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.     

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.     

Cellular kinetics of villous epithelial cells and m cells in rabbit small intestine

The cellular kinetics of villous columnar epithelial cells and M cells in the rabbit small intestine were determined by the use of 5-bromo-2'-deoxyuridine (BrdU) as a tracer. To identify M cells, vimentin antibody was used. The BrdU-labeled nuclei of columnar epithelial cells reached the base of intestinal villi in all portions at 1 day after BrdU administration. Thereafter, BrdU-labeled cells migrated toward the villous tip, but they did not move at a uniform speed. The epithelial cells which existed in intestinal villi on circular folds moved faster than those on mucosa other than circular folds. At 7 days after BrdU administration, the leading edge of BrdU-labeled epithelial cells already disappeared from the villous tip in all portions of the small intestine. In the ileal Peyer's patch, the BrdU-labeled nuclei of microvillous epithelial cells and vimentin-positive M cells appeared near the intestinal crypt orifice at 1 day after BrdU administration, and then migrated toward the luminal surface of the follicle-associated epithelium (FAE). As they moved toward the upper portion of FAE, the number of BrdU-labeled M cells on the side of the dome decreased simultaneously. The leading edge of BrdU-labeled epithelial cells disappeared from the top of the FAE within 7 days. These results suggest that M cells may differentiate from the undifferentiated cells in intestinal crypts within 1 day and disappear from the top of the FAE after the change of their form from M cells into microvillous epithelial cells.     

The identification of intestinal M cells in the sacculus rotundus and appendix of the Angora rabbit

The present study was aimed at the immunohistochemical demonstration of M cells, found in the follicle-associated epithelium (FAE) of the sacculus rotundus (SR) and appendix of the Angora rabbit, using anti-vimentin primary antibodies, and at the determination of certain fine structural characteristics. Ten adult Angora rabbits constituted the material of the study. Immunohistochemical staining revealed that many cells composing the FAE, which covered the dome regions of the SR and appendix, reacted positively with vimentin. FAE contained two different types of vimentin-positive cells. The first type surrounded intraepithelial lymphocytes (IEL) with a basolateral invagination in the apex and periphery of the dome epithelium, whilst the second type consisted of columnar cells found in the FAE near crypts. The immunoreactivity of the cells found in the FAE covering the apex and periphery of the domes was observed particularly in the perinuclear cytoplasm and the cytoplasm surrounding the IEL. Electron microscopic examination demonstrated that the M cells found in the FAE covering the apex and periphery of the dome regions of the SR and appendix did not exhibit any microvilli on their apical surface. The FAE near crypts contained columnar cells, which resembled enterocytes. The apical membrane of these cells exhibited shorter and irregular microvilli, in contrast to neighbouring enterocytes. It was determined that M cells, found in the FAE of the SR and appendix in the Angora rabbit, displayed similarities in terms of localization and fine structure. This situation may be indicative of the two lymphoid structures with different localization having similar functional properties.     

Interactions of butyric acid- and acetic acid-treated Salmonella with chicken primary cecal epithelial cells in vitro

In vitro studies of the interaction between pathogenic bacteria and the chicken intestinal epithelium are hampered by the lack of a host- and tissue-specific in vitro model. Therefore, a reproducible method for isolation and cultivation of chicken primary cecal epithelial cells was developed. Cecal crypts were isolated and cultured in vitro to form a semiconfluent layer of epithelial cells. Incubation of Salmonella enteritidis with these cells resulted in invasion. Pretreatment of the Salmonella bacteria with butyric acid resulted in a significant decrease of invasion of the bacteria in the chicken cecal epithelial cells, whereas pretreatment with acetic acid increased invasiveness. These interactions of S. enteritidis with primary chicken cecal epithelial cells were similar to the interactions with other epithelial cell types.     

The microanatomy of the palatine tonsils of the buffalo (Bos bubalus)

The palatine tonsils play a key role in initiating immune responses against antigens entering the body through the mouth. They are also replication sites of some pathogens. There is no data available about the structure of the palatine tonsils of the Egyptian water buffalo. Therefore, palatine tonsils of 14 clinically healthy buffalo bulls (2–3 years old) were examined macroscopically and microscopically using light, and transmission electron microscopes. The tonsils had an elongated kidney shape with a central invagination (tonsillar fossa) containing a single macroscopic opening leading to a small central cavity (tonsillar sinus). A number of macroscopic crypts originated from this sinus (internal crypts). Besides the tonsillar fossa, also small macroscopic crypts (external crypts) were present. The tonsils were enclosed by a thin connective tissue capsule and septa divided the tonsils into incomplete lobes. Within these encapsulated organs mucous glands were very obvious. Each crypt was highly branched and lined with stratified squamous non-keratinized epithelium. Several lymphoid cells infiltrated between the epithelial cells forming patches of lymphoepithelium. The crypt lumen contained lymphocytes, neutrophils and erythrocytes. Lymph nodules with clear germinal centers extended under the epithelial surface. Diffusely distributed lymphocytes were found in the narrow interfollicular region. High endothelial venules, interdigitating dendritic cells, macrophages and plasma cells were observed among the diffuse lymphocytes. Lymphatics filled with lymphocytes drained the tonsils.     

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.     

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.     

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.     

鸡传染性法氏囊病的病理学研究

人工接种２８日龄非免疫鸡传染性法氏囊病病毒（ＩＢＤＶ）后,对感染鸡的法氏囊、胸腺、脾、盲肠扁桃体、哈德氏腺、肝、肾进行病理组织学检查。感染后４８ｈ,法氏囊淋巴组织最早出现坏死且长久存在。其他淋巴器官的病变出现较迟,程度轻微且恢复较快。ＩＢＤＶ单抗免疫荧光检测,法氏囊及其他淋巴器官中均检测到病毒,接种后１２ｈ法氏囊中即检出病毒,持续时间也最长（攻毒后１２ｄ）,其次是盲肠扁桃体（攻毒后８ｄ）。攻毒１３ｄ以后,上述器官均未检测到病毒。法氏囊粘膜上皮的扫描电镜观察,攻毒后２ｄ,上皮细胞肿胀,微绒毛减少或消失。攻毒后３ｄ,局部上皮细胞坏死、脱落,并向整个粘膜层扩展,攻毒后１０ｄ,上皮层基本修复。     

Staining patterns for actin and villin distinguish M cells in bovine follicle-associated epithelium

M cells play a central role in the initiation of mucosal immune responses. However, a primary source of difficulty for investigations of this is the lack of an available specific marker for bovine M cells. As M cells possess irregular and short microvilli, we investigated the distribution and localization of the microvillar proteins actin and villin by immunohistochemistry of the gut of calves. In ileum of the calf, actin and villin were clearly and continuously immunostained in the brush border of the villous epithelia, however, discontinuous immunostaining with patches of no staining were observed in follicle-associated epithelium (FAE). Electron microscopy revealed that M cells had irregular microvilli and lacked the typical brush border, and it was inferred that these patches of no staining might be the intercellular crevices of M cells. As the microvilli of M cells were very sparse, there were several areas of weak immunostaining in calf jejunal FAE. These results suggest that M cells in calf FAE are detectable by the absence of staining for actin and villin.     

Are there M cells in the cecal tonsil of chickens?

A unique morphological cell type, "microfold or membranous (M) cell-like cell", was detected electron-microscopically in the cecal tonsil epithelium of the chicken. M cell-like cells possessed a few short microvilli of irregular arrangement and a large number of lymphocytes and macrophages wedged into their basal surfaces. Triticum vulgaris was found to bind to M cell-like cells. With horseradish peroxidase (HRP) treatment, M cell-like cells showed an active HRP uptake just as did the neighbouring usual absorptive epithelial cells. No uptake of colloidal carbon particles from the intestinal lumen was recognized in any part of the intestinal epithelium. These results suggest that M cell-like cells of the chicken possess some M cell-characteristic morphological and histochemical features, but that their active uptake of foreign materials is not so developed as in mammalian M cells.     

Intestinal trichomonads (Tritrichomonas mobilensis) in the natural host Saimiri sciureus and Saimiri boliviensis

A retrospective study of cecal and colonic tissues from 28 squirrel monkeys (Saimiri sciureus and Saimiri boliviensis) demonstrated enteric trichomonads within luminal crypts. Twenty-one of 28 (75%) had trichomonads in the mucosal epithelium either in cup-like depressions or intraepithelial vacuoles. Organisms were also beneath the superficial luminal mucosal epithelium and between the basement membrane and crypt epithelial cells. Immunoperoxidase staining also identified organisms within the lamina propria and submucosa. Additional histologic changes included mucosal ulceration, multifocal cryptitis, and focal epithelial necrosis. Most areas containing trichomonads did not have an associated inflammatory response.     

Colonization strategy of Campylobacter jejuni results in persistent infection of the chicken gut

Although poultry meat is now recognized as the main source of Campylobacter jejuni gastroenteritis, little is known about the strategy used by the bacterium to colonize the chicken intestinal tract. In this study, the mechanism of C. jejuni colonization in chickens was studied using four human and four poultry isolates of C. jejuni. The C. jejuni strains were able to invade chicken primary cecal epithelial crypt cells in a predominantly microtubule-dependent way (five out of eight strains). Invasion of cecal epithelial cells was not accompanied by necrosis or apoptosis in the cell cultures, nor by intestinal inflammation in a cecal loop model. C. jejuni from human origin displayed a similar invasive profile compared to the poultry isolates. Invasiveness of the strains in vitro correlated with the magnitude of spleen colonization in C. jejuni inoculated chicks. The C. jejuni bacteria that invaded the epithelial cells were not able to proliferate intracellularly, but quickly evaded from the cells. In contrast, the C. jejuni strains were capable of replication in chicken intestinal mucus. These findings suggest a novel colonization mechanism by escaping rapid mucosal clearance through short-term epithelial invasion and evasion, combined with fast replication in the mucus.     

Changes in the leucocyte subpopulations of the palatine tonsillar crypt epithelium of pigs in response to Streptococcus suis type 2 infection

The tonsils are portal of entry and a site of multiplication and persistence for a variety of pathogens, including Streptococcus suis (S. suis), which is a common cause of meningitis, septicemia and arthritis in pigs. Understanding the early changes that occur in the first barrier of the tonsil, i.e. the crypt epithelium, in response to S. suis infection is critical in clarifying the pathogenesis of this disease and for the future development of efficient methods of mucosal vaccination. In this study, we investigated the early changes, from 18 to 72 h, that occur in leucocyte subpopulations of the crypt epithelium of the palatine tonsils of 3-week-old pigs in response to S. suis type 2 infection. Monoclonal antibodies against leucocyte markers CD3, CD4, CD8, gammadelta T cell receptor, lambda-immunoglobulin light-chain, myeloid cells, and major histocompatibility complex class II molecule (MHC-II) were used in an avidin-biotin immunoperoxidase technique. An increase in the number of lambda-immunoglobulin light-chain positive cells (B cell subset) was noticed in crypts of S. suis-infected animals from 18 h after infection onwards. This increase was significant at 18 and 48 h after infection. The number of CD4 and CD8 cells was greater from 18 h onwards, with a significant increase at 24 and 72 h post-infection. No significant difference in numbers of CD3, gammadelta T cell receptor and MHC-II positive cells was detected in the crypts of infected animals compared to controls. Macrophages, neutrophils and crypt epithelial cells stained positively with the myeloid marker, and the area of crypt epithelium positive for this marker was increased in the crypts of infected animals, with a significant difference detected at 24 and 72 h after infection. These results suggest that there is participation of the innate immunity in the early phase of S. suis infection, represented by neutrophils, macrophages and likely epithelial cells, and that there is a potential for the initiation of both humoral and cellular responses against S. suis within the crypt epithelium of the palatine tonsil.     

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.     

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.     

C6, a new monoclonal antibody,reacts with the follicle-associated epithelium of calf ileal Peyer's patches

The follicle-associated epithelium (FAE) of Peyer''s patches (PPs) contains M cells that are important for reducing mucosal immune responses by transporting antigens into the underlying lymphoid tissue. We generated a monoclonal antibody (C6) that reacted with the FAE of calf ileal PPs, and analyzed the characteristics of C6 using immunohistochemistry and Western blotting. FAE of the ileal PP was stained with C6 during both late fetal developmental and postnatal stages. Neither the villous epithelial cell nor intestinal crypt basal cells were stained at any developmental stage. During the prenatal stages, FAE of the jejunal PP was C6-negative. However, a few C6-positive cells were distributed diffusely in some FAE of the jejunal PPs during the postnatal stages. The protein molecular weight of the antigen recognized by C6 was approximately 45 kDa. These data show that C6 is useful for identifying the FAE in ileal PPs and further suggest that differentiation of the FAE in these areas is independent of external antigens.     

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.     

Comparative study of colonizing and noncolonizing Campylobacter jejuni

Campylobacter jejuni A74/O and A74/C are congenic strains. An oral dose of 10(5) organisms of strain A74/C colonizes chicken intestines. Strain A74/O, from which A74/C is derived, does not colonize the chicken intestines with an oral dose of 10(5) organisms. In this study, the congenic bacteria were compared to identify possible colonization mechanisms. Differences were not observed in plasmid content or by HindIII, Pst I, Acc I, HincII, Ava I, Ava II, Xba I, and BamHI restriction enzyme digestion of total DNA. Transmission electron microscopy of negatively stained samples revealed no differences between the strains. Sections of cecal tissue from nonfed day-of-hatch chicks were cultured with each strain for 2 hours and then examined by light and electron microscopy. Both strains caused necrosis of villus epithelial cells. Immunofluorescent or silver staining revealed strain A74/C located deep in numerous epithelial crypts, but strain A74/O only was present in one sample mixed with sloughed necrotic cells. Similarly, organisms were detected by transmission electron microscopy deep in crypts in tissues cultured with A74/C, but not A74/O. Cells of A74/C detected in crypts did not appear to associate with epithelial cells. The strains did not differ in chemotactic behavior to mucin or fucose.