Lectin binding sites in the human gallbladder and cystic duct

Summary A battery of 18 fluorescein isothiocyanate (FITC) labeled lectins was used to histochemically define the features of epithelial cells in the body, neck and the cystic duct of the human gallbladder. Surface epithelium in all three anatomic locations reacted with all lectins, either diffusely or focally, except for lectin type I from Griffonia simplicifolia and type II from Ulex europaeus. No quantitative differences were noted except for the tendency of some lectins to bind more often to the neck and cystic duct epithelium rather than the body and vice versa. In the body the surface epithelium did not differe from cells lining the crypts. On the other hand, glands of the neck and the cystic duct were essentially indistinguishable one from another but differed from the overlying surface epithelium in so far as they reacted with some lectins which were unreactive with surface epithelium and vice versa. Considerable case to case variation in the expression of lectin binding sites in each of three anatomic areas was noted. No consistent differences were noticed between gallbladders removed for cholecystitis — cholelithiasis and those removed for other incidental reasons. We conclude that all epithelial cells in the cholecysto-biliary tract are rich on glyconjugates but the pattern of expression varies depending on the anatomical location and the influence of poorly understood individual determinants.     

Transdifferentiation of murine squamous vaginal epithelium in proestrus is associated with changes in the expression of keratin polypeptides

The superficial layers of the stratified squamous epithelium of the murine vagina undergo transdifferentiation into cuboidal mucinous cells during the proestrus phase of the normal estrous cycle. In contrast to their squamous progenitor cells which have the cytoskeletal characteristics of squamous epithelium, mucinous cells express keratin polypeptides typical of simple nonstratified epithelia. Accordingly, the transdifferentiation of squamous cell into mucinous cells involves not only a change in cell morphology but also a switch in the expression of keratin polypeptides. These data indicate that the stratified squamous cells of the vagina are not terminally differentiated and their phenotype can be hormonally modulated.     

Lectin binding sites in the human gallbladder and cystic duct

A battery of 18 fluorescein isothiocyanate (FITC) labeled lectins was used to histochemically define the features of epithelial cells in the body, neck and the cystic duct of the human gallbladder. Surface epithelium in all three anatomic locations reacted with all lectins, either diffusely or focally, except for lectin type I from Griffonia simplicifolia and type II from Ulex europaeus. No quantitative differences were noted except for the tendency of some lectins to bind more often to the neck and cystic duct epithelium rather than the body and vice versa. In the body the surface epithelium did not differ from cells lining the crypts. On the other hand, glands of the neck and the cystic duct were essentially indistinguishable one from another but differed from the overlying surface epithelium in so far as they reacted with some lectins which were unreactive with surface epithelium and vice versa. Considerable case to case variation in the expression of lectin binding sites in each of three anatomic areas was noted. No consistent differences were noticed between gallbladders removed for cholecystitis--cholelithiasis and those removed for other incidental reasons. We conclude that all epithelial cells in the cholecysto-biliary tract are rich on glyconjugates but the pattern of expression varies depending on the anatomical location and the influence of poorly understood individual determinants.     

Pregnancy-related changes in the mouse oviduct and uterus revealed by differential binding of fluoresceinated lectins

The binding of 20 fluorescein isothiocyanate (FITC)-labeled lectins to various portions of the pregnant and non-pregnant murine oviduct and uterus was studied by fluorescence microscopy. Five lectins (from Ricinus communis (RCA-I), Maclura pomifera (MPA), Triticum vulgare (wheat germ-WGA), Bauhinia purpurea (BPA), and Ulex europeus (UEA-I] reacted differentially with the epithelium of pregnant as compared with the non-pregnant uterus. The binding of RCA-I, MPA and WGA delineated pregnancy-related changes in the distal oviduct and colliculus tubaris. WGA recognized also pregnancy related changes in the proximal oviduct. The reactivity of the remaining 15 lectins did not distinguish the pregnant and non-pregnant oviduct and uterus, although some of them served to identify specific components of the mouse genital tract. Thus, Soybean lectin (SBA) reacted almost exclusively with the colliculus tubaris. UEA-I alone reacted exclusively with the epithelium of the non-pregnant uterus. RCA-II reacted preferentially with the epithelium of the oviduct and uterus as compared with its weak reactivity with the stroma. Two lectins (from Pisum sativum and Lens culinaris) reacted selectively with stromal cells of the uterus and oviduct. Present data indicate that the differential binding properties of these FITC-labeled lectins can be exploited to identify certain components of the mouse oviduct and uterus and to indicate changes in the cell surface and/or cytoplasm in these structures during pregnancy.     

Histochemical and ultrastructural study of the digestive tract of the tortoise Testudo graeca (Testudines)

The digestive tract of the tortoise Testudo graeca (Testudines) was investigated by means of light and electron microscopy. The esophagus of T. graeca was lined by two types of epithelium: non-keratinized stratified squamous in the upper portion and stratified columnar in the lower. The lamina propria of the esophagus contained tubular or tubuloacinar glands. The mucosa of the stomach showed similar characteristics to those of other reptiles. The small intestine exhibited longitudinal folds lined by absorptive and goblet cells. The large intestine was lined by columnar mucous cells. Within the lamina propria of the large intestine, masses of 10–15 epithelial cells connecting with the surface epithelium by means of slender cytoplasmic processes were observed. A battery of six lectins (Con-A, PNA, WGA, DBA, SBA, and LTA) was used to identify the epithelial mucins. WGA and DBA reacted with all types of mucous cells throughout the alimentary canal. PNA was only unreactive in the intestine, LTA in the esophagus, and SBA in the large intestine. These results indicate a similar lectin-binding pattern throughout the gut of T. graeca.     

Cell proliferation in the principal target organs of the dog (beagle) ovary during various periods of the estrous cycle

Cell proliferation in the principal target organs of the ovary was studied in 7 beagle dogs during proestrus, early estrus, secretory and regressive metestrus. Pulse labelling with (3H)-thymidine and autoradiography were applied and labelling indices were determined. Organs with squamous and with glandular epithelium except the mammary gland showed a proliferation peak during early proestrus, the incidence of labelling being higher in organs with squamous epithelium. During early proestrus the intensity of labelling decreased in organs with squamous epithelium in the following order: vulva, vagina, portio vaginalis, and portio supravaginalis. At this time cell replication was higher in the superficial glands of the cervix and corpus uteri as well as the circular layer of the myometrium compared with values from the basal glands or the longitudinal layer. The steepest decline of the proliferative activity occurred between the proestrous period. While some labelled cells were observed during secretory metestrus, labelling was almost absent during regressive metestrus. By contrast, the mammary gland began to develop at the periovulatory period and was at the height of its proliferative activity during secretory metestrus. It is concluded for species with a long estrous cycle that: (1) the rate of cell replication in the vulva, vagina, uterus and oviduct is at its peak at the beginning of the estrous cycle and declines during proestrus; (2) as compared to observations in species with short estrous cycles, cell proliferation is very low during metestrus.     

Differentiation-dependent expression of lectin binding sites on normal and neoplastic keratinocytes in vivo and in vitro.

We used lectins as probes to demonstrate the composition of membrane carbohydrates of canine keratinocytes in various functional stages and various degrees of differentiation. Keratinocytes during normal epidermal turnover were compared by lectin immunohistochemistry to cells of hyperplastic epidermis and neoplastic keratinocytes. Three types of epidermal tumors and oral squamous cell carcinomas were examined. In addition, two in vitro tissue culture systems for keratinocytes were studied and compared with in vivo epithelium. In normal skin, PNA reacted only weakly with basal cells, whereas in hyperplastic skin basal cells bound this lectin strongly, demonstrating increasing expression of PNA binding sites with increasing thickness of the stratified squamous epithelium. ConA bound to basal cell tumors only. In oral squamous cell carcinomas, the expression of distinct lectin binding sites correlated with certain histological growth patterns, e.g., UEA-I reacted with highly invasive tumors but not with tumors showing a solid growth pattern. Using cell surface iodination and polyacrylamide gel electrophoresis, distinct differences in cell membrane protein expression were demonstrated between normal and neoplastic keratinocytes. SDS-polyacrylamide gel electrophoresis of cultured normal and neoplastic keratinocytes revealed several cell surface proteins that are specific for either cell type. Neoplastic cells specifically express a 140 KD lectin binding cell surface glycoprotein. The results of this study show that lectin binding patterns of keratinocytes are dependent on the functional state and the degree of differentiation of the cells and demonstrate correlation of some histological growth patterns with distinct lectin binding phenotypes, suggesting association of expression of cell membrane carbohydrate moieties with growth patterns. In addition, close similarities between "lifted cultures" grown at the air-liquid interface and native tissue demonstrate the value of this culture system as a model for differentiated stratified squamous epithelium.     

Lectin binding patterns to terminal sugars of rat lung alveolar epithelial cells.

We used post-embedding cytochemical techniques to investigate the lectin binding profiles of rat lung alveolar epithelial cells. Sections from rat lung embedded in the hydrophilic resin Lowicryl K4M were incubated either directly with a lectin-gold complex or with an unlabeled lectin followed by a specific glycoprotein-gold complex. The binding patterns of the five lectins used could be divided into three categories according to their reactivity with alveolar epithelial cells: (a) the Limax flavus lectin and Ricinus communis I lectin bound to both type I and type II cell plasma membranes; (b) the Helix pomatia lectin and Sambucus nigra L. lectin bound to type II but not type I cells; and (c) the Erythrina cristagalli lectin reacted with type I cells but was unreactive with type II cells. The specificity of staining was assessed by control experiments, including pre-absorption of the lectins with various oligosaccharides and enzymatic pre-treatment of sections with highly purified glycosidases to remove specific sugar residues. The results demonstrate that these lectins can be used to distinguish between type I and type II cells and would therefore be useful probes for investigating cell dynamics during lung development and remodeling.     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Summary Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Morphology of the genital organs of the female red-rumped agouti (Dasyprocta leporina,Linnaeus, 1758) during estrous cycle phases and in advanced pregnancy

The study investigated the gross and microscopic anatomy of the genital organs of 20 agoutis at different stages of the estrous cycle and four in the final trimester of pregnancy. Specimens were euthanized and their reproductive organs were fixed in a 4% paraformaldehyde or 2.5% glutaraldehyde solution and submitted to routine histological techniques for light and scanning electron microscopy. In the ovary, during the proestrus phase, we observed developing follicles and corpus luteum (CL) in regression; during estrus, there were Graafian follicles; during metestrus, there was a hemorrhagic corpus, whereas in diestrus, there was a mature CL. The uterus was partially double because the cervix was cranially septate but caudally, the septum disappeared, forming a single ostium that opened into the vagina. Changes occurred along the estrous cycle in the uterine and vaginal epithelia, that is, an increase in the uterine epithelium height accompanied by an increase of thickness of the vaginal epithelium during the follicular phase and a decrease of thickness of both epithelia during the luteal phase. The endometrial lining was composed of a simple cuboidal epithelium to simple columnar epithelium with basal nuclei. The vaginal mucosa consisted of epithelium that varied from nonkeratinized stratified squamous (luteal phase) to keratinized stratified squamous (follicular phase). The clitoris was external to the vagina. It presented two protruding lateral keratinized spicules and a centralized urethra, with no common parts between the urinary and genital tracts. Anatomical and histological changes were observed mainly in the cervix, vagina and spicules of the clitoris during the EC.     

Change in the electrical impedance caused by cornification of the epithelial cell layer of the vaginal mucosa in the rat

The electrical impedance in the vagina (EIV) is significantly high in the proestrus stage compared with other stages of the estrous cycle in rats. Therefore, the EIV can be used to detect the optimum day for mating. The relation between the EIV and the conditions of the epithelial cell layer of vaginal mucosa were investigated. The EIV was at a high level (over 3,000 omega) in vaginas in the proestrus stage in either intact or excised vaginal mucus. But it decreased (under 3,000 omega) after the epithelium of the vaginal mucosa was removed. The EIV of ovariectomized rats was low, but increased after Estradiol administration. The cornification of the epithelial cell layer of the vaginal mucosa occurred concurrently with the high EIV in the proestrus stage and after Estradiol administration. This indicates that the cornification of the epithelial cell layer of the vaginal mucosa may cause the elevation of EIV in the proestrus stage, the optimum day for mating in the rats.     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Different binding to squamous and columnar epithelium of the uterine cervix as a marker of epithelial differentiation

Biotinylated lectins were used to investigate the expression of carbohydrate residues on columnar and squamous epithelium of the uterine cervix. Con A, WGA, RCA I, PNA, UEA I, DBA and SBA were used. In the native exocervical and in metaplastic squamous epithelium of the transformation zone, one group of lectins (Con A, WGA, RCA I and PNA) stained the cell periphery of all epithelial layers. A second group (UEA I, DBA and SBA) colored the cell periphery of the suprabasal cells. The basal layer was always negative. All lectins labeled the apical border and occasionally the cytoplasm of the endocervical columnar epithelium. Lectin-binding of metaplastic and native squamous epithelium could possibly be used as a marker of epithelial differentiation in normal and abnormal conditions.     

Ultrastructure of cyclic changes in the murine uterus,cervix, and vagina

The regional and cyclic changes in the murine genital epithelium were studied by transmission and scanning electron microscopy to provide a morphological standard to serve as a basis for investigation of host-parasite relationships in genital infections. Thus, we examined not only mucosal epithelial cell changes, but also surface mucus, normal flora and inflammatory cells. Ultrastructurally, at proestrus/estrus, we found uterine and most cervical epithelial cells covered with microvilli overlaid with mucus-like secretions and evidence of internal secretory activity. There was little normal flora anywhere in the tract. At early metestrus, we found squamous cervicovaginal epithelial cells with low discontinuous microrugae, extensive normal flora and many neutrophils beginning to migrate through the epithelium. The flora and neutrophils could explain the relative lack of susceptibility to infection at that time. At diestrus the appearance of a newly regenerated epithelium and lack of normal flora suggested that initiation of infection could occur at this stage; however, the presence of large numbers of neutrophils ready to phagocytize invading bacteria indicated a deterrent to infection. This study of cyclic changes in flora, mucus, neutrophils and epithelial cells provided ultrastructural evidence to support an earlier hypothesis that the greatest susceptibility to gonococcal infection in mice occurred at proestrus/estrus.     

Spatial relationship of the vaginal microflora to the vaginal epithelium of female rats: scanning electron microscopy.

The vaginal mucosa of ovariectomized female rats has been examined by scanning electron microscopy before and after estrogen [1,3,5(10)-estratriene-3,17beta-diol] treatment. Without estrogen stimulation vaginal colonization is minimal and the epithelium is characterized by a layer of epithelial cells covered with small microvillous-like projections. Progressive changes that were consistent with estrogenic cytoproliferative effects were seen after estrogen treatment. By post-treatment day 3 bacterial colonization was maximal and the epithelium was comprised of flat squamous cells that tended to become detached from the underlying tissue layers. Bacteria were seen in association with the intercellular borders of this tissue and occurred singly or as microcolonies. No distinct physical attachment structures were identified, although an amorphous extracellular material that may serve to attach the bacteria to the squamous epithelial cells was frequently seen.     

Pregnancy-related changes in the human endometrium revealed by lectin histochemistry

The binding of 22 fluorescein isothiocyanate (FITC) conjugated lectins to human proliferative phase and pregnant endometrium was studied histochemically. Only the lectin from Bauhinia purpurea (BPA) reacted exclusively with the epithelial cells. All the others reacted to a certain extent with glandular and/or stromal cells. Lectins from soybean (SBA), and Vicia villosa seeds (VVA) reacted with endometrial glands of pregnancy but not with the glands of the proliferative endometrium. In the proliferative endometrium SBA reacted only with cells of the surface endometrium. Lectin from peanuts (PNA) reacted only with some glands in the proliferative endometrium but was unreactive with others. In pregnant endometrium PNA reacted with all glands. Lectins from lentils (LCA) and red kidney beans (PHA-E and PHA-L) reacted with endometrial glands of the proliferative phase but not with the glands from pregnant endometrium. We thus show that FITC labeled lectins define specific carbohydrate moieties selectively expressed on either proliferative phase or pregnant endometrial glands.     

Sialic acid-specific lectin-mediated adhesion of Tritrichomonas foetus and Tritrichomonas mobilensis

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract producing infection associated with inflammatory changes, abortion, and infertility, Tritrichomonas mobilensis was isolated from squirrel monkey colon, and symptoms involve diarrheal complications. Both tritrichomonads produced hemagglutinins with the properties of sialic acid-specific lectins. Assays on the adherence of these protozoans to Chinese hamster ovary (CHO) cells and to bovine cervical and monkey colon mucus were performed to assess the function of the lectins in adhesion. Sialic acid at concentration as low as 2 mM inhibited the adhesion to CHO cells, less effectively to the mucus. Predigestion with Clostridium perfringens sialidase prevented the adhesion to both epithelial cells and the mucus. Inhibition of endogenous sialidases with 2,3-dehydro-2-deoxy-NeuAc increased the adhesion of T. mobilensis to CHO cells. Specific anti-T. foetus lectin (TFL) and anti-T. mobilensis lectin (TML) antibodies inhibited adhesion of the trichomonads to the epithelial cells and to the mucus. TFL histochemistry disclosed the presence of lectin ligands on keratinized vaginal epithelia, cervical mucosa, and mucin and on endometrial glands and their secretions. TML histochemistry showed reactivity with the luminal membranes of colonic glandular epithelium and less with the colonic mucin. Both lectins bound to the surface membrane of CHO cells. Anti-lectin antibodies showed granular cytoplasmic and strong membrane localization of the lectins in both tritrichomonads. Although the 2 tritrichomonads have different habitats, the results indicate that both these protozoa use lectins with sialic acid specificity for adhesion to mucosal surfaces.     

Human vaginal epithelial multilayer tissue culture

Summary Fragments of normal human adult vagina, when explanted onto glass slides gave rise to outgrowing sheets of pure epithelium, which had microscopic morphological features in common with normal vaginal epithelium. Infrequent fibroblast contamination was observed. Proliferating epithelial cells formedmultilayers of stratified squamous epithelium and demonstrated a progressive decrease in proliferative activity after 14 days. Continuous lines of epithelial cells were not obtained. Even in the absence of estrogens, transmission electron microscopy revealed evidence of keratinization of the superficial cells of the multilayer. Scanning electron microscopy of the surface of mature epithelial cells in culture revealed ultrastructural features that closely resembled those present on the surface of exfoliated cells obtained by scraping the vagina in vivo. This in vitro tissue culture model of human vaginal epithelium may provide a simple method of studying factors that influence vaginal epithelium growth, maturation and function.     

DIFFERENTIAL LOCALIZATION OF CELL SURFACE AND SECRETORY COMPONENTS IN RAT INTESTINAL EPITHELIUM BY USE OF LECTINS

Sections through various levels of small intestine from adult male rats were examined by fluorescence microscopy after treatment with fluorescein isothiocyanate-labeled lectins from Dolichos biflorus, Lotus tetragonolobus, Ricinus communis, and Triticum vulgare (wheat germ). The latter three lectins reacted with the microvillar portion of the epithelial cells lining the crypts and villi in sections of intestine adjacent to the pylorus. This pattern of reactivity was sharply altered along the first 15 cm of intestine so that in sections distal to this point the luminal surfaces of only those epithelial cells in the crypts and at the base of the villi reacted with the L. tetragonolobus and R. communis lectins, whereas the wheat germ lectin reacted with the surfaces of the cells lining the villi. In sections from the distal end of the small intestine, all three lectins reacted with the surfaces of cells only at the base of the villi and in the crypts. These results show a difference in surface components in cells at various portions on the villi and the dependence of these differences on the region of intestine. The D. biflorus lectin reacted with approximately 25% of the goblet cells at each level of intestine studied whereas the reactivities of the goblet cells with the other three lectins were dependent upon the region of intestine.     

Expression pattern of glycoconjugates in rat retina as analysed by lectin histochemistry

The present study sought to characterize the expression and distribution of complex glycoconjugates in the rat retina by lectin histochemistry, using a panel of 21 different lectins with different carbohydrate specificities. Paraffin sections of Carnoy-fixed Sprague-Dawley rat eyes were stained with various biotinylated lectins, followed by the streptavidin-peroxidase and glucose oxidase-diaminobenzidine-nickel staining procedures. The results showed that the retinal pigment epithelium was stained intensely with LCA, Jacalin, WFA, S-WGA, PWA, DSA, UEA-I, LTA and PHA-E, suggesting that this epithelium contained glycoconjugates with alpha-Man, alpha-Glc, alpha-Gal/GalNAc, beta-GalNAc, alpha-Fuc, NeuAc and other oligosaccharide residues. The outer and inner segments of the photoreceptor layer showed different lectin binding affinities. The outer segments reacted with S-WGA and GS-II, whereas the inner segments reacted with UEA-II, UEA-I, LTA and MAA, suggesting that the inner segments contained glycoconjugates rich in alpha-Fuc and NeuAc(alpha2,3)Gal residues. PNA labelled specifically the cones and could be used as a specific marker for these photoreceptors. RCA-I, WFA, S-WGA, DSA, MAA and PHA-E reacted with both the outer and inner plexiform layers. On the other hand, UEA-I and LTA specifically labelled the outer plexiform layer, while PNA labelled the inner plexiform layer. The retinal microglial cells were labelled specifically by GS-I-B4 and SNA. Interestingly, we also observed that WFA bound specifically to Müller cells and could be used as a novel marker for this retinal glial cell. The capillaries and larger vessels in the retina and choriocapillaris reacted intensely with GS-I-B4, RCA-I, S-WGA, PWA, DSA and PHA-E. No significant differences in lectin binding were observed in the microvessels at these two sites. In summary, the present study demonstrated the expression patterns of glycoconjugates in the rat retina and that certain lectins could be used as histochemical markers for specific structural and cellular components of the rat retina.