Regional differences in the ultrastructure of mucosal macrophages in the rat large intestine.

We previously clarified the histological characteristics of macrophages in the rat small intestine using serial block-face scanning electron microscopy (SBF-SEM). However, the regional differences in the characteristics of macrophages throughout the large intestine remain unknown. Here, we performed a pilot study to explore the regional differences in the ultrastructure of mucosal macrophages in the large intestine by using SBF-SEM analysis. SBF-SEM analysis conducted on the luminal side of the cecum and descending colon revealed macrophages as amorphous cells possessing abundant lysosomes and vacuoles. Macrophages in the cecum exhibited a higher abundance of lysosomes and a lower abundance of vacuoles than those in the descending colon. Macrophages with many intraepithelial cellular processes were observed beneath the intestinal superficial epithelium in the descending colon. Moreover, macrophages in contact with nerve fibers were more prevalent in the cecum than in the descending colon, and a subset of them surrounded a nerve bundle only in the cecum. In conclusion, the present pilot study suggested that the quantity of some organelles (lysosomes and vacuoles) in macrophages differed between the cecum and the descending colon and that there were some region-specific subsets of macrophages like nerve-associated macrophages in the cecum.

The spatial arrangement of the human large intestinal wall blood circulation.

The aim of the study was to describe and depict the spatial arrangement of the colon microcirculatory bed as a whole. Various parts of the large intestine and terminal ileum were harvested from either cadaver or section material or gained peroperatively. Samples were then injected with India ink or methylmetacrylate Mercox resin for microdissection and corrosion casting for scanning electron microscopy. The results showed that extramural vasa recta ramified to form the subserous plexus, some of them passing underneath the colon taeniae. Branches of both short and long vasa recta merged in the colon wall, pierced the muscular layer and spread out as the submucous plexus, which extended throughout the whole intestine without any interruption. The muscular layer received blood via both the centrifugal branches of the submucous plexus and the minor branches sent off by the subserous plexus. The mucosa was supplied by the mucous plexus, which sent capillaries into the walls of intestinal glands. The hexagonal arrangement of the intestinal glands reflected their vascular bed. All three presumptive critical points are only gross anatomical points of no physiological relevance in healthy individuals. Neither microscopic weak points nor regional differences were proven within the wall of the whole large intestine. The corrosion casts showed a huge density of capillaries under the mucosa of the large intestine. A regular hexagonal pattern of the vascular bed on the inner surface was revealed. No microvascular critical point proofs were confirmed and a correlation model to various pathological states was created.

Microvascular anatomy of the large intestine in adult Xenopus laevis: scanning electron microscopy of vascular corrosion casts and correlative light microscopy.

The microvascular anatomy of the large intestine of the adult South African Clawed Toad, Xenopus laevis (Daudin), was studied by scanning electron microscopy (SEM) of vascular corrosion casts (VCCs) and correlative light microscopy. Observations showed the large intestine to be supplied by the haemorrhoidal artery and the posterior mesenteric artery and drain via the posterior haemorrhoidal vein into either the left or right posterior abdominal vein. Both arteries and veins showed a bipinnate supply/draining pattern with branches running circumferentially. Vessels embraced the gut wall while arteries and veins in most cases alternated along the gut length. Many short terminal arterioles arose from the circumferential arteries at almost acute angles and capillarized after a short distance. Capillary lengths were short and continued into numerous postcapillary venules which merged either in a leaf vein-like formation or in a rosette-like formation with up to four draining sites per supplying arteriole. The microvasculature was found to be well adapted 1) to sustain blood flow under different amounts of feces in the gut and 2) to provide optimal conditions for the resorption of water and salts from the gut lumen into the blood vascular system by the high number of venules and their conspiciouos rosette-like and leaf vein-like patterns.

Functional significance of the variations in the geometrical organization of tight junction networks.

Using freeze-fracture techniques, we have examined the morpholog of tight junction networks found along the length of the alimentary tract of Xenopus laevis before and after metamorphosis. We have developed the hypothesis, based on these observations, that the geometrical organization of the network determined by the stress-induced shape changes normally experienced by the cells linked by the network. Consistent with this theory, tight junctions can be classified into two distinct types of network organization which differ in their response normal and experimentally induced stress conditions: (a) loosely interconnected networks which can stretch or compress extensively under tension, thereby adapting to stress changes in the tissue; and (b) evenly cross-linked networks which retain their basic morphology under normal stress conditions. The absorptive cells of the large intestine as well as the mucous cells of the gastrointestine or stomach are sealed by the first, flexible type of tight junction. The second type of junctional organization, the evenly cross-connected network, is found between absorptive cells of the small intestine and ciliated cells of the esophagus, and reflects in its constant morphology the relative stability of the apical region of both of these cell types. Networks intermediate between these two types arise when a cell which would normally form a lossely interconnected network borders a cell which tends to form a more evenly cross-linked network, as is found in the esophagus where ciliated and goblet cells adjoin. Despite the change in the animal's diet during metamorphosis from herbivorous to carnivorous, the basic gemetrical organization of the networks associated with each tissue of the alimentary tract remains the same.

The terminal web. A reevaluation of its structure and function.

The apical cytoplasm of epithelial cells of the small and large intestines has been examined by freeze-etch techniques as well as conventional and high voltage electron microscopy of sectioned material to gain a better understanding of the fine structural organization of the terminal web region. In the small intestine the terminal web exhibits a distinct stratification caused by the association of different sets of filaments with the three members of the junctional complex. Individual filaments of this network are closely associated with the sealing elements of the tight junctions, the surface of the core microfilament bundles, and the intermicrovillar plasma membrane. This region of the terminal web is the apical zone. The adherens zone appears as a band of interwoven filaments of two different diameters extending across the cytoplasm at the level of the intermediate junction. Within this region of the terminal web, individual 60-70 A actin-like filaments separate from the bundles of core microfilaments to interact with one another and with filaments of similar diameter from the zonula adherens. 100 A tonofilaments also contribute to the adherens zone, presumably stabilizing the orientation of the actin-like filaments. The basal zone which underlies the adherens zone consists of closely interwoven bundles of tonofilaments that are anchored to and interconnect the spot desmosomes. Within the large intestine the cytoplasmic microfilaments form a looser and less clearly stratified network which nevertheless retains the same basic organization found in the small intestine. Transmembrane linkers appear to originate within the cytoplasmic plaques of the spot desmosomes, pass through the plasma membranes, and meet in a staggered configuration in the intercellular space; these linkers may thus mediate the actual mechanical coupling between the cytoskeletal networks of tonofilament bundles of adjacent cells. This integrated system of cytoplasmic filaments and intercellular junctions endows the apical cytoplasm with both the flexibility and the stability necessary for the normal functioning of the epithelium.

The Effects of Deoxynivalenol and Zearalenone on the Pig Large Intestine. A Light and Electron Microscopy Study.

The contamination of feed with mycotoxins results in reduced growth, feed refusal, immunosuppression, and health problems. Deoxynivalenol (DON) and zearalenone (ZEN) are among the most important mycotoxins. The aim of the study was to examine the effects of low doses of these mycotoxins on the histological structure and ultrastructure of the large intestine in the pig. The study was performed on 36 immature gilts of mixed breed (White Polish Big × Polish White Earhanging), which were divided into four groups administrated per os with ZEN at 40 µg/kg BW, DON at 12 µg/kg BW, a mixture of ZEN (40 µg/kg BW) and DON (12 µg/kg BW) or a placebo. The pigs were killed by intravenous overdose of pentobarbital after one, three, and six weeks of treatment. The cecum, ascending and descending colon samples were prepared for light and electron microscopy. Administration of toxins did not influence the architecture of the mucosa and submucosa in the large intestine. ZEN and ZEN + DON significantly decreased the number of goblet cells in the cecum and descending colon. The mycotoxins changed the number of lymphocytes and plasma cells in the large intestine, which usually increased in number. However, this effect differed between the intestine segments and toxins. Mycotoxins induced some changes in the ultrastructure of the mucosal epithelium. They did not affect the expression of proliferative cell nuclear antigen and the intestinal barrier permeability. The obtained results indicate that mycotoxins especially ZEN may influence the defense mechanisms of the large intestine.

Logarithmic expansion of LGR5+ cells in human colorectal cancer.

Stem cells of the small and large intestine are marked by expression of the Wnt target gene LGR5, a leucine-rich-repeat-containing G protein-coupled receptor. Previous studies reported increased expression of LGR5 in human colorectal cancer (CRC) compared to normal tissue either by immunohistochemistry or in situ hybridization (ISH). However, as these studies were semi-quantitative they did not provide a numerical estimate of the magnitude of this effect. While we confirm that LGR5+ cells are exclusively located at the base of normal human small and large intestinal crypts, representing approximately 6% of total crypt cells, we show this cell population is 10-fold expanded in all grades of CRC, representing as much as 70% of the cells of tumor crypt-like structures. This expansion of the LGR5 compartment coincides with maintenance of crypt-like glandular structure (adenomas, and well and moderately differentiated adenocarcinomas), and is reduced in poorly differentiated CRC, where crypt-like glandular architecture is lost, accompanied by reduced epithelial terminal differentiation. Altogether these results indicate that LGR5+ cell expansion is a hallmark of CRC tumorigenesis occurring during progression to adenoma, supporting CRC as a stem cell disease with implications for CRC therapy.

Electron microscopic study of novel threadlike structures on the surfaces of mammalian organs.

The ultrastructures of novel threadlike structures (NTSs) and corpuscles on the surfaces of internal organs of rats were investigated using electron microscopy. The samples were studied in situ by using a stereomicroscope and were taken for further morphological analysis. Scanning electron microscope (SEM) images revealed a bundle structure of threadlike tissue, which was composed of several 10-micro m-thick subducts. The surfaces of the corpuscles were rather coarse and fenestrated. The corpuscles had cucumber-like shapes with an average length of about 2 mm and a thickness of about 400 micro m. Transmission electron microscope (TEM) images disclosed disordered collagen fibers, which formed the extracellular matrix of the threadlike tissue, and immune-function cells, like macrophages, mast cells, and eosinophils. Sinuses of various diameters, which were thought to be cross-sections of the lumens of the subducts, were observed in the TEM, cryo-SEM and focused-ion-beam SEM images. These SEM images were obtained for the first time to reveal the detailed structure of the NTSs that were only recently discovered.

Experimental study on the role of Brachyspira alvinipulli in intestinal spirochaetosis of geese.

Ten one-day-old goslings were inoculated orally with a Brachyspira alvinipulli strain isolated from the large intestine of geese that had died of intestinal spirochaetosis (Group A), 10 day-old goslings were inoculated orally with a B. hyodysenteriae strain (Group B), and a third group of 10 goslings (Group C) served as uninfected control. The goslings were observed daily for clinical signs. They were sacrificed on days 7, 14, 21 and 35 days postinfection (PI), and necropsied. Segments of the large intestine were subjected to histopathological, immunohistochemical, electron microscopic (TEM, SEM) and microbiological examinations. Mortality did not occur during the experimental period. However, in both groups the caecum of the goslings killed by bleeding was slightly dilated, in its lumen there was a watery, yellowish and frothy content, and the mucous membrane was slightly swollen. By histopathological, immunohistochemical and electron microscopic examination, B. alvinipulli and B. hyodysenteriae could be detected in the caecum or colon, in the lumen of the glands and sometimes among the glandular epithelial cells in goslings of the respective groups, and could be reisolated from these organs by culturing. A mild inflammation of the intestinal mucosa was also noted. In transverse section of the brachyspirae, numerous (16-22) periplasmic flagella could be detected inside the outer sheath, also depending on the plane of section.

Tight junctions in epithelial cells of human fetal hindgut, normal colon, and colon adenocarcinoma.

The structural patterns of tight junctions in normal human colon mucosa, colon adenocarcinomas, and fetal colon were studied and compared by the freeze-fracturing technique. The zonula occludens of the normal colon cells at the upper, more differentiated part of the crypts of Lieberkühn appeared as continuous belts made of about eight parallel strands. At the less differentiated bases of the crypts, the zonula occludens was less regular and contained fewer, mostly beaded strands. In the colons of 10-week fetuses, early stages of tight junction assembly were observed. At the same time, vesicles bearing remnants of tight junction elements were observed within the cytoplasm. This finding suggested that during the early development and organization of the fetal gut, mechanisms of assembly and disassembly of tight junctions are operating concomitantly. In well-differentiated adenocarcinomas, the cells in the luminal region retained their polarity and had seven or eight parallel junctional elements. In infiltrating cells, however, tight junctions appeared as fascia occludens and resembled the junctional organization of 10-week fetuses.

Insights into Campylobacter jejuni colonization and enteritis using a novel infant rabbit model.

A lack of relevant disease models for Campylobacter jejuni has long been an obstacle to research into this common enteric pathogen. Here we used an infant rabbit to study C. jejuni infection, which enables us to define several previously unknown but key features of the organism. C. jejuni is capable of systemic invasion in the rabbit, and developed a diarrhea symptom that mimicked that observed in many human campylobacteriosis. The large intestine was the most consistently colonized site and produced intestinal inflammation, where specific cytokines were induced. Genes preferentially expressed during C. jejuni infection were screened, and acs, cj1385, cj0259 seem to be responsible for C. jejuni invasion. Our results demonstrates that the infant rabbit can be used as an alternative experimental model for the study of diarrheagenic Campylobacter species and will be useful in exploring the pathogenesis of other related pathogens.

Human large intestinal epithelium: light microscopy, histochemistry, and ultrastructure.

Despite numerous reports of morphologic characteristics of premalignant and malignant large intestinal epithelium, the literature lacks comprehensive reports of the morphologic features of the epithelium of the normal large intestine, except of the rectum. Large intestinal epithelium from 41 persons was obtained, and samples from the ascending, transverse, descending, and rectosigmoid areas were studied by light microscopy, histochemical techniques, and transmission and scanning electron microscopy. The morphologic features and histochemical reactions of the various segments of the large intestine are different. Neutral mucopolysaccharide is predominant in the ascending colon, whereas the rectum has predominantly or exclusively acidic mucin. Only three basic epithelial cell phenotypes have been identified: undifferentiated cells, mucous cells, and endocrine cells. The columnar cells at the surface between the crypts appear to be a variant of mucous cells. Compared with other segments, the rectum shows an unusually high concentration of endocrine cells, positively correlating with the high incidence of carcinoid tumors in that segment of the large intestine. The mucous cells in all segments contain large mucous vacuoles and small apical vesicles. The apical vesicles show variable electron density, being most dense in the ascending colon and becoming progressively less dense at the transverse and descending colon and most electron-lucent in the sigmoid colon and rectum. Ultrastructurally, the mucin shows a variable degree of heterogeneity in the proximal segments. This study suggests that some of the previously described ultrastructural features of abnormal large-intestinal epithelium may be only the result of failure to compare the so-called abnormal cells with normal cells from the same region. Well-controlled studies of the abnormal epithelium of a particular segment of large intestine must include the normal epithelium from the identical segment as control in order to make interpretations accurate.

Alterations in mucosal morphology and permeability, but no bacterial or endotoxin translocation takes place after intestinal ischemia and early reperfusion in pigs.

Ischemia and reperfusion of the gut may be an important etiological factor in the development of multiple organ failure. We have used a hemorrhagic and a superior mesenteric artery (SMA) occlusion shock model in pigs to estimate the effect of ischemia and reperfusion on intestinal morphology, mucosal permeability, and the occurrence of bacterial or endotoxin translocation. Mucosal ulceration and necrosis were found in the SMA shock model, while the morphological changes were less pronounced in the hemorrhagic shock model. Scanning electron microscopy showed shrinkage of the villi and plugging of the colonic crypts in both shock models. Enterocyte cell kinetics was investigated using 5-bromo-2'-deoksyuridine (BrdU) incorporation and immunovisualization by anti-BrdU antibodies. Cell renewal was almost completely lost from the jejunum to the rectum in both shock models. Intramucosal pH was measured using a tonometer placed in the terminal ileum. Segments of intestinal mucosa were mounted in Ussing chambers, and permeability was measured using radiolabeled probe molecules of differing molecular weights. Augmented molecular flux of inulin (M(r) 5.000) and mannitol (M(r) 182) and loss of short circuit current (Isc) and transepithelial potential difference (PD) were found in mucosae from both shock models. Endotoxin was demonstrated in the ascitic fluid in both shock models; 9.5 (2.7-14.3) (median and 95% confidence interval) EU/mL in the SMA occlusion model and 16.0 (4.9-29.4) EU/mL in the hemorrhagic shock model), but the levels were not significantly higher than in the control model 6.5 (4.3-34.0) EU/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Different organization of intermediate filaments in columnar cells of rat large intestinal mucosa as revealed by confocal laser scanning microscopy and quick-freezing and deep-etching method.

The relationship between cell differentiation and ultrastructural changes of intermediate filaments (IF) was studied in columnar cells of large intestinal mucosa of rats by confocal laser scanning microscopy and quick-freezing and deep-etching method. A feature of the IF in immature columnar cells was minibundle formation with prominent branching, which organized the meshwork structures. The minibundles, which appeared to be formed by the attachment of two or more IF in side-to-side fashion, were loosely distributed throughout the cytoplasm. In contrast, in mature columnar cells, the IF were densely distributed under the terminal web in the cytoplasm and beneath the upper part of the lateral membrane regions, whereas the other areas of the cytoplasm contained only a small number of IF. Minibundle formation was not observed, and the branching was rarely identified. The changes in the distribution and density of IF, which are expressed in specific areas of mature columnar cells, apparently represent a characteristic of intracellular differentiation. It is suggested that the dissociation of minibundled IF, which was often observed in the immature columnar cells, is an important step in the acquisition of functional polarity in cells of this type.

Escherichia coli O157:H7 induces attaching-effacing lesions in large intestinal mucosal explants from adult cattle.

Attaching-effacing (A/E) lesions following natural and experimental infection with Escherichia coli O157:H7 have been seen in neonatal and 3-4-month-old weanling but not older cattle. To test the hypothesis that the adult bovine large intestinal epithelium is resistant to the development of A/E lesions, colonic and rectal mucosal tissue explants from 18-month-old steers were inoculated with E. coli O157:H7 and examined. Epithelial cells of inoculated explants developed A/E lesions at the bacterial attachment sites, providing evidence that the large intestinal mucosal epithelium may be a site of infection that contributes to carriage of E. coli O157:H7 in adult cattle.

Royal Society of Tropical Medicine and Hygiene meeting at Manson House, London, 14 December 1995. Enteropathogenic Escherichia coli--mucosal infection models.

The formation of attaching and effacing (A/E) lesions is central to the pathogenesis of enteropathogenic Escherichia coli (EPEC)-mediated disease in humans and Citrobacter rodentium-mediated transmissible colonic hyperplasia in mice. Closely related outer membrane proteins, known as intimins, are required for formation of the A/E lesion by both EPEC and C. rodentium. In this study we found similar ultrastructural damage in small intestinal biopsies from an EPEC-infected child and large bowel specimens from C. rodentium-infected mice. The C. rodentium-infected large bowel biopsies revealed massive hyperplastic reactions and the infected human small intestinal biopsies showed an increase in total crypt cell number and mitotic index. EPEC-infected small intestinal organ cultures revealed bacteria adhering in a localized pattern and evidence of A/E lesions. Covaspheres coated with a biologically active cell-binding domain of intimin also adhered to cells in a localized fashion but did not induce the characteristic A/E lesions.

Intestinal injuries caused by experimental palytoxicosis in mice.

Intestinal injuries in mice caused by i.p. administration of palytoxin at 1 microgram/kg were studied microscopically. Within 1 hr, bleeding started from small intestinal villi. At 6 hr, congestion in the villi and edema in the lamina propria in the crypt layer developed prominently. Edema and necrosis of lamina propria in the villus appeared after 16 hr. At 24 hr, villi lost their epithelial cells and then their length decreased to 1/4 to 1/8 of normal. Diarrhea was seen after 16 hr, accompanying severe peritonitis. Hypersecretion of mucus from the large intestine was considered to be physically stimulated by peritonitis, which then induced diarrhea.

Morphological observations of diarrhea in mice caused by experimental ciguatoxicosis.

Diarrhea and morphological influences on digestive tracts caused by ciguatoxin (CTX) were observed in mice microscopically. The lethal doses and clinical symptoms caused by i.p. administration were almost the same as those by p.o., the prominent difference being diarrhea that was caused only by i.p. route. The diarrhea was caused by a dose from 1/7 MU to 1 MU of CTX (MU: mouse unit, to kill a mouse of 15 g in 24 hr, corresponding to 7 ng of pure CTX), but not at lower or higher doses. In this study, we used an i.p. dose of 4/5 MU (10.4 ng/28 g). Diarrhea started within 10 min after administration and lasted until 30 min. The changes were observed in the large intestine; namely, CTX accelerated mucus secretion and peristalsis in the colon and stimulated defecation at the rectum, resulting in prominent diarrhea. In the colon, a large quantity of mucus was secreted from even immature goblet cells, and epithelial cell damages were observed in the upper portion of the large intestine but not in the latter half. The morphological changes caused by CTX in the upper portion of the large intestine were similar to those seen with cholera toxin.

The fate of epithelial cells in the human large intestine.

One hundred and forty biopsies of the colon and rectum, collected during routine colonoscopies of 51 patients aged 19 to 74 years, were examined using light microscopy and transmission and scanning electron microscopy. The results indicated that surface epithelial cells undergo apoptosis, passing through fenestrations in the basement membrane to where they enter the lamina propria and are taken up by macrophages; and it is hypothesized that apoptotic cells are carried through the fenestrations on a current of fluid. The study also found that epithelial cells positioned over the crypts are better attached and more robust than those more distant from the crypt opening; and it is further hypothesized that, after reaching the top of the crypts, some goblet cells cease secreting mucus and pass onto the surface compartment of absorptive cells. An unexpected finding was that the lower regions of the crypts commonly contain isolated necrotic colonocytes. Apoptotic cells were rarely observed in the crypt epithelium. The findings of this study support the "recycling" model of epithelial cell death in the surface compartment of the human colon.

Dysentery in calves caused by an atypical strain of Escherichia coli (S102-9).

Dysentery lasting 4-8 days was produced in five 4-day-old colostrum-fed calves, after inoculation with an atypical strain of Escherichia coli S102-9; peak excretion of S102-9 occurred during the period of dysentery. Two calves were killed when clinical signs were most severe and bacteria were seen attached to the surfaces of enterocytes in the large intestine; microscopic lesions were seen in these areas. The lesions were identical to those previously reported in a natural outbreak of dysentery in calves, from which E. coli S102-9 was isolated, and to those seen in gnotobiotic calves experimentally infected with S102-9. Reinfection of the three surviving calves 16-20 days later with S102-9 and primary infection of two calves aged 24 and 51 days did not cause dysentery. Four of 659 coliforms isolated from field outbreaks of calf diarrhoea resembled the atypical strain S102-9. These four isolates and S102-9 did not produce heat-stable enterotoxin, but all produced a toxin cytopathic for Vero and HeLa cells. Two of the four isolates were inoculated alone into 4-day-old gnotobiotic calves deprived of colostrum; neither calf developed dysentery but microscopic lesions identical to those produced by S102-9 were detected in the large intestines of both animals.