Whipple's Disease in the Rectum Light and Electron Microscopic Findings

Agustin Gonzalez-Licea, M.D., and John H. Yardley, M.D.

LARGE NuMBERs of PAS-positive macrophages accumulate In the jejunal mucosa of patients with Whipple's disease, and for this reason the diagnosis can be made with great accuracy by using a peroral jejunal biopsy. Since similar cells may be found in the rectum of patients with that disease,lA detection by rectal biopsy has also been suggested.2S However, the rectum differs from the small intestine in a major respect that is pertinent to Whipple's disease. Unlike the small intestine, macrophages similar to those seen in Whipple's disease are commonly present in rectal tissue, whether it is normal or altered by other conditions. This second type of macrophage, termed a muciphage by Azzopardi and Evans,5 also contains a PAS-positive substance. Muciphages in the rectum not only hamper use of rectal biopsy to detect Whipple's disease, but their occurrence also suggests that the disorder might have different ultrstructural racteristics in the rectum. The chief purposes of this study, therefore, were to emine by electron microscopy, Whipple's disease involving the rectum, while giv- ing special attention to features that might be related to muciphage formation and then to contast these findings with Whipple's disease as it is seen in the small intestine. Previous reports i- have shown that the ultrastructure of muciphages differs markedly from macrophages of Whipple's disease in the small intestine. Rectal biopsy specimens from 2 patients with Whipple's disease, one of which showed slight melanosis coli, were investigated. To provide material for comparison, rectal specimens showing muciphages and melanosis coli were also studied. Material and Methods Patients Patient 1 (JHH 117 25 97), whose clinical findings have been described previously,s was a 45-year-old male who had had polyarthralgia, fever with occasional From the Department of Pathology, The Johns Hopkins University School of Medicine and Hospital, Baltimre, Md. Supported in part by Grant AM-00660-13 from the U. S. Public Health Service. Dr. Yardley is the recipient of a Research Career Development Award from the U. S. Public Health Service. Accepted for publication Feb. 27, 1968. Address for reprint requests: Dr. Gonzalez-Licea, Centro Medico Nacional, IM.S.S., Apartado Postal 73/032, Mexico City, 73, D.F., Mexico. 1191 1192 GONZALEZ-LICEA AND YARDLEY Vol. 52, No. 6

chills, abdominal bloating, frequent bowel movements, and loss of appetite and weight for 5 years. He showed reduced serum carotene (28 mg./100 ml.), and X-ray examination revealed coarsened jejunal folds. Stool fat and D-xylose excretion were normal. Patient 2 (JHH 121 14 89) was a 40-year-old male with a 7-year history of polyarthralgia and a 1-year history of increasing visual difficulty. Gastrointestinal symptoms were minimal; although mucosal folds in the small intestine were en- larged as seen by X-ray, laboratory tests for malabsorption, performed shortly after antibiotic treatment had been instituted, were all normal. His progressive blindness was caused by an unusual ocular inflammation that was believed to have been related to his Whipple's disease. Intermittent corticosteroid and ACTH treatment had been given over a 1 %-year period for his eye symptoms, but gave only tempo- rary relief. Initial small-bowel biopsies from both patients showed numerous PAS-positive macrophages in the lamina propria. These showed no more than faint staining by the mucicarmine, Alcian blue, or colloidal iron methods. Electron microscopy on both jejunal specimens revealed macrophage granules typical of Whipple's disease 9-14 (Fig. 1). The biopsy from Patient 1 also revealed numerous extra- and intracellular bacteria; 9-14 these were absent in Patient 2, presumably because antibiotic therapy had been started in the patient 3 weeks previously.18 l4 Both patients showed marked symptomatic response to antibiotic treatment, in- cluding profound improvement in the ocular changes in Patient 2, and there was an associated disappearance of PAS-positive macrophages in subsequent jejunal biopsies from both patients. For control purposes, otherwise normal rectal biopsy specimens containing muciphages were obtained from 3 individuals. One had an Endolimax nana infestation, the second had a rectal stricture, and the third demonstrated Dientamoeba fragilis infestation. Rectal tissue was also obtained from a patient with mucous colitis who showed melanosis coli. Tissue Preparation For light microscopy, rectal biopsy tissue was fixed in either 10% phosphate- buffered formalin or a mixture of formalin, acetic acid, and alcohol. Periodic acid- Schiff (PAS), Alcian blue, mucicarmine, and hematoxylin and eosin staining was done on the tissues from all 5 patients. For electron microscopy, fresh specimens were diced and fixed in chilled 1% phosphate-buffered osmic acid 15 for 45 min. to 1 hr. The tissue was dehydrated in graded alcohols and embedded in Araldite. "Thick" sections (1-2 ,u) of the plastic- embedded material were prepared for light microscopy and stained with toluidine blue and by the PAS reaction.16"17 This permitted accurate localization of PAS- positive macrophages during final trimming of blocks. Ultrathin sections were stained with lead citrate18 or silver methenamine 19 and examined in an RCA EMU 3G or 3H microscope.

Results Light Microscopy Muciphages. A general description of these cells will be given to provide a clear definition of the problem posed by their presence in rectal tissue when studying Whipple's disease. Muciphages occur in about 50%o of all rectal specimens,5 a finding June 1968 WHIPPLE'S DISEASE 1193 we have confirmed in our own material; their number can vary from one to many. The cells have a diameter of from about 10 to 40 ,U; they are most numerous in the lamina propria but may also be found in the submucosa when infiltration is heavy. With hematoxylin and eosin staining, muciphages show a moderate-to-large amount of cytoplasm that is most often eosinophilic but is sometimes faintly basophilic and foamy. Their cytoplasm stains with the PAS reaction (Fig. 2, inset), with methods for acid mucins such as Alcian blue (Fig. 2) or colloidal iron, and with mucicarmine. The stained intracytoplasmic material often appears granular, most noticeably after use of acid fixatives. Muciphages also demonstrate yellow-green autofluorescence with ultraviolet illumination.5'7 This finding, which differs markedly from the pale-blue autofluorescence seen with goblet cell mucin, was regarded by Fisher and Hellstrom 7 as evidence that ceroid was contained in foamy cells seen by them in the rectum. The cells which they described, however, had the same staining and ultrastructural characteristics described for muciphages. Although the matter is not settled, we feel that Azzopardi and Evans 5 were correct in their view that the cytoplasmic material in muciphages is probably epithelial mucin which has passed into the rectal lamina propria and has been phagocytized by macrophages. If this is so, then it must be assumed that the mucin undergoes changes leading to altered autofluorescence after phagocytosis. All 3 rectal specimens used in the present investigation for study of muciphages by electron microscopy showed, by light microscopy, moderate-to-large numbers of cells fitting the above description (Fig. 2). Whipple's Disease. There were no specific cytologic features by which macrophages in the 2 rectal specimens from the patients with Whipple's disease could be unequivocally recognized as due to that disorder rather than to muciphage infiltration. While large numbers of macrophages containing myriads of PAS-postive granules were observed in both patients, the rectum of Patient 1 also revealed strong Alcian blue staining of its macrophages (Fig. 3). The same finding was noted in Patient 2, although to a lesser extent. In addition, a small amount of pigment resembling pseudomelanin of melanosis coli was evident in Patient 2. It is noteworthy that the observations in these 2 cases stood in sharp contrast to findings in a rectal specimen from another patient with Whipple's disease reported from this institution.2 In that patient Alcian blue staining of the numerous PAS-positive macrophages seen in the 1194 GONZALEZ-LICEA AND YARDLEY Vol. 52, No. 6 rectum was only faintly positive (Fig. 4). Thus, stains for acid mucins may at times provide a distinction between rectal involvement with Whipple's disease and muciphage infiltration. This would be expected from the fact that not all rectal biopsies show muciphages. Electron Microscopy General Considerations. Rectal macrophages in all material examined were typically of irregular shape, largely because of numerous pseudopodia. Nuclei were ovoid, and nucleoli were not prominent. The endoplasmic reticulum and mitochondria were well developed. Golgi apparatuses and inclusions resembling lysosomes were scanty in some cells and numerous in others. The macrophage cytoplasm in all specimens showed variably sized, membrane-bound bodies filled with different materials that often seemed to be undergoing digestion. The term "granule" will be applied to these compartmentalized contents of the macrophages. Muciphages. The findings in muciphages were like those previously published.57 There were numerous intracytoplasmic granules which varied in size over a wide range (from about 100 m, to 2 , in diameter). They contained pale material that appeared amorphous at high magnifi- cation (Fig. 5). A darker zone, which may have been due to lipid, was typically present just under the limiting membrane of the smaller-sized granules. Large granules frequently appeared to have been formed by fusion of the smaller ones. Other membrane-bounded electron-dense structures were also noted adjacent to or breaking into the muciphage granules in a few instances. This finding was regarded as evidence of lysozyme transport to the granules. Unidentified, dark, more homoge- neous droplets, which presumably contained lipid in many instances, were observed in the cytoplasm of some muciphages. Nothing was seen in the muciphages, apart from the granules containing amorphous material, that could be reasonably viewed as re- sponsible for the intense, granular PAS and Alcian blue staining. Melanosis Coli. The control patient demonstrating melanosis coli showed macrophages with cytoplasmic deposits consisting of collections of tangled membranes having variable density (Fig. 6). These were entirely similar to previously described findings.20 A bounding membrane was not always visible around the deposits. Variable density of the tangled network of membranes, which differed markedly in ar- rangement from the relatively parallel membranes seen in Whipple's disease, seemed to be caused by varying amounts of superimposed en- crusting material that was presumably the actual pseudomelanin pigment. June 1968 WHIPPLE'S DISEASE 1195 Whipple's Disease. A few bacteria were observed in the extracellu- lar space of the lamina propria in Patient 1, and more rarely inside phagocytic vacuoles of macrophages (Fig. 7). The organisms had di- mensions and an ultrastructural appearance similar to the bacilli found in the small intestine from this patient (Fig. 1) and in other studies of Whipple's disease.10-4 Although bacteria or structures suggesting partially digested organisms were not observed within macrophage granules, the fact that organisms were not detectable previously even in known colonic inflammatory diseases 21 is additional evidence that the bacteria noted in Patient 1 were related to the patient's Whipple's disease. Membrane-bound granules were numerous within the macrophages of both patients. Some granules resembled closely the granules seen in muciphages (Fig. 8). Others, however, showed assorted contents (Fig. 8) that in many instances included membranous material of the type that is highly typical of macrophage inclusions in the small intestine in Whipple's disease (compare with Fig. 1). Still other granules in rectal macrophages, especially from Patient 2, also showed clusters of small fibrils measuring 85-150 A in diameter (Fig. 10, inset). Fibrils of the same type were also prominent in macrophage granules of the small intestine in Patient 2, and they have been previously observed in Whipple's disease.14,22 While occasional granules contained only membranous material and fibrils like that noted in Whipple's disease, most also showed amorphous material similar to that noted in muciphage granules (Fig. 8 and 9). Patient 2 showed less amorphous material than did Patient 1, a finding that correlates with the weaker Alcian blue staining of these macrophages. Some granules in Patient 2 also contained structures like those which occur in melanosis coli (Fig. 10). We even observed occasional single granules having varied components that were individually similar to the constituents of: (1) muciphages, (2) the PAS-positive macrophages of Whipple's disease in the small intestine, and (3) the inclusion material of melanosis coli (pseudomelanin). A few dark, well-demarcated droplets that were presumed to be lipid were noted in granules from both patients with Whipple's disease (Fig. 8 and 9). The silver methenamine method, as adapted for electron microscopy, extended the above observations. This stain localizes to areas that are, in general, stained by the PAS reaction.19 Muciphage granules from the control subjects and the amorphous material within macrophages from Whipple's disease showed a dense, uniform deposition of silver grains (Fig. 11). In Whipple's disease the silver grains were also lo- 1196 GONZALEZ-LICEA AND YARDLEY Vol. 52, No. 6 cated on typical membranous inclusions (Fig. 12). The latter finding is in agreement with previously described observations in the small intestine.13 Still other granules, or areas within granules, showed no silver deposition. Most unstained regions within granules probably repre- sented lipid material or pseudomelanin deposits. Discussion The present ultrastructural study has demonstrated that rectal macrophages in Whipple's disease can contain granules showing membranous material characteristic of the disorder. Furthermore, the phagocytosis of bacilli by macrophages noted in rectal tissue suggests that Whipple's disease develops there in the manner postulated for the small intestine. Numerous observers 1-4,423-26 have concluded from ultrastructural studies that there is invasion of the small intestine by as yet unidentified (but usually gram-positive) bacilliform microorganisms that are phagocytized by macrophages. These cells appear to subsequently accumulate membranous material in their phagocytic vacuoles, leading to granule formation. The accumulation of membranous material may come about because of incomplete digestion of one or more bacterial components, perhaps those derived from cell walls. Membranous material probably accounts for most of the PAS-positive staining seen in untreated Whipple's disease of the small intestine. Similar observations and conclusions have been made from ultrastructural studies of lymph nodes.9'24 On the other hand, our findings in the rectum in Whipple's disease differed from those in the small intestine, especially in its untreated state, in two major respects: (1) macrophage granules in the rectum very frequently contained substances other than the characteristic membranous material, and (2) the rectum showed only small numbers of microorganisms. Trier et al.14 has paid particular attention to those substances other than membranous material which can occur in macrophage granules of Whipple's disease in the small intestine. They noted other substances chiefly in the deepest parts of the mucosa before treatment, but saw them throughout the mucosa after antibiotic therapy had led to disappearance of bacilli and reduction in the amount of membranous material. A major finding among these other substances was amorphous material. An amorphous substance has also been seen in the small intestine by other investigators,25 and comparable findings were noted by us in the small-intestinal biopsies from the 2 patients of this study. In addition, peculiar fibrillar material of the type which we saw in the June 1968 WHIPPLE'S DISEASE 1197 rectum has been described in the small intestine in Whipple's disease, especially after antibiotic 14 or steroid 22 treatment. The latter authors 22 suggested that the fibrils may be glycoprotein. Small osmiophilic droplets, which were possibly lipid, and other ill-defined structures not unlike some of those seen in the rectum, in addition to the membranous material, were also noted by Trier et al.14 In view of the above observations, it is obvious that unambiguous identification of those substances which occur only in the rectum in Whipple's disease, especially with regard to the amorphous material, is not feasible. There is evidence, however, suggesting that much of the amorphous substance seen by us in rectal macrophages in Whipple's disease was different from that often seen when studying the disorder in the small intestine, and that it was in all likelihood equivalent to the amorphous material found in muciphages: (1) The amorphous material seen by us in Whipple's disease of the rectum and in muciphages appeared similar by electron microscopy, using both conventionally prepared and methenamine silver-stained tissue. (2) While Alcian blue stains were strongly positive for muciphages and the rectal macrophages of Whipple's disease, they were negative or faint in simultaneous biopsies from the small intestine in the same patients. Indeed, although there have been exceptions,26 Alcian blue staining of macrophages in the small intestine in Whipple's disease has usually been described as negative or faint.23'27,28 Hence, neither membranous nor amorphous material of Whipple's disease of the type seen in the small intestine accounts as well for Alcian blue staining in the rectum as does the material found in muciphages. Results with mucicarmine stainingB bring one to the same conclusion. (3) Finally, amorphous material was extremely prominent in macrophages throughout the rectal mucosa prior to antibiotic treatment in Patient 1, unlike the comparable small- bowel biopsies described by Trier et al.'4 The occurrence of structures resembling pseudomelanin, in conjunc- tion with membranous material in the macrophages of Patient 2, gives further weight to the conclusion that rectal macrophages in Whipple's disease also continue functioning as muciphages. In fact, after examin- ing available data there is no reason to believe that macrophages in Whipple's disease are functionally altered in any basic way. Enzyme histochemical studies done on small intestine from patients with this disorder,28-30 which have not shown that function is abnormal, are in agreement with the above conclusion. Since knowledge of local and general factors that may be critical to the genesis of Whipple's disease is essentially nonexistent, any com- 1198 GONZALEZ-LICEA AND YARDLEY Vol. 52, No. 6 ment as to why so few bacilli were found in the rectum from the patient who had received no antibiotics is necessarily highly specula- tive. We only suggest that the rectum in Whipple's disease may be less heavily exposed and/or naturally more resistent to the infection. This study confirms and further explains the fact that rectal biopsy in Whipple's disease is of limited diagnostic usefulness because of pos- sible confusion with ordinary muciphage infiltration. One might suspect Whipple's disease in those instances where a rectal biopsy shows many PAS-positive macrophages that do not stain significantly with Alcian blue or other methods for acid mucins. However, jejunal biopsy remains the best source of tissue for reliably detecting the disease and making a definitive diagnosis. Summary Rectal specimens which contained PAS- and Alcian blue-positive macrophages from 2 patients with Whipple's disease were studied by light and electron microscopy and compared with control specimens showing muciphages and melanosis coli. Bacteria were occasionally seen in and among rectal macrophages in the 1 patient with Whipple's disease who had not received antibiotics. Only in Whipple's disease did rectal macrophages show ultrastructural findings typical of that disorder. On the other hand, amorphous material, comparable in several ways to that seen in muciphages, was also present in abundance. Melanosis coli pigment was additionally identified by electron microscopy in 1 patient. It was concluded that while rectal involvement in Whipple's disease probably develops because of infection with an unidentified bacillus in a manner similar to that postulated for the small intestine, macrophages in the rectum can continue to function as muciphages and to accumulate melanosis coli pigrnent in the disorder. The study also confirms and further explains why rectal biopsy is not suitable for reliable detection of Whipple's disease using routine microscopy. References 1. SIERACKI, J. C., and FINE, G. Whipple's disease: Observations on systemic involvement. II. Gross and histologic observations. Arch Path (Chicago) 67:81-93, 1959. 2. FLEMING, W. H., YARDLEY, J. H., and HENDRIX, T. R. Diagnosis of Whipple's disease by rectal biopsy. New Eng J Med 267:33-34, 1962. 3. CARAVATI, C. M., LrrCH, M., WEISIGER, B. B., RAGLAND, S., and BERLINER, H. Diagnosis of Whipple's disease by rectal biopsy with a report of three additional cases. Ann Intern Med 58:166-170, 1963. June 1968 WHIPPLE'S DISEASE 1199

4. HAUBRICH, W. S., WATSON, J. H. L., and SIERACKI, J. C. Unique morphologic features of Whipple's disease: A study by light and electron microscopy. Gastroenterology 39:454-468, 1960. 5. AzzoPARDI, J. G., and EVANS, D. J. Muciprotein-containing histiocytes (muciphages) in the rectum. J Clin Path 19:368-374, 1966. 6. PITTMAN, F. E., SMrIH, W. T., MIzRARI, A., BLANC, W. A., and PrrTMAN, J. C. Clinical, histochemical and electron microscopic study of colonic histiocytosis. Gut 7:458-467, 1966. 7. FISHER, E. R., and HELLSTROMM, H. R. Ceroid-like colonic histiocytosis. Amer I Clin Path 42:581-592, 1964. 8. CHARACHE, P., BAYLESS, T. M., SHELLEY, W. M., and HENDRIX, T. R. Atyp- ical bacteria in Whipple's disease. Trans Ass Amer Physicians 79:399-408, 1966. 9. PAPADIMITRIOU, J. M. Electron microscopic demonstration of organisms in the intestine and mesenteric lymph node in a case of Whipple's disease. Aust Ann Med 14:155-161, 1965. 10. COHEN, A. S. An electron microscopic study of the structure of the small intestine in VVhipple's disease. J Ultrastruct Res 10:124-144, 1964. 11. YARDLEY, J. H., and HENDRIX, T. R. Combined electron and light microscopy in Whipple's disease: Demonstration of "bacillary bodies" in the intestine. Bull Hopkins Hosp 109:80-98, 1961. 12. CHEARS, W. C., JR., and ASHWORTH, C. T. Electron microscopic study of the intestinal mucosa in Whipple's disease: Demonstration of encapsulated bacilliform bodies in the lesion. Gastroenterology 41:129-138, 1961. 13. KENT, T. H., LAYTON, J. M., CLIFTON, J. A., and SCKEDL, H. P. Whipple's disease: Light and electron microscopic studies combined with clinical studies suggesting an infective nature. Lab Invest 12:1163-1178, 1963. 14. TRIER, J. S., PHELPS, P. C., EIDELMAN, S., and RUBIN, C. E. Whipple's disease: Light and electron microscopic correlation of jejunal mucosal his- tology with antibiotic treatment and clinical status. Gastroenterology 48: 684-707, 1965. 15. MILLONIG, C. "Further Observations on a Phosphate Buffer for Osmium Fixation." In Electron Microscopy, Proceedings of the 5th International Congress of Electron Microscopy (Vol. 2), BREESE, S. S., JR., Ed. Acad Press, New York, 1962, p. P8. 16. REVEL, J. P., and HAY, E. D. An autoradiographic and electron microscopic study of collagen synthesis in differentiating cartilage. Z Zellforsch 61:110- 144, 1963. 17. CARDNO, S. S., and STEINER, J. W. Improvement of staining techniques for thin sections of epoxy-embedded tissue. Amer J Clin Path 43:1-8, 1965. 18. REYNOLDS, E. S. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208-212, 1963. 19. MOVAT, H. Z. Silver impregnation methods for electron microscopy. Amer J Clin Path 35:528-537, 1961. 20. SCHRODT, G. R. Melanosis coli: A study with the electron microscope. Dis Colon Rectum 6:277-283, 1963. 21. GONZALEZ-LICEA, A., and YARDLEY, J. H. A comparative ultrastructual study of the mucosa in idiopathic ulcerative colitis, shigellosis and other human colonic diseases. Bull Hopkins Hosp 118:444-461, 1966. 22. ADAMs, W. R., WOLFSOHN, A. W., and SPImo, H. M. Some morphologic characteristics of Whipple's disease. Amer J Path 42:415-429, 1963. 1200 GONZALEZ-LICEA AND YARDLEY Vol. 52, No. 6

23. ASHWORTH, C. T., DOUGLAS, F. C., REYNOLDS, R. C., and THOMAS, P. J. Bacillus-like bodies in Whipple's disease; disappearance with clinical remis- sion after antibiotic therapy. Amer J Med 37:481-490, 1964. 24. KOJECKY, Z., MALINSKY, J., KODONSEK, R., and MARSALEK, E. Frequence of occurrence of microbes in the intestinal mucosa and in the lymph nodes during a long term observation of a patient suffering from Whipple's disease. Gastroenterologia (Basel) 101: 163-172, 1964. 25. KURTZ, S. M., DAVIS, T. D., and RUFFIN, J. M. Light and electron microscopic studies of Whipple's disease. Lab Invest 11:653-665, 1962. 26. ROSTGAARD, J. Bacteria in Whipple's disease. Combined electron and light microscopy. Acta Path Microbiol Scand 60:450-461, 1964. 27. SANDER, S. Whipple's disease associated with amyloidosis. Acta Path Micro- biol Scand 61:530-536, 1964. 28. FISHER, E. R. Whipple's disease: pathogenetic considerations: Electron microscopic and histochemical observations. JAMA 181:396-403, 1962. 29. HOLLENBERG, M. Whipple's disease: A case report, with enzyme histochemical and electron microscopic findings. Amer I Med 32:448-459, 1962. 30. SOBEL, H. J., and AVRIN, E. The histogenesis of Whipple's disease a cyto- chemical, electron microscopic, and electron histochemical study. (abst.) Amer J Clin Path 42:519-520, 1964. The authors are grateful to Drs. Theodore Bayless and Thomas Hendrix for providing biopsy material. Technical assistance was given by Mrs. Gertrude Brown, Mr. Michael Friedman, Miss Martha Gray, Mrs. Joan Uhrin, Miss Pamela Varner, Mr. Edward Walker, and Mrs. Catherine Wichhart. Light micrographs were prepared by Mr. Raymond E. Lund.

Legends for Figures Fig. 1. Whipple's disease (Patient 1). Macrophage in jejunal mucosa showing typical cytoplasmic inclusion (WI). Membranous material typical of the disorder is present. Another granule contains partially digested bacteria (B). Lipid droplets (L) are also seen. X 48,600. Fig. 2. Muciphages (arrow) in lamina propria of rectum (control patient). Note similar staining of goblet cell mucin (G). Alcian blue. X 450. Inset: Muciphages in same specimen. Periodic acid-Schiff. X 450. Fig. 3. Rectal tissue from Whipple's disease (Patient 1). Macrophages (arrows) show finely granular staining for mucin. Compare with Fig. 2. Alcian blue. X 450. Inset: Macrophages also stain heavily with periodic acid-Schiff method. X 450. Fig. 4. Rectal tissue from Whipple's disease (previously published 2). This patient differs from the present 2 in that macrophages (arrow) show only very slight staining for acid mucin. Alcian blue. X 450. Inset: Macrophages are stained strongly positive with periodic acid-Schiff. X 450. June 1968 WHIPPLE'S DISEASE 1201

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Fig. 5. Muciphage (control patient). Several typical granules (G) are shown; they are filled with amorphous material, have a darker peripheral zone, and are membrane bound. Lipid droplet (L) is noted within a granule. Mitochondrion (M) and presumed lysosomes (arrows) are also indicated. X 56,700. Fig. 6. Melanosis coli (control patient). Appearance of pigmented granule within a macrophage. Granule is dark at top and less dense in middle and at bottom where tangled meshwork of membranes is more apparent. X 47,000. Fig. 7. Whipple's disease (Patient 1). Rectal macrophage demonstrating bacteria (B) within phagocytic vacuoles. X 56,700. June 1968 WHIPPLE'S DISEASE 1203

Fig. 8. Whipple's disease (Patient 2). Rectal macrophage with intracytoplasmic granules showing variable contents. Some contain amorphous material (AM) and resemble muciphage granules. Others contain membranous material indicative of Whipple's disease (WI) and of pigment of melanosis coli (P). Lipid droplets (L) are in cytoplasm and within some granules. Nucleus (N) is indicated. X 14,800. Inset: Detail of area indicated by arrow in Fig. 8 shows distinction between amorphous (AM) and adjacent membranous material. X 40,100. 1204 GONZALEZ-LICEA AND YARDLEY Vol. 52, No. 6

Fig. 9. Whipple's disease (Patient 1). Contents of this macrophage granule include membranous material (WI) which reflects Whipple's disease and associated amorphous material (AM) which might result from Whipple's disease. In view of the fact that these cells stain for acid mucins, however, amorphous material may well represent muciphage inclusion material. Lipid droplets (L) are again noted in an adjacent granule and in cytoplasm. X 64,800. Fig. 10. Whipple's disease (Patient 2). In addition to amorphous (AM) and membranous (WI) material, this granule contains a large amount of pigment (P) consistent with melanosis coli (compare with Fig. 6). X 48,600. Inset: Portion of another granule from this patient. Shows appearance of fibrillar substance. X 67,000. June 1968 WHIPPLE'S DISEASE 1205

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Fig. 11. Silver methenamine reaction in muciphage granules (control patient). Note ran- dom distribution of deposited silver and variable intensity of its deposition in different mucin granules. The greater size of silver grains seen here as compared with Fig. 12 is due to longer exposure to silver methenamine. X 64,800. Fig. 12. Whipple's disease (Patient 2). Granule in rectal macrophage. Silver is randomly deposited in amorphous (AM) component but in an aligned fashion along membranous material (WI) of Whipple's disease. Silver methenamine. X 64,800.