The Ultrastructure of Transmissible Murine Colonic Hyperplasia Elizabeth Johnson, MS, and Stephen W. Barthold, DVM, PhD
Transmissible murine colonic hyperplasa was mined ultrastucturally by sequential sampling after inoulation with the etiologic agent, Citrobacter fieundiL Light-micro. scopic changes in the descending colon of inoculated mice were correlated with scaning and transmission electron-microscopic findings Bacteria were attachd to the surfkce of the mucosa between 4 and 10 days after inculation. Hyperplaia was most severe at 16 days and thereafter underwent regression. Regression was e ed by extrssion of infected cells from the surface mucosa and replacement by immature hyperplastic epithelium. Hyperplastic epithelium throughout the crypt resembled u ferentiated crypt cells of controls. By 45 days, the mucosa had reverted to near normal structure. ITe results suggest that severe mucosal prolifertion with minimal inflammatory change resulted from attachment of bacteria to the surface musl epithelium. Ihe hyperplastic reponse appeared to be a defense mechanism of replacing infected cells with newly migrated, uninfected epithelium. (Am J Pathol 97:291-314, 1979)
TRANSMISSIBLE MURINE COLONIC HYPERPLASIA is an infectious disease of mice that provides insight into the mechanisms of mucosal proliferation in the intestine. The disease is caused by a specific variant of Citrobacter freundii (4280) interacting with presently undefined dietary constituents and host genetic factors."2 Infection with C freundii 4280 results in profound hyperplasia of the distal colonic mucosa and minimal inflammation. The lesion is most severe 2-3 weeks after inoculation and then undergoes regression. During its zenith, the hyperplastic mucosal epithelium has a greatly expanded proliferative zone, even encompassing the surface epithelium. The cell cycle time and S phase are prolonged, and the cell migration rate is accelerated.3 The cytokinetics resemble those seen in colonic neoplasia and in human colonic disorders that predispose to neoplasia.34'5 Transmissible murine colonic hyperplasia provides a promoting effect on experimental chemical carcinogenesis, in a manner possibly analogous to the increased susceptibility and early onset of colorectal cancer in patients suffering from mucosal proliferative disease. It reduces the latent period for the appearance of neoplastic changes and enhances the susceptibility of mucosa to the carcinogen 1,2-dimethlhydrazine.6 From the Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut. Supported by the National Large Bowel Cancer Project, USPHS Research Grant 5-R-26-CA15405, National Cancer Institute. Accepted for publication June 14, 1979. Address reprint requests to Dr. Stephen W. Barthold, Section of Comparative Medicine, Yale University School of Medicine, 375 Congress Avenue, New Haven, CT 06510.
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The mechanisms of interaction of C freundii 4280 with the colonic mucosa and subsequent lesion regression are not well understood. The present study explored these aspects of the disease by electron microscopy. Materials and Methods Outbred NIH Swiss [N:(S)] mice were obtained from a Cesarean-derived, barrier-maintained production colony at Yale. The colony was determined free of infectious disease, including colonic hyperplasia, by periodic monitoring. Mice were housed in groups of 5-6 with hardwood-chip bedding in 29x 19X 13-cm polystyrene cages covered with filter caps. They were fed Purina Laboratory Chow and hyperchlorinated water (9 ppm) ad libitum. Fifty-eight mice were inoculated orally at 4 weeks of age with 2-3 drops of an 18-hour culture of C freundii 4280 ' in thioglycollate broth. Seven control mice were inoculated with an equal volume of sterile broth. Mice were killed with CO2 on various days after inoculation. Refer to Table 1 for the number of mice killed and the intervals sampled. The choice of intervals was based upon previous pathogenesis studies.' An additional 9 mice were inoculated with Cfreundii and examined at 1 and 2 days after inoculation. Colons were perfused in situ with McDowell's fixative,7 opened longitudinally, and spread on filter paper. Segments of ascending, transverse, and descending colon were minced and fixed for 4 hours, rinsed in 2 changes of 0.1 M Millonig's buffer with sucrose, pH 7.3, and postfixed in 2% osmium tetroxide in 0.1 M Millonig's buffer for 1 hour. Following dehydration through graded ethanols and propylene oxide, the tissues were embedded in Epon-Araldite 8 and sectioned with an LKB Ultratome III. Thick sections (1-2 m,u) were stained with toluidine blue, and thin sections were stained in alcoholic uranyl acetate and lead citrate.9 Transmission electron microscopy (TEM) was performed with a Philips 201 electron microscope. Pieces of descending colon were also processed for scanning electron microscopy (SEM). Following aldehyde and osmium fixation, tissues were rinsed 10 times in 15 minutes in 0.1 M Millonig's buffer, incubated for 10 minutes in saturated aqueous thiocarbohydrizide, rinsed 10 times in distilled water, and fixed a second time in 2% osmium tetroxide. Samples were dehydrated to 100% ethanol, critical-point dried, coated with gold-paladium, and viewed with an ETEC scanning electron microscope. Tissues from the 3 segments of colon were also processed for light microscopy by placement in Bouin's fixative after original McDowell's fixation. They were embedded in paraffin, sectioned at 5 u, and stained with hematoxylin and eosin (H&E). Crypt cell column heights were determined for the descending colon of each mouse by obtaining the mean height of both sides of 5 complete crypts from H&E sections. These findings were correlated with TEM and SEM findings in the descending colon.
Results The mice developed typical signs and lesions of transmissible murine colonic hyperplasia following C freundii inoculation."l' Crypt cell column heights indicated that hyperplasia was apparent as early as 4 days after inoculation, was maximal at 16 days, and then underwent regression (Table 1). Five mice of the 58 inoculated with C freundii died on Day 16 and were not used in the study. The colons of all control mice were histologically normal. Crypts of the descending colon were composed of columnar cells undergoing a gradual transition from immature, mitotically active vacuolated cells in the lower
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Table 1-Number of Mice Sampled and Mean Crypt Cell Column Heights (± SE) at Selected Intervals After C freundii 4280 Inoculation
Days after inoculation
ot
4
6 10
16f
24 35 45
Number of mice
Crypt height*
7 10 5 8 10 7 7 6
27.3 ± 2.6 38.6+5.1 38.4+2.4 45.3 + 4.8 81.4 + 15.8 43.0 + 4.6 48.8 ± 5.2 45.1 +5.4
* Crypt height = the mean number of cells lining five crypts from the midpoint of the crypt base to the crypt mouth (descending colon). t Day 0 = compendium of all control mice sampled at time 0 (2 mice); Day 4 (1 mouse); Day 16 (2 mice), and Day 45 (2 mice). f Five died and were discarded.
half of the crypt to mature goblet or columnar absorptive cells at the crypt neck. Mature or depleted goblet cells and tall columnar absorptive cells with centrally located nuclei formed a uniform mucosal surface. Crypt units were distributed in a roughly hexagonal pattern when viewed by SEM. Boundaries of surface cells were discernible, but the surface was smooth and covered with an even coat of well-developed microvilli interrupted by occasional goblet cells (Figure 1). Days 4 and 6
Hyperplasia began in most mice inoculated with Cfreundii at these intervals, as evidenced by the slight elongation of the crypt columns (Table 1). The organisms were not readily visible with paraffin sections, but 2-,u toluidine-blue-stained Epon sections revealed numerous bacteria attached to the plasma membrane of surface mucosal epithelium of 8 of 10 mice killed at 4 days and 2 of 5 mice killed at 6 days (Figure 2). In a retrospective study, mice killed at 1 and 2 days after inoculation with C freundii showed no bacterial attachment, and the mucosa resembled that of control mice. Occasionally, bacteria also penetrated crypt lumens and attached to the surface of immature as well as maturing cells along the length of the crypt. Bacterial attachment was also apparent in the transverse colon and less commonly in the ascending colon. The bacteria in these segments were scant and not associated with detectable hyperplasia. When viewed with SEM, myriads of short rod-shaped bacteria were seen attached to each epithelial cell and uniformly covered the surface mucosa (Figure 3). They were identical morphologically to the Cfreundii organisms grown in broth culture. Bacteria were attached to cup-like
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areas on the plasma membrane. Infected cells had few microvilli, bulged into the lumen, and partially distorted the hexagonal crypt pattern. In some mice, particularly on Day 6, many crypt openings were surrounded by a collar of uninfected epithelial cells. These cells appeared to be displacing infected cells to the outer margins of each crypt unit as they migrated onto the surface (Figure 4). Ultrastructurally, bacterial attachment was associated with a number of changes in surface epithelium. Smooth-surfaced bacteria were randomly attached to plaque-like sites on the plasma membrane. Effacement of microvilli occurred at the attachment site, and the numbers of microvilli were reduced along intervening areas of plasma membrane (Figure 5). The underlying cytoplasm contained only remnants of the terminal web (Figure 6). Surface cells were rounded, were separated from neighboring cells by dilated intercellular spaces, and often appeared to be exfoliating. Vacuoles, lipid-like inclusions, and small dense bodies, possibly lysosomes, developed in infected cells, mitochondria degenerated, and the cytoplasm condensed. Bacteria were occasionally observed intracellularly in exfoliating cells and occasionally between cells near the basement membrane, but they were never observed beneath the basement membrane (Figure 7). When crypt elongation was slight, cells at all crypt levels resembled comparable levels in control crypts. Hyperplastic crypts had cellular crowding at the crypt base. Cells in the basal and mid-crypt regions were compressed and elongated (Figure 8). Mitotic figures were increased and usually were limited to the lower half of the crypt. Vacuoles in cells in the lower crypt were small, and the development of mucin droplets in goblet cells occurred over more cell positions than in cells from control mice; so cells with normal amounts of mucus were present only in the upper crypt and crypt neck regions. The lamina propria was compressed in hyperplastic colons by thickening crypt columns but contained a cell population similar to that of controls. Globule leukocytes, seen infrequently in control mice, were observed regularly in mid-crypt regions in infected mice (Figure 9). Day 10
Crypts in the descending colon were moderately hyperplastic (Table 1). Bacteria were attached to surface epithelium in all 8 mice examined, but the pattern of attachment was no longer diffuse. Infected cells were restricted to the margins of the crypt units. SEM views of the surface mucosa showed that there was a diffuse honeycomb pattern of raised inter-
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connected ridges formed by crowded epithelial cells at the interface of adjacent crypt units (Figure 10). Bacterial attachment was restricted to these raised ridges and usually to necrotic or extruding cells at the tops of the ridges. Many of the cells composing these ridges were uninfected. Intervening areas of surface mucosa were composed of uninfected goblet and absorptive cells migrating from crypt openings onto the mucosal surface (Figure 11). Crypt orifices were usually slit-like, in contrast to control crypts, which had round orifices. Bacteria also colonized occasional areas of eroded mucosa. The lower two-thirds of crypts were lined by hyperplastic columnar epithelium with basal nuclei. Large cytoplasmic vacuoles characteristic of cells in the lower crypt region of controls were noticeably absent, but intracytoplasmic Golgi bodies, mitochondria, rough endoplasmic reticulum, and free ribosomes were characteristic of partially differentiated cells. Differentiated cells were located in the upper third of the crypt and migrated onto the surface. Rarely, crypt lumens were distended and filled with neutrophils and large numbers of partially degraded bacteria (Figure
12). Mild hyperplastic changes were also observed in the transverse colon in 2 of the mice in association with moderate numbers of bacteria attached to the surface mucosa. Day 16
Maximal hyperplasia occurred at this time (Table 1). Bacteria were no longer attached to the mucosal surface. The colonic mucosa was composed of severely elongated crypt columns that were often branched at the base and lined by closely packed epithelium. Mitotic figures were found throughout the entire crypt epithelium. Some inflammation occurred in the lamina propria of most mice. Remnants of the honeycomb pattern of ridges remained over much of the mucosal surface. Fewer necrotic cells were located along the ridges, crypt openings were slit-like, and cells migrating from the crypt openings were small. Mature goblet cells were not observed (Figure 13). Ultrastructurally, there was no maturational transition from crypt to surface epithelial cells. Hyperplastic cells lined the entire crypt (Figure 14). They were tall columnar cells with basal nuclei. The cytoplasm contained many free ribosomes with moderate numbers of mitochondria, 1 or 2 supranuclear flattened Golgi bodies, and a few short profiles of rough endoplasmic reticulum. Lateral interdigitations and microvilli were diminished but were more prominent in upper crypt regions. Cells at crypt
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openings and along the luminal surface were electron-dense. Cells in the remaining surface folds and ridges were often separated from the underlying vascular network. These surface cells were cuboidal, and microvilli were absent (Figure 15). Days 24 and 35
Lesions had partially regrfessed. Crypts were still elongated (Table 1), but many crypt cells appeared morphologically normal. Vacuoles reappeared in basal crypt cells. The brush border and associated fuzzy coat were present on upper crypt cells. Surface cells were morphologically normal (Figure 16). Mature goblet cells were present along the crypt column and surface mucosa and often in relatively greater frequency than in control colons. Day 45
Crypts were still slightly elongated (Table 1), but crypt and surface cells were comparable to controls. SEM of the luminal surface showed a flat surface punctuated by round or slit-like crypt openings at slightly irregular intervals. Discussion
Transmissible murine colonic hyperplasia is caused by infection with a specific variant of C freundii. This organism apparently induces hyperplasia through its influence on the surface mucosal epithelium. Bacterial attachment onto surface mucosa occurs within the first 4 days following oral inoculation and results in ultrastructural modifications of the cell membrane and, presumably, cellular function. Hyperplasia of crypt epithelium follows, and the crypts are lined by undifferentiated epithelium. Bacterial attachment persists until about Day 10; then the remaining infected cells are extruded and are replaced by uninfected cells. Hyperplasia intensifies until about Day 16 and then diminishes. The mucosa reverts to normal after the loss of the inciting bacteria and the cellular alterations that they seem to induce. Morphologic changes that occur during development and regression of hyperplasia can be related to altered cytokinetics. Following bacterial attachment around Day 4, there is expansion of the proliferative zone. At 23 weeks after inoculation, the proliferative zone in moderately hyperplastic colons includes the basal half of the crypt, as compared with the lower third in control mice. In severely hyperplastic colons, mitotic activity occurs along the entire crypt and frequently at the luminal surface.
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This corresponds to the spread of incompletely differentiated cells upward from crypt bases. The rate of cell migration is also greatly accelerated as hyperplasia develops.3 The abnormal pattern of surface mucosa, characterized by raised ridges that develop at crypt unit interfaces, apparently results from an imbalance between cell migration from crypts and cell extrusion at the surface. Incompletely differentiated cells are apparently retained and accumulate in these ridges. Transmissible murine colonic hyperplasia resembles certain other proliferative large bowel lesions. The proliferative zone is expanded and the cellular migration rate is accelerated in patients with chronic ulcerative colitis and celiac sprue.5'0 As immature cells migrate onto the luminal surface in ulcerative colitis, the surface mucosa becomes irregular and is composed of furrows of corrugated cells. Goblet cells are absent, as in the peak phase of transmissible murine colonic hyperplasia." In contrast, human colonic hyperplastic polyps are characterized by hypermaturation of crypt epithelium. There is an absence of mitotic activity at the surface.'2 Cell migration and turnover occur at a slower than normal rate.'3 In kinetics and structure murine colonic hyperplasia more closely resembles human colonic adenomatous lesions 14 and 1,2-dimethylhydrazine-induced neoplasia in the mouse.'5 Changes in the luminal surface have been noted in rats following 8 weeks of 1,2-dimethylhydrazine treatment.'6 Protruding crypts with slit-like openings resembled those seen in murine colonic hyperplasia. Attachment of Cfreundii to the plasma membrane appears to be an essential step in the pathogenicity of this organism. Intestinal colonization is enhanced with other enterobacteria by adhesive factors located in filamentous structures (pili) on the surface of the organisms.17 Pili were not found on unattached C freundii organisms in broth culture or those located at the cell surface, although Sedlak reports that several strains of C freundii have pili.'8 C freundii was found in intimate contact with the plasma membrane but, unlike certain strains of Escherichia coli, Salmonella, and Shigella,'9-2' did not penetrate beyond this point. Several other types of protozoa and bacteria have also been found on the mucosal surface of mouse intestines. They include the bacterium Streptobacillus monoliformis and the protozoans Cryptosporidium muris and Hexamita muris.22'23 C freundii, like Salmonella typhimurium in the mouse and Shigella flexneri in the monkey and guinea pig, produces immediate cytoplasmic alterations in infected epithelial cells. Colonized cells bulge into the lumen, possibly because of the breakdown of the terminal web, and separate from neighboring cells. Lipid-like inclusions, vacuoles, and small
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dense bodies, possibly lysosomes, develop in the cytoplasm. Similar cellular changes are associated with S flexneri infection in colonic epithelium of monkeys.2' Certain species of Citrobacter have an "endotoxin with a biological activity roughly equivalent to that of other enterobacteria."'8 Cellular changes in murine colonic hyperplasia may either be related to an endotoxin or to interruption of the normal processes of cellular metabolism related to the distortion of surface epithelium brush border induced by Cfreundii. C freundii appears to attach preferentially to the mucosa of the descending colon, although extension of hyperplasia to the transverse and ascending colons as well as the cecum have been reported.' When hyperplasia was observed in the transverse and ascending colon, the degree of hyperplasia seemed to be correlated with the number of bacteria attached to the surface epithelium. The predilection for the descending colon may reflect differences in receptor sites, concentration of enhancing luminal factors, or bacteria themselves. Diet and host genetics are known to influence the severity of this disease, possibly by one or more of these mechanisms.2 Enteropathogenic E coli colonize posterior regions of the small intestine in vivo,17 while work with ligated intestinal loops indicates that bacteria attach to jejunal epithelium as well as to ileal epithelium,24 suggesting that luminal factors may be important determinants in bacterial attachment. Infected cells were displaced by newly arising epithelium and eventually were completely extruded. The sequential observations in this study indicate that bacteria attach to the epithelium only during the first 4 days, after which time the number of bacteria in the lumen may decline or changes in the luminal environment may interfere with further attachment. Luminal antibody or antibody at surfaces of migrating cells after Day 4 may render these cells resistant to further bacterial attachment. Although the surface absorptive epithelium appears to be preferentially involved, cellular maturity does not appear to affect bacterial attachment in transmissible murine colonic hyperplasia. In instances where the organism had penetrated crypts, bacteria were found attached along the entire crypt column. Similarly, in studies of E coli attachment to isolated epithelial cells, no differences in attachment to "crypt" or "tip" cells were detected.25 Intestinal mucosal proliferation is modified by a variety of physiologic factors, including establishment of normal microflora, bile acids, hormones, nutrition, age, and general state of health.3 Pathologic insults to the intestinal mucosa also stimulate proliferation. Infection without exten-
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sive cell death can result in mucosal hyperplasia, as seen in ileal hyperplasia in hamsters 2 and ileal adenomatosis of swine,27 both of which are associated with the presence and replication of intracellular bacteria. Injury to the mucosa by organisms such as Shigella, Salmonella, some strains of E coli, and some intestinal viruses"'-' induces rapid regeneration of mucosal epithelium via transient crypt hyperplasia. Cfreundii appears to induce hyperplasia via an incompletely understood effect upon the surface mucosal epithelium and resulting feedback to cryptal proliferative cells. References 1. Barthold SW, Coleman GL, Bhatt PN, Osbaldiston GW, Jonas AM: The etiology of transmissible murine colonic hyperplasia. Lab An Sci 26:889-894, 1976 2. Barthold SW, Osbaldiston GW, Jonas AM: Dietary, bacterial, and host genetic interactions in the pathogenesis of transmissible murine colonic hyperplasia. Lab An Sci 27:938-945, 1977 3. Barthold SW: Autoradiographic cytokinetics of colonic mucosal hyperplasia in mice. Cancer Res 39:24-39, 1979 4. Deschner EE, Lipkin M: Proliferative patterns in colonic mucosa in familial polyposis. Cancer 35:413-418, 1975 5. Eastwood GL, Trier JS: Epithelial cell renewal in cultured rectal biopsies in ulcerative colitis. Gastroenterology 64:383-390, 1973 6. Barthold SW, Jonas AM: Morphogenesis of early 1,2-dimethylhydrazine-induced lesions and latent period reduction of colon carcinogenesis in mice by a variant of Citrobacterfreundii. Cancer Res 37:4352-4360, 1977 7. McDowell EM, Trump BF: Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 100:405-414, 1976 8. Mollenhauer HH: Plastic embedding mixtures for use in electron microscopy. Stain Technol 39:111-114, 1964 9. Venable JH, Coggeshall R: A simplified lead citrate stain for use in electron microscopy. J Cell Biol 25:407-408, 1965 10. Barthold SW, Coleman GL, Jacoby RO, Livestone EM, Jonas AM: Transmissible murine colonic hyperplasia. Vet Pathol 15:223-236, 1978 11. Kavin H, Hamilton DG, Greasley RE, Eckart JD, Zuidema G: Scanning electron microscopy: A new method in the study of rectal mucosa. Gastroenterology 59:426432, 1970 12. Lane N, Lev R: Observations on the origin of adenomatous epithelium of the colon. Serial section studies of minute polyps in familial polyposis. Cancer 16:751-764, 1963 13. Hayashi T, Yatani R, Apostol J, Stemmermann GN: Pathogenesis of hyperplastic polyps of the colon: A hypothesis based on ultrastructure and in vitro cell kinetics. Gastroenterology 66:347-356, 1974 14. Kaye GI, Fenoglio CM, Pascal RR, Lane N: Comparative electron microscopic features of normal, hyperplastic, and adenomatous human colonic epithelium: Variations in cellular structure relative to the process of epithelial differentiation. Gastroenterology 64:926-945, 1973 15. Toth B, Malick L, Shimizu H: Production of intestinal and other tumors by 1,2-
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dimethylhydrazine dihydrochloride in mice: I. A light and transmission electron microscopic study of colonic neoplasms. Am J Pathol 84:69-86, 1976 16. Barkla DH, Tutton PJM: Surface changes in the descending colon of rats treated with dimethylhydrazine. Cancer Res 37:262-271, 1977 17. Hohmann A, Wilson MR: Adherence of enteropathogenic Escherichia coli to intestinal epithelium in vivo. Infect Immun 12:866-880, 1975 18. Sedlak J: Present knowledge and aspects of Citrobacter. Curr Topics Microbiol Immun 62:41-59, 1973 19. Staley TE, Jones EW, Corley LD: Attachment and penetration of Escherichia coli into intestinal epithelium of the ileum in newborn pigs. Am J Pathol 56:371-392, 1969 20. Takeuchi A: Electron microscope studies of experimental Salmonella infection: I. Penetration into the intestinal epithelium by Salmonella typhimurium. Am J Pathol 50:109-136, 1967 21. Takeuchi A, Formal SB, Sprinz H: Experimental acute colitis in the rhesus monkey following peroral infection with Shigella flexneri: An electron microscope study. Am J Pathol 52:503-529, 1968 22. Hampton JC, Rosario B: The attachment of microorganisms to epithelial cells in the distal ileum of the mouse. Lab Invest 14:1464-1481, 1965 23. Hampton JC, Rosario B: The attachment of protozoan parasites to intestinal epithelial cells of the mouse. J Parasitology 52:939-949, 1966 24. Nagy B, Moon HW, Isaacson RE: Colonization of porcine small intestine by Escherichia coli: Ileal colonization and adhesion by pig enteropathogens that lack K88 antigen and by some acapsular mutants. Infect Immun 13:1214-1220, 1976 25. Wilson MR, Hohmann AW: Immunity to Escherichia coli in pigs: Adhesion of enteropathogenic Escherichia coli to isolated intestinal epithelial cells. Infect Immun 10:776-782, 1974 26. Johnson EA, Jacoby RO: Transmissible ileal hyperplasia of hamsters: II. Ultrastructure. Am J Pathol 91:451-468, 1978 27. Rowland AC, Lawson GHK: Intestinal adenomatosis in the pigs Immunofluorescent and electron microscopic studies. Res Vet Sci 17:323-330, 1974 28. Abrams, GD, Schneider H, Formal SB, Sprinz H: Cellular renewal and mucosal morphology in experimental enteritis: Infection with Salmonella typhimurium in the mouse. Lab Invest 12:1241-1248, 1963 29. Biggers DC, Kraft LM, Sprinz H: Lethal intestinal virus infection of mice (LIVIM): An important new model for study of the response of the intestinal mucosa of injury. Am J Pathol 45:413-422, 1964 30. Takeuchi A, Sprinz H, Labrec EH, Formal SB: Experimental bacillary dysentery: An electron microscopic study of the response of the intestinal mucosa to bacterial invasion. Am J Pathol 47:1011-1044, 1965 31. Wilson MR, Hohmann AW: Immunity to Escherichia coli in pigs: Adhesion of enteropathogenic Escherichia coli to isolated intestinal epithelial cells. Infect Immun 10:776-782, 1974
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Figure 1-Scanning electron micrograph of the colon of a control mouse. One polygonal crypt unit is depicted. (x 1 600) Figure 2-Light micrograph of the upper portions of crypts and surface mucosa 6 days after inoculation with C freundii 4280. Organisms are attached to the plasma membrane of surface mucosal epithelial cells and crypt neck cells. Epon-Araldite section. (Methylene blue and Azure II, x 1 100)
Figure 3-Scanning electron micrograph of colonic crypt unit following bacterial attachment on Day 6. The crypt opening is enlarged and randomly oriented bacteria cover the surface of rounded epithelial cells. (x 1 050)
1
3
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2
Figure 4-Scanning electron micrograph of a colonic crypt opening 6 days after inoculation with C freundii. The newly migrated cells that form a ring around the crypt mouth are, for the most part, free of bacteria. Many bacteria colonize marginal areas. (x 1 680)
Figure 5-Electron micrograph depicting bacteria along the colonic mucosal surface 4 days after inoculation with C freundii 4280. The brush border is disrupted as bacteria attach to the plasma membrane, and few microvilli remain in intervening areas. (x51 00) Figure 6-Electron micrograph of surface epithelial cell 6 days after inoculation with C freundii 4280. The surface of this heavily infected cell is devoid of microvilli. Organisms are attached to plaque-like areas of plasma membrane. Only remnants of the terminal web persist in the underlying cytoplasm. (x 15,200)
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Figure 8-Electron micrograph of hyperplastic cells in mid-crypt region in moderately hyperplastic colon 6 days after inoculation with C freundii 4280. The elongated cells have a rudimentary brush border, and some contain small amounts of mucin. (x5300)
Figure 9-Globule leukocyte located between immature cells near the crypt base in the mucosa of a mouse 6 days after C freundii 4280 inoculation. (x 1 1,400) Figure 10-Scanning electron micrograph of the luminal surface 10 days after inoculation of C freundii 4280. There is a honeycomb pattern of interconnecting ridges between adjacent crypts. Cells at the apices of ridges are necrotic and detaching from the surface. Recessed crypt openings are slit-like. (x670) Figure 11-Scanning electron micrograph of one crypt unit in the colon 10 days after inoculation with C freundii 4280. Necrotic cells occur at the surface of the ridge. The crypt opening is slit-like, and small goblet and absortive cells have migrated onto the luminal surface. (x 1 1 60)
9
10
I11
Figure 12-Neutrophils within a colonic crypt lumen 10 days after C freundii 4280 inoculation. Cells contain intact and partially degraded bacteria. (x5300)
Figure 13-Scanning electron micrograph of colonic surface mucosa 16 days after inoculation with C freundii 4280. Surface ridges have diminished, but the surface remains convoluted. Fewer necrotic cells are found along surface ridges, and crypt openings are narrow slits. (x330) Figure 14-Transmission electron micrograph of colonic epithelial cells in the upper crypt 16 days after inoculation with C freundii 4280. The cells are elongated and have basal nuclei, and, occasionally, small accumulations of mucus are seen just below the plasma membrane. They have a reduced surface volume along the crypt lumen, a rudimentary brush border, and large numbers of unattached ribosomes in the cytoplasm. (x 10,500)
12
14
Figure 15-Transmission electron micrograph of colonic surface mucosa 16 days after C freundii 4280 inoculation. Cells along surface folds (right) at the peak of hyperplasia are cuboidal and have few microvilli. (x 5900) Figure 16-Electron micrograph of colonic surface mucosa 24 days after C freundii4280 inoculation. Epithelial absorptive and goblet cells are fully developed and morphologically normal. An intact brush border covers the surface mucosa. (x4500)
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Transmissible murine colonic hyperplasa was mined ultrastucturally by sequential sampling after inoulation with the etiologic agent, Citrobacter fieundiL Light-micro. scopic changes in the descending colon of inoculated mice were correlated with scaning and transmission electron-microscopic findings Bacteria were attachd to the surfkce of the mucosa between 4 and 10 days after inculation. Hyperplaia was most severe at 16 days and thereafter underwent regression. Regression was e ed by extrssion of infected cells from the surface mucosa and replacement by immature hyperplastic epithelium. Hyperplastic epithelium throughout the crypt resembled u ferentiated crypt cells of controls. By 45 days, the mucosa had reverted to near normal structure. ITe results suggest that severe mucosal prolifertion with minimal inflammatory change resulted from attachment of bacteria to the surface musl epithelium. Ihe hyperplastic reponse appeared to be a defense mechanism of replacing infected cells with newly migrated, uninfected epithelium. (Am J Pathol 97:291-314, 1979)
TRANSMISSIBLE MURINE COLONIC HYPERPLASIA is an infectious disease of mice that provides insight into the mechanisms of mucosal proliferation in the intestine. The disease is caused by a specific variant of Citrobacter freundii (4280) interacting with presently undefined dietary constituents and host genetic factors."2 Infection with C freundii 4280 results in profound hyperplasia of the distal colonic mucosa and minimal inflammation. The lesion is most severe 2-3 weeks after inoculation and then undergoes regression. During its zenith, the hyperplastic mucosal epithelium has a greatly expanded proliferative zone, even encompassing the surface epithelium. The cell cycle time and S phase are prolonged, and the cell migration rate is accelerated.3 The cytokinetics resemble those seen in colonic neoplasia and in human colonic disorders that predispose to neoplasia.34'5 Transmissible murine colonic hyperplasia provides a promoting effect on experimental chemical carcinogenesis, in a manner possibly analogous to the increased susceptibility and early onset of colorectal cancer in patients suffering from mucosal proliferative disease. It reduces the latent period for the appearance of neoplastic changes and enhances the susceptibility of mucosa to the carcinogen 1,2-dimethlhydrazine.6 From the Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut. Supported by the National Large Bowel Cancer Project, USPHS Research Grant 5-R-26-CA15405, National Cancer Institute. Accepted for publication June 14, 1979. Address reprint requests to Dr. Stephen W. Barthold, Section of Comparative Medicine, Yale University School of Medicine, 375 Congress Avenue, New Haven, CT 06510.
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The mechanisms of interaction of C freundii 4280 with the colonic mucosa and subsequent lesion regression are not well understood. The present study explored these aspects of the disease by electron microscopy. Materials and Methods Outbred NIH Swiss [N:(S)] mice were obtained from a Cesarean-derived, barrier-maintained production colony at Yale. The colony was determined free of infectious disease, including colonic hyperplasia, by periodic monitoring. Mice were housed in groups of 5-6 with hardwood-chip bedding in 29x 19X 13-cm polystyrene cages covered with filter caps. They were fed Purina Laboratory Chow and hyperchlorinated water (9 ppm) ad libitum. Fifty-eight mice were inoculated orally at 4 weeks of age with 2-3 drops of an 18-hour culture of C freundii 4280 ' in thioglycollate broth. Seven control mice were inoculated with an equal volume of sterile broth. Mice were killed with CO2 on various days after inoculation. Refer to Table 1 for the number of mice killed and the intervals sampled. The choice of intervals was based upon previous pathogenesis studies.' An additional 9 mice were inoculated with Cfreundii and examined at 1 and 2 days after inoculation. Colons were perfused in situ with McDowell's fixative,7 opened longitudinally, and spread on filter paper. Segments of ascending, transverse, and descending colon were minced and fixed for 4 hours, rinsed in 2 changes of 0.1 M Millonig's buffer with sucrose, pH 7.3, and postfixed in 2% osmium tetroxide in 0.1 M Millonig's buffer for 1 hour. Following dehydration through graded ethanols and propylene oxide, the tissues were embedded in Epon-Araldite 8 and sectioned with an LKB Ultratome III. Thick sections (1-2 m,u) were stained with toluidine blue, and thin sections were stained in alcoholic uranyl acetate and lead citrate.9 Transmission electron microscopy (TEM) was performed with a Philips 201 electron microscope. Pieces of descending colon were also processed for scanning electron microscopy (SEM). Following aldehyde and osmium fixation, tissues were rinsed 10 times in 15 minutes in 0.1 M Millonig's buffer, incubated for 10 minutes in saturated aqueous thiocarbohydrizide, rinsed 10 times in distilled water, and fixed a second time in 2% osmium tetroxide. Samples were dehydrated to 100% ethanol, critical-point dried, coated with gold-paladium, and viewed with an ETEC scanning electron microscope. Tissues from the 3 segments of colon were also processed for light microscopy by placement in Bouin's fixative after original McDowell's fixation. They were embedded in paraffin, sectioned at 5 u, and stained with hematoxylin and eosin (H&E). Crypt cell column heights were determined for the descending colon of each mouse by obtaining the mean height of both sides of 5 complete crypts from H&E sections. These findings were correlated with TEM and SEM findings in the descending colon.
Results The mice developed typical signs and lesions of transmissible murine colonic hyperplasia following C freundii inoculation."l' Crypt cell column heights indicated that hyperplasia was apparent as early as 4 days after inoculation, was maximal at 16 days, and then underwent regression (Table 1). Five mice of the 58 inoculated with C freundii died on Day 16 and were not used in the study. The colons of all control mice were histologically normal. Crypts of the descending colon were composed of columnar cells undergoing a gradual transition from immature, mitotically active vacuolated cells in the lower
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Table 1-Number of Mice Sampled and Mean Crypt Cell Column Heights (± SE) at Selected Intervals After C freundii 4280 Inoculation
Days after inoculation
ot
4
6 10
16f
24 35 45
Number of mice
Crypt height*
7 10 5 8 10 7 7 6
27.3 ± 2.6 38.6+5.1 38.4+2.4 45.3 + 4.8 81.4 + 15.8 43.0 + 4.6 48.8 ± 5.2 45.1 +5.4
* Crypt height = the mean number of cells lining five crypts from the midpoint of the crypt base to the crypt mouth (descending colon). t Day 0 = compendium of all control mice sampled at time 0 (2 mice); Day 4 (1 mouse); Day 16 (2 mice), and Day 45 (2 mice). f Five died and were discarded.
half of the crypt to mature goblet or columnar absorptive cells at the crypt neck. Mature or depleted goblet cells and tall columnar absorptive cells with centrally located nuclei formed a uniform mucosal surface. Crypt units were distributed in a roughly hexagonal pattern when viewed by SEM. Boundaries of surface cells were discernible, but the surface was smooth and covered with an even coat of well-developed microvilli interrupted by occasional goblet cells (Figure 1). Days 4 and 6
Hyperplasia began in most mice inoculated with Cfreundii at these intervals, as evidenced by the slight elongation of the crypt columns (Table 1). The organisms were not readily visible with paraffin sections, but 2-,u toluidine-blue-stained Epon sections revealed numerous bacteria attached to the plasma membrane of surface mucosal epithelium of 8 of 10 mice killed at 4 days and 2 of 5 mice killed at 6 days (Figure 2). In a retrospective study, mice killed at 1 and 2 days after inoculation with C freundii showed no bacterial attachment, and the mucosa resembled that of control mice. Occasionally, bacteria also penetrated crypt lumens and attached to the surface of immature as well as maturing cells along the length of the crypt. Bacterial attachment was also apparent in the transverse colon and less commonly in the ascending colon. The bacteria in these segments were scant and not associated with detectable hyperplasia. When viewed with SEM, myriads of short rod-shaped bacteria were seen attached to each epithelial cell and uniformly covered the surface mucosa (Figure 3). They were identical morphologically to the Cfreundii organisms grown in broth culture. Bacteria were attached to cup-like
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areas on the plasma membrane. Infected cells had few microvilli, bulged into the lumen, and partially distorted the hexagonal crypt pattern. In some mice, particularly on Day 6, many crypt openings were surrounded by a collar of uninfected epithelial cells. These cells appeared to be displacing infected cells to the outer margins of each crypt unit as they migrated onto the surface (Figure 4). Ultrastructurally, bacterial attachment was associated with a number of changes in surface epithelium. Smooth-surfaced bacteria were randomly attached to plaque-like sites on the plasma membrane. Effacement of microvilli occurred at the attachment site, and the numbers of microvilli were reduced along intervening areas of plasma membrane (Figure 5). The underlying cytoplasm contained only remnants of the terminal web (Figure 6). Surface cells were rounded, were separated from neighboring cells by dilated intercellular spaces, and often appeared to be exfoliating. Vacuoles, lipid-like inclusions, and small dense bodies, possibly lysosomes, developed in infected cells, mitochondria degenerated, and the cytoplasm condensed. Bacteria were occasionally observed intracellularly in exfoliating cells and occasionally between cells near the basement membrane, but they were never observed beneath the basement membrane (Figure 7). When crypt elongation was slight, cells at all crypt levels resembled comparable levels in control crypts. Hyperplastic crypts had cellular crowding at the crypt base. Cells in the basal and mid-crypt regions were compressed and elongated (Figure 8). Mitotic figures were increased and usually were limited to the lower half of the crypt. Vacuoles in cells in the lower crypt were small, and the development of mucin droplets in goblet cells occurred over more cell positions than in cells from control mice; so cells with normal amounts of mucus were present only in the upper crypt and crypt neck regions. The lamina propria was compressed in hyperplastic colons by thickening crypt columns but contained a cell population similar to that of controls. Globule leukocytes, seen infrequently in control mice, were observed regularly in mid-crypt regions in infected mice (Figure 9). Day 10
Crypts in the descending colon were moderately hyperplastic (Table 1). Bacteria were attached to surface epithelium in all 8 mice examined, but the pattern of attachment was no longer diffuse. Infected cells were restricted to the margins of the crypt units. SEM views of the surface mucosa showed that there was a diffuse honeycomb pattern of raised inter-
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connected ridges formed by crowded epithelial cells at the interface of adjacent crypt units (Figure 10). Bacterial attachment was restricted to these raised ridges and usually to necrotic or extruding cells at the tops of the ridges. Many of the cells composing these ridges were uninfected. Intervening areas of surface mucosa were composed of uninfected goblet and absorptive cells migrating from crypt openings onto the mucosal surface (Figure 11). Crypt orifices were usually slit-like, in contrast to control crypts, which had round orifices. Bacteria also colonized occasional areas of eroded mucosa. The lower two-thirds of crypts were lined by hyperplastic columnar epithelium with basal nuclei. Large cytoplasmic vacuoles characteristic of cells in the lower crypt region of controls were noticeably absent, but intracytoplasmic Golgi bodies, mitochondria, rough endoplasmic reticulum, and free ribosomes were characteristic of partially differentiated cells. Differentiated cells were located in the upper third of the crypt and migrated onto the surface. Rarely, crypt lumens were distended and filled with neutrophils and large numbers of partially degraded bacteria (Figure
12). Mild hyperplastic changes were also observed in the transverse colon in 2 of the mice in association with moderate numbers of bacteria attached to the surface mucosa. Day 16
Maximal hyperplasia occurred at this time (Table 1). Bacteria were no longer attached to the mucosal surface. The colonic mucosa was composed of severely elongated crypt columns that were often branched at the base and lined by closely packed epithelium. Mitotic figures were found throughout the entire crypt epithelium. Some inflammation occurred in the lamina propria of most mice. Remnants of the honeycomb pattern of ridges remained over much of the mucosal surface. Fewer necrotic cells were located along the ridges, crypt openings were slit-like, and cells migrating from the crypt openings were small. Mature goblet cells were not observed (Figure 13). Ultrastructurally, there was no maturational transition from crypt to surface epithelial cells. Hyperplastic cells lined the entire crypt (Figure 14). They were tall columnar cells with basal nuclei. The cytoplasm contained many free ribosomes with moderate numbers of mitochondria, 1 or 2 supranuclear flattened Golgi bodies, and a few short profiles of rough endoplasmic reticulum. Lateral interdigitations and microvilli were diminished but were more prominent in upper crypt regions. Cells at crypt
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openings and along the luminal surface were electron-dense. Cells in the remaining surface folds and ridges were often separated from the underlying vascular network. These surface cells were cuboidal, and microvilli were absent (Figure 15). Days 24 and 35
Lesions had partially regrfessed. Crypts were still elongated (Table 1), but many crypt cells appeared morphologically normal. Vacuoles reappeared in basal crypt cells. The brush border and associated fuzzy coat were present on upper crypt cells. Surface cells were morphologically normal (Figure 16). Mature goblet cells were present along the crypt column and surface mucosa and often in relatively greater frequency than in control colons. Day 45
Crypts were still slightly elongated (Table 1), but crypt and surface cells were comparable to controls. SEM of the luminal surface showed a flat surface punctuated by round or slit-like crypt openings at slightly irregular intervals. Discussion
Transmissible murine colonic hyperplasia is caused by infection with a specific variant of C freundii. This organism apparently induces hyperplasia through its influence on the surface mucosal epithelium. Bacterial attachment onto surface mucosa occurs within the first 4 days following oral inoculation and results in ultrastructural modifications of the cell membrane and, presumably, cellular function. Hyperplasia of crypt epithelium follows, and the crypts are lined by undifferentiated epithelium. Bacterial attachment persists until about Day 10; then the remaining infected cells are extruded and are replaced by uninfected cells. Hyperplasia intensifies until about Day 16 and then diminishes. The mucosa reverts to normal after the loss of the inciting bacteria and the cellular alterations that they seem to induce. Morphologic changes that occur during development and regression of hyperplasia can be related to altered cytokinetics. Following bacterial attachment around Day 4, there is expansion of the proliferative zone. At 23 weeks after inoculation, the proliferative zone in moderately hyperplastic colons includes the basal half of the crypt, as compared with the lower third in control mice. In severely hyperplastic colons, mitotic activity occurs along the entire crypt and frequently at the luminal surface.
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This corresponds to the spread of incompletely differentiated cells upward from crypt bases. The rate of cell migration is also greatly accelerated as hyperplasia develops.3 The abnormal pattern of surface mucosa, characterized by raised ridges that develop at crypt unit interfaces, apparently results from an imbalance between cell migration from crypts and cell extrusion at the surface. Incompletely differentiated cells are apparently retained and accumulate in these ridges. Transmissible murine colonic hyperplasia resembles certain other proliferative large bowel lesions. The proliferative zone is expanded and the cellular migration rate is accelerated in patients with chronic ulcerative colitis and celiac sprue.5'0 As immature cells migrate onto the luminal surface in ulcerative colitis, the surface mucosa becomes irregular and is composed of furrows of corrugated cells. Goblet cells are absent, as in the peak phase of transmissible murine colonic hyperplasia." In contrast, human colonic hyperplastic polyps are characterized by hypermaturation of crypt epithelium. There is an absence of mitotic activity at the surface.'2 Cell migration and turnover occur at a slower than normal rate.'3 In kinetics and structure murine colonic hyperplasia more closely resembles human colonic adenomatous lesions 14 and 1,2-dimethylhydrazine-induced neoplasia in the mouse.'5 Changes in the luminal surface have been noted in rats following 8 weeks of 1,2-dimethylhydrazine treatment.'6 Protruding crypts with slit-like openings resembled those seen in murine colonic hyperplasia. Attachment of Cfreundii to the plasma membrane appears to be an essential step in the pathogenicity of this organism. Intestinal colonization is enhanced with other enterobacteria by adhesive factors located in filamentous structures (pili) on the surface of the organisms.17 Pili were not found on unattached C freundii organisms in broth culture or those located at the cell surface, although Sedlak reports that several strains of C freundii have pili.'8 C freundii was found in intimate contact with the plasma membrane but, unlike certain strains of Escherichia coli, Salmonella, and Shigella,'9-2' did not penetrate beyond this point. Several other types of protozoa and bacteria have also been found on the mucosal surface of mouse intestines. They include the bacterium Streptobacillus monoliformis and the protozoans Cryptosporidium muris and Hexamita muris.22'23 C freundii, like Salmonella typhimurium in the mouse and Shigella flexneri in the monkey and guinea pig, produces immediate cytoplasmic alterations in infected epithelial cells. Colonized cells bulge into the lumen, possibly because of the breakdown of the terminal web, and separate from neighboring cells. Lipid-like inclusions, vacuoles, and small
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dense bodies, possibly lysosomes, develop in the cytoplasm. Similar cellular changes are associated with S flexneri infection in colonic epithelium of monkeys.2' Certain species of Citrobacter have an "endotoxin with a biological activity roughly equivalent to that of other enterobacteria."'8 Cellular changes in murine colonic hyperplasia may either be related to an endotoxin or to interruption of the normal processes of cellular metabolism related to the distortion of surface epithelium brush border induced by Cfreundii. C freundii appears to attach preferentially to the mucosa of the descending colon, although extension of hyperplasia to the transverse and ascending colons as well as the cecum have been reported.' When hyperplasia was observed in the transverse and ascending colon, the degree of hyperplasia seemed to be correlated with the number of bacteria attached to the surface epithelium. The predilection for the descending colon may reflect differences in receptor sites, concentration of enhancing luminal factors, or bacteria themselves. Diet and host genetics are known to influence the severity of this disease, possibly by one or more of these mechanisms.2 Enteropathogenic E coli colonize posterior regions of the small intestine in vivo,17 while work with ligated intestinal loops indicates that bacteria attach to jejunal epithelium as well as to ileal epithelium,24 suggesting that luminal factors may be important determinants in bacterial attachment. Infected cells were displaced by newly arising epithelium and eventually were completely extruded. The sequential observations in this study indicate that bacteria attach to the epithelium only during the first 4 days, after which time the number of bacteria in the lumen may decline or changes in the luminal environment may interfere with further attachment. Luminal antibody or antibody at surfaces of migrating cells after Day 4 may render these cells resistant to further bacterial attachment. Although the surface absorptive epithelium appears to be preferentially involved, cellular maturity does not appear to affect bacterial attachment in transmissible murine colonic hyperplasia. In instances where the organism had penetrated crypts, bacteria were found attached along the entire crypt column. Similarly, in studies of E coli attachment to isolated epithelial cells, no differences in attachment to "crypt" or "tip" cells were detected.25 Intestinal mucosal proliferation is modified by a variety of physiologic factors, including establishment of normal microflora, bile acids, hormones, nutrition, age, and general state of health.3 Pathologic insults to the intestinal mucosa also stimulate proliferation. Infection without exten-
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sive cell death can result in mucosal hyperplasia, as seen in ileal hyperplasia in hamsters 2 and ileal adenomatosis of swine,27 both of which are associated with the presence and replication of intracellular bacteria. Injury to the mucosa by organisms such as Shigella, Salmonella, some strains of E coli, and some intestinal viruses"'-' induces rapid regeneration of mucosal epithelium via transient crypt hyperplasia. Cfreundii appears to induce hyperplasia via an incompletely understood effect upon the surface mucosal epithelium and resulting feedback to cryptal proliferative cells. References 1. Barthold SW, Coleman GL, Bhatt PN, Osbaldiston GW, Jonas AM: The etiology of transmissible murine colonic hyperplasia. Lab An Sci 26:889-894, 1976 2. Barthold SW, Osbaldiston GW, Jonas AM: Dietary, bacterial, and host genetic interactions in the pathogenesis of transmissible murine colonic hyperplasia. Lab An Sci 27:938-945, 1977 3. Barthold SW: Autoradiographic cytokinetics of colonic mucosal hyperplasia in mice. Cancer Res 39:24-39, 1979 4. Deschner EE, Lipkin M: Proliferative patterns in colonic mucosa in familial polyposis. Cancer 35:413-418, 1975 5. Eastwood GL, Trier JS: Epithelial cell renewal in cultured rectal biopsies in ulcerative colitis. Gastroenterology 64:383-390, 1973 6. Barthold SW, Jonas AM: Morphogenesis of early 1,2-dimethylhydrazine-induced lesions and latent period reduction of colon carcinogenesis in mice by a variant of Citrobacterfreundii. Cancer Res 37:4352-4360, 1977 7. McDowell EM, Trump BF: Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 100:405-414, 1976 8. Mollenhauer HH: Plastic embedding mixtures for use in electron microscopy. Stain Technol 39:111-114, 1964 9. Venable JH, Coggeshall R: A simplified lead citrate stain for use in electron microscopy. J Cell Biol 25:407-408, 1965 10. Barthold SW, Coleman GL, Jacoby RO, Livestone EM, Jonas AM: Transmissible murine colonic hyperplasia. Vet Pathol 15:223-236, 1978 11. Kavin H, Hamilton DG, Greasley RE, Eckart JD, Zuidema G: Scanning electron microscopy: A new method in the study of rectal mucosa. Gastroenterology 59:426432, 1970 12. Lane N, Lev R: Observations on the origin of adenomatous epithelium of the colon. Serial section studies of minute polyps in familial polyposis. Cancer 16:751-764, 1963 13. Hayashi T, Yatani R, Apostol J, Stemmermann GN: Pathogenesis of hyperplastic polyps of the colon: A hypothesis based on ultrastructure and in vitro cell kinetics. Gastroenterology 66:347-356, 1974 14. Kaye GI, Fenoglio CM, Pascal RR, Lane N: Comparative electron microscopic features of normal, hyperplastic, and adenomatous human colonic epithelium: Variations in cellular structure relative to the process of epithelial differentiation. Gastroenterology 64:926-945, 1973 15. Toth B, Malick L, Shimizu H: Production of intestinal and other tumors by 1,2-
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dimethylhydrazine dihydrochloride in mice: I. A light and transmission electron microscopic study of colonic neoplasms. Am J Pathol 84:69-86, 1976 16. Barkla DH, Tutton PJM: Surface changes in the descending colon of rats treated with dimethylhydrazine. Cancer Res 37:262-271, 1977 17. Hohmann A, Wilson MR: Adherence of enteropathogenic Escherichia coli to intestinal epithelium in vivo. Infect Immun 12:866-880, 1975 18. Sedlak J: Present knowledge and aspects of Citrobacter. Curr Topics Microbiol Immun 62:41-59, 1973 19. Staley TE, Jones EW, Corley LD: Attachment and penetration of Escherichia coli into intestinal epithelium of the ileum in newborn pigs. Am J Pathol 56:371-392, 1969 20. Takeuchi A: Electron microscope studies of experimental Salmonella infection: I. Penetration into the intestinal epithelium by Salmonella typhimurium. Am J Pathol 50:109-136, 1967 21. Takeuchi A, Formal SB, Sprinz H: Experimental acute colitis in the rhesus monkey following peroral infection with Shigella flexneri: An electron microscope study. Am J Pathol 52:503-529, 1968 22. Hampton JC, Rosario B: The attachment of microorganisms to epithelial cells in the distal ileum of the mouse. Lab Invest 14:1464-1481, 1965 23. Hampton JC, Rosario B: The attachment of protozoan parasites to intestinal epithelial cells of the mouse. J Parasitology 52:939-949, 1966 24. Nagy B, Moon HW, Isaacson RE: Colonization of porcine small intestine by Escherichia coli: Ileal colonization and adhesion by pig enteropathogens that lack K88 antigen and by some acapsular mutants. Infect Immun 13:1214-1220, 1976 25. Wilson MR, Hohmann AW: Immunity to Escherichia coli in pigs: Adhesion of enteropathogenic Escherichia coli to isolated intestinal epithelial cells. Infect Immun 10:776-782, 1974 26. Johnson EA, Jacoby RO: Transmissible ileal hyperplasia of hamsters: II. Ultrastructure. Am J Pathol 91:451-468, 1978 27. Rowland AC, Lawson GHK: Intestinal adenomatosis in the pigs Immunofluorescent and electron microscopic studies. Res Vet Sci 17:323-330, 1974 28. Abrams, GD, Schneider H, Formal SB, Sprinz H: Cellular renewal and mucosal morphology in experimental enteritis: Infection with Salmonella typhimurium in the mouse. Lab Invest 12:1241-1248, 1963 29. Biggers DC, Kraft LM, Sprinz H: Lethal intestinal virus infection of mice (LIVIM): An important new model for study of the response of the intestinal mucosa of injury. Am J Pathol 45:413-422, 1964 30. Takeuchi A, Sprinz H, Labrec EH, Formal SB: Experimental bacillary dysentery: An electron microscopic study of the response of the intestinal mucosa to bacterial invasion. Am J Pathol 47:1011-1044, 1965 31. Wilson MR, Hohmann AW: Immunity to Escherichia coli in pigs: Adhesion of enteropathogenic Escherichia coli to isolated intestinal epithelial cells. Infect Immun 10:776-782, 1974
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Figure 1-Scanning electron micrograph of the colon of a control mouse. One polygonal crypt unit is depicted. (x 1 600) Figure 2-Light micrograph of the upper portions of crypts and surface mucosa 6 days after inoculation with C freundii 4280. Organisms are attached to the plasma membrane of surface mucosal epithelial cells and crypt neck cells. Epon-Araldite section. (Methylene blue and Azure II, x 1 100)
Figure 3-Scanning electron micrograph of colonic crypt unit following bacterial attachment on Day 6. The crypt opening is enlarged and randomly oriented bacteria cover the surface of rounded epithelial cells. (x 1 050)
1
3
0.J
2
Figure 4-Scanning electron micrograph of a colonic crypt opening 6 days after inoculation with C freundii. The newly migrated cells that form a ring around the crypt mouth are, for the most part, free of bacteria. Many bacteria colonize marginal areas. (x 1 680)
Figure 5-Electron micrograph depicting bacteria along the colonic mucosal surface 4 days after inoculation with C freundii 4280. The brush border is disrupted as bacteria attach to the plasma membrane, and few microvilli remain in intervening areas. (x51 00) Figure 6-Electron micrograph of surface epithelial cell 6 days after inoculation with C freundii 4280. The surface of this heavily infected cell is devoid of microvilli. Organisms are attached to plaque-like areas of plasma membrane. Only remnants of the terminal web persist in the underlying cytoplasm. (x 15,200)
6
5
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Figure 7-Degenerative changes in surface mucosa following bacterial attachment. The cells at the left are dense, the mitochondria are swollen, and the cytoplasm contains small vacuoles. (x81 00) (With a photographic reduction of 4%)
9.'
'9
Figure 8-Electron micrograph of hyperplastic cells in mid-crypt region in moderately hyperplastic colon 6 days after inoculation with C freundii 4280. The elongated cells have a rudimentary brush border, and some contain small amounts of mucin. (x5300)
Figure 9-Globule leukocyte located between immature cells near the crypt base in the mucosa of a mouse 6 days after C freundii 4280 inoculation. (x 1 1,400) Figure 10-Scanning electron micrograph of the luminal surface 10 days after inoculation of C freundii 4280. There is a honeycomb pattern of interconnecting ridges between adjacent crypts. Cells at the apices of ridges are necrotic and detaching from the surface. Recessed crypt openings are slit-like. (x670) Figure 11-Scanning electron micrograph of one crypt unit in the colon 10 days after inoculation with C freundii 4280. Necrotic cells occur at the surface of the ridge. The crypt opening is slit-like, and small goblet and absortive cells have migrated onto the luminal surface. (x 1 1 60)
9
10
I11
Figure 12-Neutrophils within a colonic crypt lumen 10 days after C freundii 4280 inoculation. Cells contain intact and partially degraded bacteria. (x5300)
Figure 13-Scanning electron micrograph of colonic surface mucosa 16 days after inoculation with C freundii 4280. Surface ridges have diminished, but the surface remains convoluted. Fewer necrotic cells are found along surface ridges, and crypt openings are narrow slits. (x330) Figure 14-Transmission electron micrograph of colonic epithelial cells in the upper crypt 16 days after inoculation with C freundii 4280. The cells are elongated and have basal nuclei, and, occasionally, small accumulations of mucus are seen just below the plasma membrane. They have a reduced surface volume along the crypt lumen, a rudimentary brush border, and large numbers of unattached ribosomes in the cytoplasm. (x 10,500)
12
14
Figure 15-Transmission electron micrograph of colonic surface mucosa 16 days after C freundii 4280 inoculation. Cells along surface folds (right) at the peak of hyperplasia are cuboidal and have few microvilli. (x 5900) Figure 16-Electron micrograph of colonic surface mucosa 24 days after C freundii4280 inoculation. Epithelial absorptive and goblet cells are fully developed and morphologically normal. An intact brush border covers the surface mucosa. (x4500)
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