Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine.

The parasympathetic control of goblet cell secretion and the membrane events accompanying accelerated mucus release were studied in large intestinal mucosal biopsies maintained in an organ culture system. The secretory response of individual goblet cells to 10(-6) M acetylcholine chloride with 3 x 10(-3) M eserine sulfate (a cholinesterase inhibitor) was assessed by light microscopy and autoradiography, by scanning and transmission electron microscopy, and by freeze-fracture. Goblet cells on the mucosal surface are unaffected by acetylcholine. In crypt goblet cells acetylcholine-eserine induces rapid fusion of apical mucous granule membranes with the luminal plasma membrane (detectable by 2 min), followed by sequential, tandem fission of the pentalaminar, fused areas of adjacent mucous granule membranes. These events first involve the most central apical mucous granules, are then propagated to include peripheral granules, and finally spread toward the most basal granules. By 60 min, most crypt cells are nearly depleted. The apical membrane, although greatly amplified by these events, remains intact, and intracellular mucous granules do not coalesce with each other. During rapid secretion membrane-limited tags of cytoplasm are observed attached to the cavitated apical cell surface. These long, thin extensions of redundant apical membrane are rapidly lost, apparently by being shed into the crypt lumen.

Inhibition of endotoxin-induced bacterial translocation in mice.

The primary functions of the gut are to absorb nutrients and exclude bacteria and their products. However, under certain circumstances the gut may lose its barrier function and serve as a reservoir for systemic microbial infections. These experiments were performed to determine the mechanisms whereby endotoxin causes bacteria to escape (translocate) from the gut. Bacteria translocated from the gut to the mesenteric lymph nodes of mice challenged with nonlethal doses of Escherichia coli 026:B6 or E. coli 0111:B4 endotoxin. Physical disruption of the gut mucosal barrier appears to be the primary mechanism whereby endotoxin promotes bacterial translocation. Mucosal injury and endotoxin-induced bacterial translocation were reduced by inhibition (allopurinol) or inactivation (tung-sten diet) of xanthine oxidase activity (P less than 0.01), but were not affected by the platelet-activation factor antagonists, SRI 63-441 or BN 52021. Because the inhibition or inactivation of xanthine oxidase activity reduced both the extent of mucosal injury and endotoxin-induced bacterial translocation, the effect of endotoxin on the gut appears to be mediated, at least to some degree, by xanthine oxidase-generated, oxygen-free radicals.

Sulfasalazine or enteral diets containing fish oil or oligosaccharides attenuate chronic colitis in rats.

: Recent studies have suggested that n-3 fatty acids from fish oil (FO) as well as short-chain fatty acids may attenuate some of the gut injury and inflammationassociated ulcerative colitic (UC). The objectives of this study were to (a) assess the antiinflammatory activity of sulfasalazine (SAZ), a drug known to be effective in the treatment of human UC in a model of chronic granulomatous colitis in rats and (b) determine whether enteral diets supplemented with either FO or two indigestible oligosaccharides (fructooligosaccharide, FOS; xylooligosaccharide, XOS) could attenuate the inflammation observed in a model of chronic granulomatous colitis. In one series of experiments, female Lewis rats were randomized into three groups consisting of a sham-operated control group, a colitic group, and a colitic group in which rats were given oral sulfasalazine (SAZ) immediately after induction of colitis and continued for 3 weeks. Chronic granulomatous colitis with liver and spleen inflammation was induced by subserosal (intramural) injection of purified peptidoglycan-polysaccharide (PG/PS) into the distal colon. Sham-operated rats were injected with human serum albumin. All rats received standard lab chow. In a second series of experiments, female Lewis rats were randomized into six groups consisting of four colitic groups fed enteral diets, a colitic group fed chow, and a sham-operated group fed a control enteral diet. Enteral diets (300 kcal/kg/day) contained either FO, FOS/ gum arabic, XOS/gum arabic, or no bioactive ingredient (control diet). All rats were fed for 1 week before induction of colitis. Rats consumed the diets for 3 additional weeks before being killed. SAZ significantly attenuated the PG/PS-induced increases in myeloperoxidase (MPO) activity as well as significantly reduced the PG/PS-induced increases in liver and spleen weights. Control (enteral diet) as well as the FO and XOS diets significantly attenuated the increase in colon weight when compared with chow-fed rats. We also found that the FO and XOS diets significantly attenuated the PG/PS-induced increases in colonic MPO activity and colon weight. The FOS and XOS diets significantly attenuated the PG/PS-induced increases in liver weights when compared with PG/PS + chow-fed animals. The antiinflammatory activity of these diets was confirmed by means of histological inspection showing an inhibition of inflammation and maintenance of crypt cell integrity. These results demonstrate that a complete enteral diet supplemented with either FO, FOS, or XOS exhibited antiinflammatory activity that was similar in efficacy to the known antiinflammatory drug SAZ in this model of colitis.

Role of mucin, mannose, and beta-1 integrin receptors in Escherichia coli translocation across Caco-2 cell monolayers.

Our previous work suggests that Caco-2 cells play an active role in bacterial translocation (BT). Since bacterial enterocyte interactions may be receptor-mediated, the current study was performed to investigate the role of beta 1 integrin and mannose receptors as well as the general protective effect of the mucous layer in this process. Caco-2 cells grown to confluence on semipermeable membranes contained in the upper compartment of a two compartment system were utilized. BT was assessed by quantitating the number of Escherichia coli crossing the monolayers after challenge with 10(8) E. coli C25. Pretreatment of the Caco-2 cells with the beta 1 integrin receptor competitive inhibitors fibronectin or RGD did not inhibit BT; while pretreatment of Caco-2 cells with the LFA-1 (lectin) receptor competitive inhibitor mannose (12 mg/ml) or purified mucin (8 mg/ml) decreased BT compared to control membranes (p < .001). Transepithelial resistance was similar among all the groups indicating maintenance of tight junction integrity. These studies suggest that E. coli BT in the Caco-2 system can be reduced by mannose and that intestinal mucin contributes to the barrier function of the monolayer.

Food without fiber promotes bacterial translocation from the gut.

To determine whether the route and/or composition of nutritional support alters intestinal barrier function (measured as bacterial translocation), rats were divided into three groups: food (controls), intravenous total parenteral nutrition (IV-TPN) fed, and oral total parenteral nutrition (ORAL-TPN) fed. Bacterial translocation did not occur in the rats that were fed normally, but did occur in 60% of the rats fed the IV-TPN or the ORAL-TPN diets for 7 days (p less than 0.05). Since both the IV-TPN and ORAL-TPN diets induced bacterial translocation and the TPN solution (28% glucose and 4.5% amino acids) lacks fiber, two additional groups of rats were fed orally 2.5 gm cellulose powder/day plus TPN solution by either the intravenous or the oral route. The addition of cellulose powder decreased the incidence of bacterial translocation to 8% in the group fed the ORAL-TPN diet and to 0% in the group fed the IV-TPN diet. Cellulose improved intestinal barrier function, even though it did not prevent bacterial overgrowth or the loss of mucosal mass in the rats fed the IV-TPN or ORAL-TPN diets. Cellulose powder appears to have prevented bacterial translocation primarily by preventing IV-TPN- or ORAL-TPN-induced alterations in mucosal structure. Thus the oral administration of this fiber maintains intestinal barrier function and prevents bacterial translocation even in the absence of oral nutrients.

Endotoxin-induced bacterial translocation: a study of mechanisms.

Previously, we documented that nonlethal doses of endotoxin cause the translocation (escape) of bacteria from the gut to systemic organs. The purpose of this study was to determine which portion(s) of the endotoxin molecule induces bacterial translocation and to examine the role of xanthine oxidase activity in the pathogenesis of endotoxin-induced bacterial translocation. Nonlethal doses of Salmonella endotoxin preparations (wild type, Ra, or Rb), containing the terminal portion of the core polysaccharide, induced bacterial translocation, whereas those preparations lacking the terminal-3 sugars (Rc, Rd, Re, or lipid A) did not induce bacterial translocation. Additionally, only those endotoxin preparations that induced bacterial translocation injured the gut mucosa, increased ileal xanthine dehydrogenase and oxidase activity, and disrupted the normal ecology of the gut flora, resulting in overgrowth with enteric bacilli. Inhibition of xanthine oxidase activity by allopurinol prevented endotoxin (Ra)-induced mucosal injury and reduced the incidence of bacterial translocation from 83% to 30% (p less than 0.01). These results suggest that endotoxin-induced bacterial translocation requires the presence of the terminal core lipopolysaccharide moiety and that xanthine oxidase-generated oxidants are important in the pathogenesis of endotoxin-induced mucosal injury and bacterial translocation.

Hemorrhagic shock-induced bacterial translocation is reduced by xanthine oxidase inhibition or inactivation.

Experiments were performed to determine whether bacterial translocation (BT) after hemorrhagic shock is due to a reperfusion injury mediated by xanthine oxidase-derived oxidants. Rats were subjected to 30 minutes of shock (30 mm Hg) followed by reinfusion of shed blood. Twenty-four hours after hemorrhage and reinfusion, the mesenteric lymph node, liver, and spleen were harvested from each animal for bacterial culture, and the ileum and cecum were examined histologically. Sham-shocked (control) rats were instrumented, but blood was not withdrawn. The incidence of BT was higher in the shocked rats (61%) than in the sham-shocked animals (7%) (p less than 0.01). Allopurinol (50 mg/kg, administered orally), a competitive inhibitor of xanthine oxidase, reduced the incidence of shock-induced BT to 14% (p = 0.02). Similarly, rats fed a tungsten-supplemented molybdenum-free diet, which inactivates xanthine oxidase, reduced shock-induced BT to 10% (p = 0.02). The histologic damage cause by hemorrhagic shock was prevented by blocking xanthine oxidase activity. Thus hemorrhagic shock-induced bacterial translocation from the gut appears to be mediated by oxidants generated by activation of the xanthine oxidase system.

Obstructive jaundice promotes bacterial translocation from the gut.

Experiments were performed to determine if obstructive jaundice promotes the translocation of bacteria from the gastrointestinal tract to visceral organs. Three groups of mice were studied: control (n = 20), sham ligated (n = 28), and bile duct ligated (n = 33). The sham-ligated group underwent laparotomy and manipulation of the portal region, whereas the ligated group had their common bile ducts ligated. Seven days later, the mice were killed, their organs cultured, and the gastrointestinal tract examined histologically. The bilirubin levels of the ligated group (18.7 mg/dL) were elevated compared with the other groups (0.5 mg/dL) (p less than 0.05). The incidence of bacterial translocation was higher in the ligated (33%) than in the control (5%) or sham-ligated (7%) groups (p less than 0.05). Since bile is important in binding endotoxin and maintaining a normal intestinal microflora, cecal bacterial populations were quantitated. The cecal levels of gram-negative, enteric bacilli were 100-fold higher in the bile duct-ligated mice in which bacterial translocation occurred (p less than 0.05), indicating that intestinal bacterial overgrowth was a major factor responsible for bacterial translocation. The mucosal appearance of the intestines from the control and sham-ligated groups was normal. In contrast, subepithelial edema involving the ileal villi was present in the ligated group. In conclusion, the absence of bile within the gastrointestinal tract allows intestinal overgrowth with enteric bacilli and the combination of bacterial overgrowth and mucosal injury appears to promote bacterial translocation.

Obstructed intestine as a reservoir for systemic infection.

Intestinal motility, absorption, and secretion are altered after intestinal obstruction, but at what point the normal bacterial barrier function of the viable gut fails after intestinal obstruction is unclear. Thus, we tested whether after simple intestinal obstruction bacteria would translocate across the viable intestinal wall to cause systemic infection. Within 6 hours of intestinal ligation 1 cm proximal or distal to the ileocecal valve, bacteria had translocated to the mesenteric lymph nodes, and by 24 hours after intestinal obstruction, bacteria had spread to the liver, spleen, and blood stream. Bacterial translocation rarely occurred in the animals undergoing laparotomy and sham intestinal ligation, indicating that bacterial translocation was not due to surgical stress. Based on the results of these studies, bacterial translocation induced by intestinal obstruction appears to be due to disruption of the ecology of the normal gut microflora, leading to intestinal overgrowth with certain enteric bacilli and mucosal damage. Although the exact mechanism(s) by which simple mechanical obstruction injures the intestine is not known, the fact that mucosal injury did not occur in germ-free mice suggests that bacteria may play a role in the pathogenesis of obstruction-induced intestinal injury.

Effects of protein malnutrition and endotoxin on the intestinal mucosal barrier to the translocation of indigenous flora in mice.

Since protein malnourished or endotoxemic patients are at increased risk of developing nosocomial infections with enteric organisms, we investigated the effects of these risk factors alone and in combination on the intestinal mucosal barrier to bacteria. Protein malnutrition resulted in severe ileal atrophy that was directly related to the length of time the mice were protein malnourished. Although protein malnutrition did not promote bacterial translocation from the gut to systemic organs, the protein-malnourished mice were more susceptible to endotoxin-induced bacterial translocation than normally nourished mice (p less than 0.01). Since the gross epithelial damage documented after endotoxin administration in normally nourished mice was diminished after protein malnutrition, there was no correlation between the gross appearance of the epithelial mucosal barrier and the extent of endotoxin-induced bacterial translocation. These results suggest that the synergistic effect of endotoxin plus protein malnutrition on bacterial translocation is not primarily related to failure of the gut mucosal barrier. Nonetheless, it appears that protein-malnourished mice are less able to clear translocating bacteria than normally nourished mice.

Bulk prevents bacterial translocation induced by the oral administration of total parenteral nutrition solution.

The effects of a fat and glutamine-free orally administered total parenteral nutrition (TPN) solution on intestinal mucosal mass, morphology, barrier function, and cecal bacterial population levels were measured in CD-1 mice. Ileal mucosal protein content decreased by 63% (p less than 0.01) in the oral TPN-fed mice, although they gained weight on this diet. These TPN-fed mice also exhibited changes in mucosal structure and the normal ecology of their cecal microflora was disrupted leading to overgrowth with Gram-negative enteric bacilli. These changes in intestinal mucosal mass, morphology, and gut bacterial ecology were associated with an increased incidence of bacterial translocation (BT) (TPN group 70% BT vs control group 15% BT: p less than 0.01). The administration of cellulose fiber or kaolin (bulk-forming agents), but not of citrus-pectin (a fully-fermentable, nonresidue fiber) reduced the incidence of BT in the TPN-fed mice to control levels. The beneficial effects of these bulk-forming agents appeared to be due to their ability to prevent TPN-induced disruption of the intestinal microflora and alterations in intestinal morphology, even though they did not prevent ileal mucosal protein levels from decreasing. These results suggest that the administration of bulk forming agents will prevent the loss of intestinal barrier function against luminal bacteria that occurs in mice fed an oral TPN solution.

Protein malnutrition alone and in combination with endotoxin impairs systemic and gut-associated immunity.

Because protein-malnourished or endotoxemic patients are at an increased risk of developing nosocomial infections, this study was performed to investigate the effects of protein malnutrition and endotoxemia, alone and in combination, on systemic and intestinal immunity. Protein malnutrition was created by feeding the animals a solid diet containing 0.03% protein. Subgroups of these protein-malnourished mice were killed after being challenged with saline or endotoxin on days 0, 7, 14, or 21. At death, the animals were weighed, tissues were harvested for histologic analysis (ileum, mesenteric lymph node [MLN], liver, and spleen), mitogen responsiveness (MLN, Peyer's patches, and spleen), and xanthine oxidase measurements (ileum and cecum). Separate groups were evaluated for survival. Both the saline and endotoxin-challenged mice had lost about 30% of their body weight after 21 days on the low-protein diet. The protein-malnourished mice were more susceptible to endotoxin-induced mortality (70% at 21 days) than the normally nourished mice (0%) (p less than .001). The mitogen responsiveness of the protein-malnourished mice to the T-cell mitogens (PHA and Con-A) progressively decreased the longer the mice were protein malnourished, and this decreased in blastogenic responsiveness was associated with histologic evidence of lymphoid atrophy. In contrast, the blastogenic response to the primarily B-cell mitogen, PWM, was largely preserved. The endotoxin challenge further depressed the immune state of mice tested after 0, 7, or 14 (but not 21) days of protein malnutrition. Thus, both protein malnutrition and endotoxin impaired systemic and gut-associated immune responsiveness to mitogens. However, in the protein-malnourished mice, the degree of immune suppression did not correlate with endotoxin-induced mortality.

Role of neutrophils in acetic acid-induced colitis in rats.

Intrarectal administration of 4% acetic acid produces diffuse inflammation that ultimately results in erosions and ulcerations of the rat colon. Although this model of colitis has been used extensively over the past several years, there are no quantitative data available regarding the relationship between neutrophil infiltration and mucosal injury during times of active inflammation. Therefore, the objective of this study was to define the role of extravasated neutrophils as mediators of mucosal injury and inflammation in acetic acid-induced colitis. We found the intrarectal administration of 4% acetic acid produced an 11-fold increase in colonic mucosal permeability, a 9-fold increase in colonic MPO activity, and a 1.6-fold increase in colon weight at 48 h following administration of acetic acid. In addition, we found significant correlations between colonic MPO activity and mucosal permeability and between colonic MPO activity and colon weight (P less than 0.01 for both). These data suggested that inflammatory neutrophils may mediate mucosal injury and inflammation in this model of colitis. To assess the role of circulating neutrophils, rats were rendered neutropenic for 48 h by the intraperitoneal administration of antiserum directed toward rat neutrophils (ANS). Although ANS treatment reduced both the number of circulating neutrophils and colonic MPO activity to less than 10% of control values, it did not attenuate the increases in colonic mucosal permeability nor did it attenuate the increases in colon weight produced by acetic acid. Histological inspection confirmed that ANS treatment was not effective in attenuating the injury to the epithelial barrier. These data demonstrate that infiltrating neutrophils do not mediate the mucosal injury and inflammation observed in acetic acid-induced colitis.

Mechanisms of acute and chronic intestinal inflammation induced by indomethacin.

The objective of this study was to characterize the mechanisms of acute and chronic intestinal mucosal injury and inflammation induced by subcutaneously injected indomethacin (Indo). One injection of Indo (7.5 mg/kg) produced acute injury and inflammation in the distal jejunum and proximal ileum that were maximal at three days and completely resolved within one week. Two daily subcutaneous injections of Indo produced a more extensive and chronic inflammation that lasted in an active form in more than 75% of the rats for at least two weeks. Epithelial injury, as measured by enhanced mucosal permeability, was significantly elevated only at one day in the acute model (one injection) but was persistently elevated in the chronic model (two injections). Bile duct ligation completely attenuated increased mucosal permeability in the acute model, however, depletion of circulating neutrophils had no effect. Neither Indo (0-0.1 mg/ml) nor normal bile was cytotoxic to cultured rat intestinal epithelial cells; however, they synergistically promoted significant cytotoxicity. Bile collected from rats treated with Indo was cytotoxic towards the epithelial cells in a dose-dependent manner. Sulfasalazine and metronidazole (100 mg/kg/day, both) attenuated enhanced mucosal permeability in the chronic model. Massive bacterial translocation into the mesenteric lymph nodes, liver, and spleen following two injections of Indo was significantly attenuated by metronidazole. We conclude that: (1) a single injection of Indo produces acute intestinal mucosal injury and inflammation that resolve completely within three to seven days, whereas two daily injections of Indo produce both acute and chronic injury and inflammation, (2) enterohepatic circulation of Indo is important in promoting the acute phases of injury and inflammation, (3) circulating neutrophils do not play a role in the pathogenesis of this model, and (4) endogenous bacteria play an important role in exacerbating and/or perpetuating the chronic phases of injury and inflammation.

Morphologic assessment of leukocyte-endothelial cell interactions in mesenteric venules subjected to ischemia and reperfusion.

Intravital microscopic studies of the mesenteric microcirculation have demonstrated that leukocyte adherence and emigration in postcapillary venules are a characteristic feature of tissues exposed to ischemia-reperfusion. The objectives of this study were to determine whether: (1) neutrophils are the predominant leukocytes that adhere and emigrate in postischemic mesenteric venules, and (2) leukocyte adherence and/or emigration are a prerequisite for reperfusion-induced increases in venular permeability. Leukocyte kinetics in cat mesenteric venules (25-35 microns diameter) were evaluated using both intravital microscopy and quantitative morphometry. The intestine and mesentery were exposed to 60 min of ischemia, followed by 60 min reperfusion. Some animals were pretreated with a monoclonal antibody (MoAb IB4) against the leukocyte adhesion glycoprotein, CD11/CD18. Vessels observed by intravital microscopy and adjacent venules of similar diameter were excised and processed for light (LM) and electron microscopy (EM). Horseradish peroxidase (HRP), administered intravenously, was used to assess vascular permeability by EM. By LM, the control (nonischemic) mesentery is sparsely populated by plasma cells, mast cells, and leukocytes; 30-50% of the resident population is neutrophils. Ischemia-reperfusion led to a significant increase in the number of extravascular cells, with neutrophils accounting for greater than 80% of the total cell population. Control and ischemic venules demonstrated no leakage of HRP into the interstitium. However, venules exposed to ischemia and reperfusion demonstrated HRP leakage between endothelial cells and into the surrounding interstitium; neutrophils were adherent to the luminal surface of the endothelium, transmigrating the vessel wall, and in the surrounding interstitium. Animals pretreated with MoAb IB4 presented the same cell profile as nonischemic controls, with no adherent or transmigrating neutrophils. However, some HRP leakage was noted following reperfusion in venules treated with MoAb IB4. The results of this study indicate that: (1) neutrophils are the predominate leukocytes that adhere and emigrate in postischemic venules, and (2) inhibition of leukocyte adhesion does not completely prevent the venular dysfunction associated with ischemia-reperfusion.

Absence of intestinal bile promotes bacterial translocation.

Previously, the authors documented that extrahepatic biliary obstruction promotes the systemic translocation of bacteria from the intestine to visceral tissues. The current experiments were performed to determine whether it was the absence of intestinal bile or the presence of biliary obstruction that promoted bacterial translocation. Four groups of rats were studied: 1) nonoperated controls (n = 20), sham common bile duct-ligated (n = 22), common bile duct-ligated (n = 25), and common bile duct-diverted (choledochovesical bypass) (n = 23). The sham-ligated group underwent laparotomy and manipulation of the portal region; whereas the ligated group had their common bile ducts ligated, while the choledochovesical group had a silastic tube placed from the common bile duct to the bladder. Seven days later, at death, the incidence of bacterial translocation was higher in the groups of rats subjected to common bile duct ligation (41%) or diversion (32%) than in the control (3%) or sham-ligated (5%) groups (P less than 0.05). Histologic sections of ileums of ligated and diverted animals both showed subepithelial edema. These findings suggest that it is primarily the absence of bile in the intestine that promotes mucosal injury and bacterial translocation and not biliary obstruction.

The value of cuticular fine structure in identification of juvenile anisakine nematodes.

Juveniles of the ascarid nematodes Anisakis sp. and Phocanema sp. from marine teleosts, have been implicated as major causative agents of human anisakiasis. Whereas juveniles of the related genus Thynnascaris sp. have not been reported from humans, this nematode may occur in fish concurrently with the aforementioned species. The juvenile anisakine nematodes are not readily identified, and reliable morphological markers would provide a valuable aid in identification of potentially pathogenic individuals. In this study of cuticular fine structure, the following characteristic difference appeared to be most significant: a wide electron lucent area containing partitions and previously unreported electron dense rods occurred in the basal layer of the Phocanema sp. cuticle, but not in that of Anisakis sp. or of Thynnascaris sp. Juveniles of Thynnascaris sp. possessed a cuticle that was structurally similar to that of Anisakis sp., but only about one-half as thick. Comparatively, the third stage Ascaris lumbricoides cuticle was poorly developed, and less definitively layered than those of the anisakines.

A unicellular endocrine gland in cestodes.

Unicellular glands are reported from the scolex and anterior neck region of Hymenolepis diminuta and H. nana. Despite positive staining reactions with the presumptive neurosecretory stains, paraldehyde-fuchsin and chrome-alum-hematoxylin, ultrastructurally these glands exhibit many non-neural characteristics. Glandular cell processes are frequently found in close proximity to muscular tissue, particularly in the suckers, suggesting a regulatory role in muscle modulation as a possible function. Two types of putative, neurosecretory cells are reported from the cephalic ganglia and the lateral nerve cords. Neurosecretory regulation of the unicellular endocrine glands is postulated based on the lack of direct innervation of the glands and the frequent close proximity of axons containing putative, neurosecretory granules.

Unique crystalline inclusions in neonatal pig small intestine epithelial cells.

In this study we describe the presence of novel crystalline inclusions in the jejunal and ileal epithelium of newborn pigs. Hampshire/Yorkshire piglets (0-3 days) were anesthetized with pentobarbital and tissues were recovered following laparotomy. Sections of jejunum and ileum were prepared with a variety of fixatives for paraffin and methacrylate embedment for the light and electron microscopy. The characteristics of these crystalline structures have been demonstrated by cytochemical staining, immunohistochemical staining, and ultrastructural studies. Using light microscopy, it was determined that 10.8% of villus epithelial cells in the jejunum contained inclusions, whereas inclusions were present in 26.6% of the villus epithelial cells in the ileum. Inclusions were absent in crypt epithelial cells of both areas of the intestine, and the percentage of cells with inclusions increased towards the villus tip. Preliminary histochemical analysis has been unrevealing. The inclusions are periodic acid/Schiff's (PAS) reagent-negative, and do not stain for the presence of concentrated proteins. Using electron microscopy, the inclusions are found to be clustered in the supra-nuclear region of the cells and are generally oriented with the long axis of the cell. The crystals vary in size and have a dense core region surrounded by a regular less dense periphery.