Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.     

Apoptosis of villous epithelial cells and follicle-associated epithelial cells in chicken cecum

The process of the disappearance of epithelial cells was examined in chicken cecal villi and follicle-associated epithelium (FAE). The apoptotic epithelial cells with intense DNA-fragmentation and their exfoliation were found in the villous tips. The epithelial cells with weak DNA-fragmentation were seen in the upper portion of the villi and their sparse exfoliations were also found there. Numerous epithelial cells in the intestinal lumen expressed the apoptotic features. A row of apoptotic epithelial cells with DNA-fragmentation was also found in the apical FAE, whereas no M cells exhibited any apoptotic signs. In all cecal regions, CD3+, CD8+, and TCR2+ lymphocytes were predominant in the epithelium at the upper portion of the villi and the FAE. CD4+ lymphocytes were mainly seen in the lamina propria. TCR1+ lymphocytes were not abundant in comparison with TCR2+ lymphocytes in the epithelium. TCR3+ T lymphocytes were rarely detected. These results suggest that the chicken cecal epithelial cells exfoliated into the lumen after the induction of the apoptosis, and that the induction may be involved with CD3+, CD8+, and TCR2+ lymphocytes. No death in M cells suggests that M cells may transform into microvillous epithelial cells.     

Differences in lectin-binding properties between the common mucosal epithelium and follicle-associated epithelium in the rabbit small intestine

Differences in sugar distribution between the villous epithelium and follicle-associated epithelium (FAE) were compared using lectins in the rabbit small intestine. In every portion, villous columnar epithelial cells primarily exhibited a positive reaction to the GalNAc, GlcNAc, galactose, and oligosaccharide. In the ileal Peyer's patch (PP), whereas microvillous epithelial cells exhibited positive reactions, M cells tended to be negative. The villous epithelial reaction to the fucose group was negative, but M cells and microvillous epithelial cells showed a positive to the fucose. No epithelium had a positive reaction to the mannose and glucose. The variety of lectin-binding properties of villous epithelial cells and M cells may reflect specificity for the recognizing luminal substances such as antigenic molecules and bacterial elements.     

Distribution of the pores of epithelial basement membrane in the rat small intestine

The distribution and diameter of the pores of epithelial basement membrane in the intestinal villi and the lymph nodules of ileal Peyer's patches were investigated in the rat small intestine by scanning electron microscopy after the removal of the overlying epithelial cells with OsO(4) maceration. In the duodenum, jejunum and ileum, the pores were mainly distributed at the upper three fourths of the villi, but were scarce around the top of the villi. The diameter of some of the pores in the upper three fourths of the villi was larger than that of those in the lower portion. The protrusion of lymphocytes and the cytoplasmic processes of macrophages were also seen at the orifices of the pores. In ileal Peyer's patches, in contrast, pores were densely distributed in the lower one third of the follicle-associated epithelium (FAE) where M cells were mainly seen. Furthermore, these pores were larger than those found in the upper two thirds. Lymphocytes or cytoplasmic processes of macrophages were frequently seen in the lower one third of FAE. These results suggest that the pores at the basement membrane correspond to the passage of the immunocompetent cells which are in contact with M cells or villous columnar epithelial cells and that the abundance of pores is a sign of aggressive interaction between the particular epithelial cells and the immunocompetent cells at the upper three fourths of intestinal villi and the lower one third of FAE in the rat small intestine.     

Ultrastructural study on the differentiation and the fate of M cells in follicle-associated epithelium of rat Peyer's patch

The differentiation process of immature microvillous epithelial cells to M cells and the fate of M cells in the follicle-associated epithelium (FAE) of the mucosa-associated lymphoid tissues are still unclear. In this study, the differentiation process and the fate of M cells were clarified in rat Peyer's patches under a transmission electron microscope. Almost all immature epithelial cells were found to possess long, slender microvilli, which gradually shortened, thickened and dispersed as the immature epithelial cells migrated away from the crypt orifices. These morphological changes started in the centers and moved to the peripheries of the apical surfaces of epithelial cells, accompanied by the protrusion of apical cytoplasm out of the terminal web. During these changes, the bundles of microfilaments of microvilli never shortened, and both small vesicles in the apical cytoplasm and tiny invaginations of the apical membranes were found. The intraepithelial migrating cells gradually accumulated to form typical intraepithelial pockets. In all FAE, there was no morphological sign of cell death in M cells. The rearrangement of microfilament bundles, the reconstruction of microvilli and the disappearance of pockets resulted in the transformation of M cells into microvillous epithelial cells. These serial ultrastructural changes suggest that M cells are a temporal and transitional cell type caused by the active engulfment of luminal substances and that when the engulfment ceases, the M cells transform into mature microvillous epithelial cells.     

The cellular differentiation of M cells from crypt undifferentiated epithelial cells into microvillous epithelial cells in follicle-associated epithelia of chicken cecal tonsils

To clarify the cellular origin and the fate of M cells, detailed distributions of the epithelial cells were investigated scanning electron microscopically on the follicle-associated epithelia (FAE) of chicken cecal tonsils. The distribution of M cells was closely related with the situation of the crypt orifices in chicken cecal tonsils. In undeveloped cecal tonsils, the intestinal crypts were localized at the periphery of the FAE. In these tonsils, M cells without microvilli (M(0)) were predominantly populated in the basal region of the FAE, whereas goblet cells and microvillous epithelial cells (MV) were more distributed in the middle to the apical region of the FAE. A few M cells with short microvilli were dispersed throughout the FAE. Significantly shrunk MV (MVs) clustered together in transitional portions from the lateral face to the roof of the FAE. In well-developed cecal tonsils, the crypts also opened at the lateral surface in addition to the periphery of the FAE. In these tonsils, the M(0) accumulated densely in the small areas around the crypt orifices exclusively. No sign of exfoliation of apoptotic epithelial cells was found in the M(0)-accumulated areas and at their peripheral boundaries. The MVs were often clustered in the central regions among the crypt orifices in addition to the roof of the FAE. These findings suggest that M cells are directly derived from the undifferentiated crypt epithelial cells, not fall into apoptotic cell death and further differentiate into MV in the FAE of chicken cecal tonsils.     

Pathology of infectious diarrhea of infant rats (IDIR) induced by an antigenically distinct rotavirus

Suckling rats were inoculated with a group B rotavirus to determine the progression of the morphologic changes induced in the intestine by this virus. Several changes were observed by light microscopy 1 day after viral inoculation: shortening of small intestinal villi, villous epithelial necrosis, and villous epithelial syncytia. The lesions were most often present in the distal small intestine, although other small intestinal segments were affected to a lesser degree. By day 3 post-inoculation, epithelial necrosis, and syncytia were no longer present; however, the villous epithelium was disorganized and irregularly vacuolated, and intestinal crypt epithelium was hyperplastic. Alterations in villous height to crypt depth ratios were present in portions of the small intestine for the remainder of the 12-day study period. Epithelial syncytia appeared to form by the breakdown of the lateral interdigitating membranes of the absorptive villous epithelium. Viral particles, abundant in the syncytia, appeared to form from amorphous or reticular arrays of viral precursor material. Group B rotaviral antigens, as detected by indirect immunofluorescence, were present in large amounts in the small intestinal villous epithelium only on the first day after viral inoculation. These studies show that two important diagnostic features of group B rotaviral infections of rats, epithelial syncytia and viral antigen as determined by immunofluorescence, are present only on the first day of disease. These findings should be taken into consideration when attempting to diagnose disease induced by this agent.     

Differentiation of epithelial cells to M cells in response to bacterial colonization on the follicle-associated epithelium of Peyer's patch in rat small intestine

To clarify the relationship between M cells and intestinal microflora, histoplanimetrical investigation into the bacterial colonization and the differentiation to M cells was carried out in rat Peyer's patch under physiological conditions. The follicle-associated epithelium (FAE), except for the narrow area of apical region, was closely covered with both neighboring intestinal villi and a thick mucous layer, the latter of which also filled the intervillous spaces as well as the space between the FAE and the neighboring intestinal villi. Indigenous bacteria adhered almost constantly to the narrow areas of apical regions of both intestinal villi and the FAE. Bacterial colonies were occasionally located on the basal to middle region of FAE, where M cells also appeared, forming large pockets. When bacterial colonies were located on the basal to middle region of FAE, bacteria with the same morphological characteristics also proliferated in the intervillous spaces neighboring the Peyer's patch. In cases with no bacterial colonies on the basal to middle region of FAE, however, M cells were rare in the FAE. Histoplanimetrical analysis showed the similar distribution pattern of bacterial colonies on the FAE and M cells in the FAE. M cells ultrastructurally engulfed indigenous bacteria, which were then transported to the pockets. These results suggest that indigenous bacterial colonization on the FAE stimulates the differentiation of M cells in the FAE under physiological conditions. The uptake of bacteria by M cells might contribute the regulation of the development of indigenous bacterial colonies in the small intestine.     

Ultrastructural study on the epithelial responses against attachment of indigenous bacteria to epithelial membranes in peyer's patches of rat small intestine

The ultrastructure of epithelial responses against the membrane adhesion of indigenous bacteria was investigated in the follicle-associated epithelium (FAE) of rat small intestine. The most frequent adherence of the various morphological types of bacteria to the epithelial membranes was found at the apex of the FAE. The attachment sites were deeply invaginated, and their bottoms were deformed into a sharp cone shape. Four layers with different electron densities were formed just beneath the apical membranes by microfilaments which surrounded the invaginations. The electron density of each layer was gradually decreased as being apart from the invaginations. The extremities of some bacteria in the invaginations were deformed into sharpened shapes. The cell walls of the extremities of the bacteria were occasionally dissolved in the invaginations, and their cytoplasms were slightly swollen with low electron densities. In some invaginations, the attached bacteria were eliminated to leave their fragments such as filamentous debris and a part of cell walls. Finally these remnants disappeared completely. When the bacterial colonies existed in the middle region of the FAE, the attachment of bacteria resulted in the engulfment of bacteria by M cells. The degenerated bacteria whose cytoplasmic matrices were separated into high electron dense materials and cleared materials were occasionally engulfed by ordinary microvillous columnar epithelial cells or goblet cells throughout the FAE. These findings suggest that the epithelial cells reject the attachment of live indigenous bacteria and that the M cells absorb indigenous bacteria in rat Peyer's patches.     

Special sugar expression on apoptotic epithelial cells of Peyer's patches and intestinal villi in rat small intestine

Our previous study clarified that the apical regions of both the follicle-associated epithelium (FAE) of Peyer's patches and the intestinal villi are the only adhesion sites of indigenous bacteria in rat jejuno-ileum. To survey the ligands against bacterial lectins, sugar expression patterns on epithelial cells were lectin-histochemically investigated using 21 lectins in the jejuno-ileal Peyer's patches of rats. As a result, (D-glcNAc)(2-4), detected by Solanum tuberosum (STL) and by Lycopersicon esculentum (LEL), and beta-D-gal(1-3)-D-galNAc detected by Peanut agglutinin (PNA), were strongly expressed on the brush borders of the apical regions of the FAE and the intestinal villi. On the other hand, neither sugar was expressed on the brush borders of the basal regions of both FAE and intestinal villi. The positive intensities for the lectins correlated with the progression of epithelial apoptosis in the FAE and in the intestinal villi. Moreover, the double staining with lectin histochemical method and the in situ nick end-labeling method could simultaneously detect the strong expression of both sugars and nuclear DNA fragmentation in epithelial cells at the late apoptotic stage. Other sugar expression patterns in the intestinal villi were similar with those in the FAE. There were no lectins specific for M cells in the FAE. From these findings, the possible sugars of ligands against some indigenous bacterial lectins, expressing specially on the apoptotic epithelial cells, might be narrowed down in rat jejuno-ileum.     

Persorption of bovine lactoferrin from the intestinal lumen into the systemic circulation via the portal vein and the mesenteric lymphatics in growing pigs

The absorption and the transportation of intestinally administrated bovine lactoferrin (LF) were immunohistochemically and physiochemically investigated in the small intestine of growing pigs. At the apical halves of the small intestinal villi, bovine LF was absorbed by transcytosis as small vesicles through villous columnar epithelial cells. The presence of bovine LF-positive membranes of transcytotic vesicles suggests that the absorption was mediated by LF-binding factors on the epithelial cell membranes. Almost all of the absorbed bovine LF was demonstrated to be transported via the lymphatics and the portal vein into the systemic circulation. The LF-concentration in systemic circulation was significantly higher at 1 hr following intestinal administration of bovine LF. Bovine LF-positive lymphocytes also were transferred into the systemic circulation from intestine via the lymphatics and the portal vein.     

Persorption mechanisms of luminal antigenic particulates via apoptotic epithelial cells of intestinal villi into systemic blood circulation in orally immunized rats

The possibility of persorption of prefixed bovine serum albumin-coated sheep erythrocytes (BSA-SEs) from mucous epithelial cells and its mechanisms were investigated in rats orally immunized by BSA for 14 consecutive days. On the day after the final oral immunization, the rats were duodenally perfused by BSA-SEs or non-coated SEs. BSA-SEs were also duodenally perfused in non-immunized rats. Thirty min after perfusion, BSA-SEs were significantly more engulfed by late-apoptotic-stage villous columnar epithelial cells in the orally immunized rats than those in other experiments. The specific antibody (SpAb) was detected on the surfaces of BSA-SEs in rats with oral immunization. In Peyer's patches of all animals, no SEs reached the follicle-associated epithelium, because of the close attachment of follicle-associated intestinal villi and the thick mucous layer. BSA-SEs were more frequently persorbed into portal blood in the orally immunized rats than in other rats. Small numbers of BSA-SEs or SEs were detected in the systemic blood of all animals. BSA-SEs were also histologically found in the blood vessels of the liver, but not in mesenteric lymph nodes. These findings suggest that sensitized antigenic particulates are taken up by late-apoptotic-stage villous columnar epithelial cells in the small intestine and are finally persorbed into the systemic blood circulation. The uptake of antigenic particulates might be mediated by its luminal SpAb.     

Scanning electron microscopic observations on the intestinal villi in growing White Leghorn and broiler chickens from 1 to 30 days of age

1. To obtain intestinal morphological data demonstrating the faster growth rate in broiler (BR) than in White Leghorn (WL) chickens, villi of the duodenum, jejunum and ileum were examined from 1 to 30 d of age by scanning electron microscopy. 2. At the first day after hatching, villi of each intestinal segment showed a finger-like shape in both breeds. Villi developed to a plate-like shape in the duodenum, a wave-like shape in the jejunum and a tongue-like shape in the ileum at 30 d of age via the common plate-like villi at 10 d of age. The fundamental villous shape and arrangement seem to be accomplished by 10 d of age; two types of obliquely elongated plate-like villi showed a zigzag arrangement, adjoining at an angle of 40 degrees to 60 degrees like an oblique T-shape. It is suggested that such a villous arrangement would be more effective for nutrient absorption by inducing a long zigzag flow of ingesta. 3. Compared with WL, even at the first day after hatching BR had many more developed epithelial cell protrusions over the whole apical surface of the duodenal villi. In WL the protrusions were not so apparent and located only in the central area of the villous tip. Furthermore, at 10 d of age BR showed more developed and larger villi, many wider microvilli at the apical portion of villi and more active extrusions of epithelial cells from the tip of the duodenal and jejunal villi. 4. These morphological characteristics of villi in early life in BR suggest a greater absorptive surface area and a more active intestinal function, permitting the faster growth rate of BR immediately after hatching.     

Ultrastructural changes in the small intestine of neonatal calves with enteric colibacillosis

The small intestines of calves inoculated orally with the enteropathogenic strain of Escherichia coli 0101:K'B41',K99 were examined by electron microscopy at 3, 6, 12, 16, 21, 36, 69, 70 and 72 hours after inoculation. The challenge organism adhered to the mucosa of the distal small intestine from six hours post-inoculation. Bacteria were separated from the microvillous brush border by a gap of 200 to 300 nm in which bacterial fimbriae and the microvillous glycocalyx were seen. Bacteria never were found in epithelial cells but were present in macrophages in the lamina propria from 12 hours. At three and six hours, cytopathic changes were not seen in the small intestine, but from 12 hours epithelial cells on affected villi had blunt and thick microvilli and contained cytoplasmic inclusions. Epithelial cells were seen frequently in the process of extrusion from the villi, either singly, in small groups, or as ribbons of cells. Intervillous bridges, characteristic of villous fusion, were seen frequently from 69 hours.     

Histology, immunohistochemistry and ultrastructure of the equine tubal tonsil

The tubal tonsil of the horse surrounds the pharyngeal opening of the eustachian tube and is lined by pseudostratified columnar ciliated epithelium interspersed with areas of follicle-associated epithelium (FAE) heavily infiltrated by lymphocytes but devoid of goblet and ciliated cells. Scanning and transmission electron microscopy revealed microvillous cells and cells with features characteristic of M cells such as reduced microvilli or depressed bare surface, more numerous mitochondria, small vesicles and lysosomes, as well as vimentin filaments and epitopes specific for GS 1-B4 as previously seen in the nasopharyngeal tonsil. M cells were also identified in areas of respiratory epithelium not associated with lymphoid follicles and appeared to be the nasal mucosal counterparts of recently described intestinal villous M cells in the mouse. The underlying lymphoid tissue of the FAE was generally organized as solitary lymphoid follicles without germinal centres in contrast to the diffuse and large amount of organized lymphoid follicles with germinal centres that characterize the nasopharyngeal tonsil. CD8+ T and B-lymphocytes were much fewer than in the nasopharyngeal tonsil. High endothelial venules were mainly oriented towards the parafollicular area and contained much fewer endothelial pores and vesiculo-vacuolar organelles. Finally, scattered small clusters of mucus acini and striated muscles were other features that differentiated the tubal and nasopharyngeal tonsils.     

Further contribution on the fine structure of fetal goblet cells. Investigations on the small intestine of cattle

Early in the fetal development, i.e. at the time both of the growth of intestinal villi and crypts and the epithelial cell differentiation the goblet cells also appear. The maturation of goblet cells progresses during their migration from the base of the villi respectively of the crypts to the villous top. As in the bovine large intestine there are vesicles and capsulated vacuoles, which contain vesicles, as single elements or as conglomerates in the immature goblet cells of the small intestine. These images deliver a scenario of the mechanism of secretory granule production and probably play a role in the formation of mucus.     

实验性流行性腹泻仔猪小肠粘膜上皮细胞及肠系膜淋巴结的超微结构

给8头生后3d的哺乳仔猪经口感染猪流行性腹泻病毒(PEDV)“吉”毒株,于感染后18、30、45和96h各扑杀2头,以透射电镜和扫描电镜观察了小肠粘膜上皮细胞及肠系膜淋巴结的超微结构。结果表明,小肠上皮细胞的病变因感染时间不同而有明显差异。上皮细胞的脱落和残留上皮细胞超微结构的破坏,以感染后30h最严重,病毒在这些上皮细胞内的增殖最显著。感染后45h,见有大量新生上皮细胞修补损伤的肠绒毛。感染后96h,小肠绒毛短缩、粗大乃至发生融合。实验仔猪肠系膜淋巴结内巨噬细胞和淋巴细胞的超微结构均遭到破坏,在巨噬细胞内见有PED冠状病毒粒子。     

Lysosomal structures in the intestinal epithelium of mammals during their pre- and postnatal development. A micromorphometric-functional synopsis]

The first lysosomes appear in the stratified embryonic intestinal epithelium during its transition into the simple columnar form. This occurs concurrently with the initial villogenesis. Lysosomes situated basally in the epithelium are presumably the precursor of the first giant lysosomes in the lower small intestine of rodents. Immediately after establishment of the simple configuration a special form of secondary lysosomes can be observed, i.e. glycogenosomes, in the ephemerally existing huge glycogen containing areas. During subsequent fetal intestinal development one observes two events in the epithelial cells, which are the same in principle but differ in one essential point, while they exhibit partially impressive structures. On the one hand there are autophagic degenerative lysosomal processes in the villous epithelium until birth, that lead to a surface without villi in the large intestine, where they occur particularly frequently. On the other hand giant lysosomes originate perinatally in the lower small intestine as well as in the caecum and colon ascendens, in which protein molecules, which were transported by a system of inframicrovillar membranes, are lysosomally degraded, which can be defined as a heterophagic event.     

Immunohistochemical and histoplanimetrical study on the endothelial receptor involved in transportation of minute chylomicrons into subepithelial portal blood in intestinal villi of the rat jejunum

A portion of the minute chylomicrons less than 75 nm in diameter are transcytosed from the extravascular tissue into the subepithelial blood capillaries (sBC) in the villous apices of the rat jejunum. However, the details of the transportation mechanism have not been clarified. In this study, the endothelial receptor involved in the transportation of minute chylomicrons into the sBC’s lumina was immunohistochemically and histoplanimetrically examined in intestinal villi of the rat jejunum. Immunopositivity for very low density lipoprotein (VLDL) receptor was detected on the luminal and basal surfaces of the endothelial cells of sBC in approximately 68% of those apices of jejunal villi that possessed numerous chylomicrons in the lamina propria, while VLDL receptor was detected on the endothelial cells of sBC in only approximately 8% of intestinal villi that possessed few or no chylomicrons in the lamina propria. No immunopositivity for LDL receptor was detected in the sBC of all intestinal villi. These findings suggest that VLDL receptor is expressed by the endothelial cells of the sBC in conjunction with the filling of the lamina propria of jejunal villi with many chylomicrons produced by the villous columnar epithelial cells and that the VLDL receptor mediates the transportation of minute chylomicrons, maybe VLDL, into the subepithelial portal blood from the extravascular tissue of the rat jejunal villi.     

Age-dependent resistance to transmissible gastroenteritis of swine. III. Effects of epithelial cell kinetics on coronavirus production and on atrophy of intestinal villi.

Coronavirus titers in small intestine, degree of villous atrophy and apparent rates of regeneration of intestinal villi were compared in newborn, 3-week-old and adult pigs for 1 week after they were exposed to the transmissible gastroenteritis virus of swine. The response within the newborn group was homogeneous, resulting in high virus titers, maximal villous atrophy and comparatively slow regeneration. In general, virus titers were lower, villous atrophy was less severe and regeneration more rapid in both older groups than in the newborn pigs. However, the response varied greatly in the older groups. The 3-week-old group was divided into two populations. The major population had low virus titers and developed partial villous atrophy, whereas the minor population had marked villous atrophy and virus titers comparable to those of the newborn pigs. These observations support the hyposthesis that the accelerated replacement of villous epithelium in the small intestine of pigs during the first 3 weeks contributes to the innate age-dependent resistance to transmissible gastroenteritis. The accelerated replacement of villous epithelial cells in older pigs contributes to resistance in two ways. The increased proliferative capacity of crypt epithelium results in a more rapid regeneration of atrophic villi; and the comparatively young villous absorptive cells resulting from accelerated replacement produce less virus per cell than the older ones of the newborn pig.