than loss of absorptive area are very probably involved. The evidence suggestive of a block in epithelial transport or synthesis of lipids in celiac disease has been discussed previously.17 This interpretation was based on (a) the accumulation of predominantly large droplet lipid within the surface epithelium, (b) an associated marked decrease to absence of ATPase from the surface epithelium, and (c) prompt disappearance of lipid from the surface epithelium and its appearance in lamina propria lymphatic channels, together with a return of ATPase activity in the epithelium within 3 to 5 days after initiation of a gluten free diet, without any apparent change in the morphologic pattern of the villi. In tropical sprue the distribution of lipid principally in the basement membrane suggests a different mechanism of lipid malabsorption from that proposed in celiac disease. Only i of the 4 patients reported in this communication showed lipid accumulation in the surface epithelium and this was in the form of small droplets. Although it is tempting to spec- ulate that a primary block of lipid transport exists at a thickened basement membrane, and when found in the surface epithelial cells lipid represents a "backing up" process, such a proposal would undoubtedly be a gross oversimplification for a number of reasons: (i) In a recent study we have shown that folate repletion may cause the pattern of lipid absorption to return to normal within a short period of time without producing any appreciable change in the thickness of the basement membrane.22 This type of therapy might be expected to have major ef- 776 SCHENK, SAMLOFF AND KLIPSTEIN Vol. 47, No. 5 fect on epithelial function rather than on the metabolic activity or struc- ture of the basement membrane connective tissue. (2) There is no apparent difference in the degree of basement membrane thickening in tissue from patients with celiac disease or with tropical sprue. Although this has not been emphasized previously in tropical sprue, Curran and Creamer23 have described an accumulation of a structureless hyaline substance separating lamina propria capillaries from the epithelial basement membrane in celiac disease. In advanced lesions this substance has been described as "fibrous." A review of the illustrations available strongly suggests that, as in our specimens, the basement membrane it- self becomes thickened and stains as collagen. (3) There is also no apparent relationship between the degree of basement membrane thickening and the accumulation of lipid within it in tropical sprue. (4) No evidence is as yet available that the lipid demonstrated in the basement membrane is of dietary origin. Lipid accumulation in various types of connective tissue is a well-known expression of degenerative changes, and it is possible that the basement membrane in tropical sprue may show ultrastructural and biochemical evidence of such an alteration. Although the observations of lipid distribution permit speculation as to possible mechanisms of malabsorption, it must be emphasized that these changes are of a static nature. The relative importance of various structural changes will be elucidated only when the dynamic pattern of lipid absorption in control cases and in celiac disease and tropical sprue patients can be compared. SUMMARY Peroral jejunal biopsy specimens from patients with untreated celiac disease and tropical sprue show distinctive and characteristic morphologic changes. These consist of differences in mucosal height, the pattern of lipid distribution and the activity of certain epithelial enzymes. In both conditions, lipid is absent from lamina propria lymphatic channels. In celiac disease varied sized lipid droplets appear within the surface epithelium, while in tropical sprue lipid is present within a thickened basement membrane which separates the surface epithelium and lamina propria. The morphologic changes described provide diagnostic criteria for the differentiation of these two diseases and suggest that different mechanisms of malabsorption are involved. REFERENCES i. TOWNLEY, R.R.W.; CASS, M. H., and ANDERSON, C. M. Small intestinal mucosal patterns of coeliac disease and idiopathic steatorrhoea seen in other situations. Gut, I964, 5, 5I-55. Nov., 1965 JEJUNAL BIOPSY 777

2. BURSTONE, M. S. Enzyme Histochemistry and its Application in the Study of Neoplasms. Academic Press, New York and London, I962, 62I Pp. 3. NACHLAS, M. M.; Tsou, K.-C.; DE SOUZA, E.; CHENG, C.-S., and SELIGMAN, A. M. Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem., I 957, 5, 420-436. 4. HEss, R.; SCARPELLI, D. G., and PEARSE, A.G.E. The cytochemical localiza- tion of oxidative enzymes. II. Pyridine nucleotide-linked dehydrogenases. J. Biophys. & Biochem. Cytol., I958, 4, 753-760. 5. WACHSTEIN, M., and MEISEL, E. Histochemistry of hepatic phosphatases at a physiologic pH. With special reference to the demonstration of bile canaliculi. Amer. J. Clin. Path., I957, 27, I3-23. 6. PADYKULA, H. A., and HERMAN, E. Factors affecting the activity of adenosine triphosphatase and other phosphatases as measured by histochemical techniques. J. Histochem. Cytochem., I955, 3, I6I-I69. 7. BURSTONE, M. S. Modifications of histochemical techniques for the demonstration of cytochrome oxidase. J. Histochem. Cytochem., I96I, 9, 59-65. 8. SCARPELLI, D. G.; HEss, R., and PEARSE, A.G.E. The cytochemical localiza- tion of oxidative enzymes. I. Diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase. J. Biophys. & Biochem. Cytol., I958, 4, 747-752. 9. BAKER, S. J.; IGNATIUS, M.; MATHAN, V. I.; VAISH, S. K., and CHACKO, C. C. Intestinal Biopsy in Tropical Spruce. In: Intestinal Biopsy. Ciba Foundation Study Group No. I4. Little, Brown & Co., Boston, I962, pp. 84-IOI. io. THAYSON, T.E.H. Non-trophical Sprue. A Study in Idiopathic Steatorrhoea. Humphrey Milford. Oxford University Press, London, I932, 258 pp. II. MANSON-BAHR, P. The riddle of tropical sprue. J. Trop. Med. & Hyg., I960, 63, 49-55. I2. DONIACH, I., and SHINER, M. Duodenal and jejunal biopsies. II. Histology. Gastroenterology, I957, 33, 7I-86. I3. FONE, D. J.; MEYNELL, M. J.; HARRIS, E. L.; COOKE, W. T.; BREWER, D. B., and Cox, E. V. Jejunal biopsy in adult coeliac disease and allied disorders. Lancet, I960, I, 933-939. I4. REm, A., and BRUNSER, 0. Pathogenesis of small intestine changes in celiac disease. Arch. Path. (Chicago), I964, 77, 525-528. I5. KLIPSTEIN, F. A.; SAMLOFF, I. M., and SCHENK, E. A. Tropical sprue in Haiti. To be published. i6. CREAMER, B. Variations in small-intestinal villous shape and mucosal dynamics. Brit. Med. J., I964, 2, I37I-I373. I7. SAMLOFF, I. M.; DAVIS, J. S., and SCHENK, E. A. A clinical and histochemical study of celiac disease before and during a gluten-free diet. Gastroenterology, I965, 48, I55-I72. I8. PADYKULA, H. A.; STRAUSS, E. W.; LADMAN, A. J., and GARDNER, F. H. A morphologic and histochemical analysis of the human jejunal epithelium in nontropical sprue. Gastroenterology, I96I, 40, 735-765. I9. SpIRO, H. M.; FILIPE, M. I.; STEWART, J. S.; BOOTH, C. C., and PEARSE, A.G.E. Functional histochemistry of the small bowel mucosa in malabsorptive syn- dromes. Gut, I964, 5, I45-I54. 20. SCHENK, E. A., and SAMLOFF, I. M. Unpublished observations. 2I. ISSELBACHER, K. J., and SENIOR, J. R. The intestinal absorption of carbo- hydrate and fat. Gastroenterology, I964, 46, 287-298. 778 SCHENK, SAMLOFF AND KLIPSTEIN Vol. 47, No. 5 22. KLIPSTEIN, F. A.; SCHENK, E. A., and SAMLOPF, I. M. The role of tetracycline and folate repletion in the hematologic and intestinal remission of tropical sprue. (To be published.) 23. CURRAN, R. C., and CREAMER, B. Ultrastructural changes in some disorders of the small intestine associated with malabsorption. J. Path. Bact., I963, 86, i-8.

LEGENDS FOR FIGURES Figures i to 9: Sections were stained with oil red 0. FIG. I. Normal jejunal mucosa. Red staining lipid droplets appear exclusively in lymphatic channels in the lower lamina propria. X 40. FIG. 2. Untreated celiac disease. Lipid droplets appear in the surface epithelium and are absent in the lamina propria. Although the mucosa is completely flat with no villous pattern evident, the height of the mucosa is the same as that of the normal (Fig. I). X 40. FIG. 3. Untreated tropical sprue. Lipid is present almost exclusively in the basement membrane separating the surface epithelium from the lamina propria. The mucosa is flat, and the mucosal height is approximately 6o per cent of that in the normal and in celiac disease. X 40. FIG. 4. Untreated celiac disease. The lipid droplets range from o.5 to 4 ,u and are located predominantly in the supranuclear portion of the epithelium. X IOO. FIG. S. Untreated tropical sprue. The lipid droplets in the thickened basement membrane (Fig. I2) are of uniform small size. X 1oo. FIG. 6. Untreated tropical sprue. A biopsy from a patient with clinical tropical sprue. The mucosal villous pattern is normal and shows lipid in the basement membrane. X IOO. Figures 7 to 9: Untreated tropical sprue. These three sections stained with oil red 0 illustrate certain variations in lipid distribution. FIG. 7. Small lipid droplets are evident in the surface epithelium as well as in the basement membrane. X IOO. FIG. 8. There is slight focal lipid in the basement membrane and surface epithelium, and in histiocytes in the lamina propria. X IOO. FIG. 9. Small lipid droplets are shown in the basement membrane (and perhaps basal portion of the epithelium) lining a crypt. X IOO. Nov., I965 JEJUNAL BIOPSY 779

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Figures io to I2: Gomori connective tissue stain. FIG. iO. Normal jejunal mucosa. The basement membrane which stains as collagen (green) is thin and delicate. x ioo. FIG. II. Untreated celiac disease. There is focal thickening of the basement membrane, especially beneath the surface epithelium. An increased amount of connective tissue is apparent within the lamina propria. The surface epithelium shows marked atypism. X IOO. FIG. I2. Untreated tropical sprue. The changes in the basement membrane are similar to those described in Figure ii. The surface epithelium is more regular than that seen in Figure I I, and there is a prominent chronic inflammatory cell infiltrate among the epithelial cells. X ioo. Figures I3 to I5: Alcian blue-periodic acid-Schiff stain. FIG. I3. Normal jejunal mucosa. Goblet cells principally containing acid mucopolysaccharide (blue) are evenly distributed in the villous surface and crypt epithelium. The brush border of the epithelium stains as neutral mucopolysaccharide (red). X 40. FIG. I4. Untreated celiac disease. Goblet cells are virtually inapparent in the surface epithelium, and appear in increased numbers in the crypts. Staining reaction of the acid and neutral mucopolysaccharide is unchanged from the normal. X 40. FIG. I5. Untreated tropical sprue. Changes are similar to those described in Figure 14. X 40. Figures I6 to i8: Stained for ATPase. FIG. I6. Normal jejunal mucosa. Enzyme activity is present in the apical area of the surface epithelium. X 40. FIG. I 7. Untreated celiac disease. Enzyme activity is absent in the surface epithelium. X 40. FIG. I8. Untreated tropical sprue. Enzyme activity in the surface epithelium is de- tectable but decreased. X 40. Nov., I965 JEJUNAL BIOPSY 78I

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