Cellular heterogeneity in normal human urothelium: Quantitative studies of lectin binding

Summary A panel of 10 FITC-labelled lectins (MPA, PNA, ConA, DBA, SBA, RCA-120, WGA, UEA, GS-I, GS-II) was applied to cryosections of seven specimens of normal urothelium. Seven of the lectins (MPA, ConA, RCA, WGA, UEA, GS-I and GS-II) showed a pattern of increasing fluorescence intensity from basal to superficial cells of the urothelium whereas PNA, DBA and SBA showed more uniform binding throughout the urothelium. Urothelial cell suspensions labelled with FITC-lectins were studied by flow cytometry to quantify the variation in binding to different cells types. Three cellular subpopulations were identified in normal urothelium on the basis of their optical properties. Fluorescence intensity due to specific lectin binding was then measured separately for each subpopulation. Although there was some variation among individual cases, a general pattern emerged in this small series. WGA, RCA, and GS-II bind in large quantities to all urothelial cells while PNA, SBA, ConA and DBA show little binding. MPA, RCA, UEA and GS-I showed the most marked increase in fluorescence intensity from basal to superficial cells as observed microscopically and quantified by flow cytometry.     

Cellular heterogeneity in human transitional cell carcinoma: an anlysis of optical properties and lectin binding

Summary Flow cytometry was used to measure the binding of a panel of ten fluorescein isothiocyanate(FITC)-conjugated lectins to fifteen samples of normal and neoplastic human urothelium. Concurrent measurement of light scattering and fluorescence permitted the quantification of lectin binding to cellular subpopulations defined by their light-scattering properties. In normal urothelium, we previously demonstrated levels of lectin binding to the cellular subpopulations derived from the superficial and intermediate cell layers which were higher than levels which bound to the subpopulation derived from the basal cell layer (Wardet al., 1987). This difference was most marked withMaclura pomifera agglutinin (MPA),Ricinus communis agglutinin (RCA) andUlex europus agglutinin (UEA). We now report a similar correlation between the degree of differentiation of a cellular subpopulation and the level of lectin binding in human transitional cell carcinomas (TCCs). Morphological differentiation in human TCCs is accompanied by alterations in cell-surface carbohydrates which are similar to those which accompany cellular differentiation in the corresponding normal tissue. No systematic difference in lectin binding was observed between the corresponding subpopulations of normal and neoplastic urothelial cells.     

Charge heterogeneity of the folate binding protein in normal human leukocytes

The DEAE-Sepharose CL-6B chromatographic profile of supernatant from homogenized normal human leukocytes containing large amounts of folate binder revealed two peaks of binding activity. A minor binder (I) eluted with the equilibrating buffer (1 mM sodium phosphate of pH 6.0), while the major binder (II) first eluted after the initiation of a linear phosphate gradient with 200 mM sodium phosphate of pH 7.6 as the limiting buffer. Binder II was thus a more acidic protein since it required elution by a salt-pH gradient. Binding of [3H] folate to binder II was of a high-affinity type (K = 10(10) M-1) and displayed positive cooperativity.     

Differences in lectin binding patterns of normal human endometrium between proliferative and secretory phases

Summary The biological fate of a bovine collagen implant (Zyderm Collagen Implant ZCI), injected subcutaneously into rats, was studied by the immunoperoxidase technique using specific antibodies against the bovine implant and against types I, III, IV, V collagens, fibronectin and elastin. The implant remained in the animals until the end of the experiment (90 days), with no visible modification, as demonstrated by immunoperoxidase labelling and scanning electron microscopy. A slight inflammatory reaction was visible around the implant 24 h after injection and within the implant 3 days after injection. Fibroblast invasion began 7 days after injection. The chronology of the deposition in the implant of the host (rat) extracellular matrix components was as follows: by 24 h after injection, fibronectin was observed throughout the implant; types I and V collagens appeared on the 7th day, and, in contrast to surrounding connective tissue, type V collage labelling was obtained without acid pretreatment of the section. Types III and IV collagens were detected inside the implant only 30 days after injection. At the end of the experiment (90 days), there was abundant types I and V collagens after fibroblast migration, and abundant type IV collagen demonstrating an important vascularization. No elastic fibres could be detected inside the implant but they appeared as a dense network around the implant in host connective tissue.     

Differences in lectin binding patterns of normal human endometrium between proliferative and secretory phases

Lectin binding patterns in normal human endometrium were examined by light and electron microscopy using seven different lectins (ConA, WGA, RCA, PNA, UEA-1, DBA, and SBA). For light microscopic observations, criteria based on the incidence and intensity of cells positive for the lectin staining were adopted to evaluate the different staining patterns of the proliferative and secretory endometria obtained by the avidin-biotin-peroxidase complex (ABC) technique. At the light microscopic level, ConA, WGA, and RCA stained endometrial glandular cells in both phases. The number of PNA-positive cells with the binding sites entirely limited to the apical surface tended to be reduced slightly in the secretory phase. UEA-1 weakly stained the apical surface of glandular cells in the proliferative phase but not in the secretory phase. Among the lectins used in this study, DBA and SBA displayed remarkable changes between the phases. That is, in the proliferative phase they produced only a faint or slight positive stain at the apical surface, but the incidence and intensity of DBA- and the SBA-positive glandular cells increased in the secretory phase. By electron microscopy, the reaction product of ConA was observed in the plasma membrane, endoplasmic reticulum, nuclear envelope, and the Golgi apparatus, and the binding sites of RCA and DBA were observed in the plasma and Golgi membranes. Between both phases, the reactivity of ConA and RCA showed almost no change. However, the secretory endometrial cells containing the DBA-positive Golgi apparatus were markedly increased in number compared with the proliferative ones bearing the lectin-positive organelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular heterogeneity in human epithelial neoplasms

Tumor cell heterogeneity has in recent years been the subject of numerous excellent review articles, but comprehensive reviews may not always distinguish between that which is known about tumors from direct observation and that which is inferred from the study of analagous systems. The purpose of this review is to describe what is known about cellular heterogeneity in human tumors and to discuss current models of the pathogenesis of cellular heterogeneity in light of the evidence available from the study of human cancer.     

Cellular heterogeneity of human fallopian tubes in normal and hydrosalpinx disease states identified using scRNA-seq

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In silico analysis of the cyanobacterial lectin scytovirin: new insights into binding properties

Scytovirin is a lectin isolated from the cyanobacterium Scytonema varium that has shown activity against HIV, SARS coronavirus and Zaire Ebola virus. Its 95 amino acids are divided into two structural domains (SD), the first spanning amino acids 1–48 (SD1) and the second 49–95 (SD2). Interestingly, the domains are nearly identical but differ in their affinities for carbohydrates. With the aim of enhancing understanding of the binding properties of scytovirin, we performed molecular dynamics (MD) simulations of scytovirin complexed with Man4. We set up three systems: (i) Man4 bound to both domains (SD1?+?SD2) using the full-length protein; (ii) Man4 bound to an incomplete protein, containing only SD1 and (iii) Man4 bound to an incomplete protein containing only SD2. Contrary to other reports, binding free energy results suggest that Man4 can bind simultaneously to SD1 and SD2 binding regions, but SD1 individually has the best values of energy and the best affinity for Man4. Decomposition of the binding free energy showed that the residues that interact with Man4 were different in the three systems, suggesting that the binding mechanism of Man4 varies between full-length protein, SD1 and SD2. The results presented here may help to formulate strategies to use scytovirin and promote mutagenesis studies to improve the antiviral activity of scytovirin.     

Urothelium-specific antibody and lectin surface mapping of bladder urothelium

Summary Coupled ligand-colloidal gold complexes were found to provide a convenient approach for the localization by scanning electron microscopy of cell surface membrane antigens and lectin-binding sites on bladder urothelium and for the immunocytochemical identification of urothelial cell populations at different stages of differentiation. The ligands used to probe the membrane were a urothelium-specific rabbit antibody raised to a urothelial membrane-associated antigen (UMA), and two lectins: Concanavalin A (Con A) and peanut agglutinin (PNA). A complex luminal surface distribution pattern was demonstrated by the UMA antigen related to the stage of urothelial cell maturation and differentiation. UMA could be detected on the surface of immature and early differentiating intermediate cells, but was absent from the late differentiation stage, becoming re-expressed as the cells matured and was found in greatest abundance on the terminally differentiated superficial cells. It was absent on cells in benign hyperplasia of the urothelium. Cellular and regional differences in lectin binding to the urothelial cell surface was suggested with Con A receptors localized uniformly over the superficial cells, and PNA receptors confined to linear arrays or occasional clusters over the apical surface but evenly dispersed over the lateral surface of these cells.     

Histochemical investigations on lectin binding in normal and irradiated mouse embryos

Lectin binding in normal and irradiated embryonic mouse tissues on day 10 of gestation was studied by peroxidase techniques. Specific binding ofDolichos biorus lectin (DBA) was detected in the mesodermal blood vessels and in the otic vesicles. The amount of DBA as well as that ofsoybean agglutinin (SBA) andpeanut agglutinin (PNA) increased after exposure to low doses of radiation (0.25, 0.50 and 0.75 Gy). The modifying influence of ionizing radiation was observed in the pituitary region, in the otic vesicles and in the blood vessel endothelium. The greatest effect appeared in the pituitary region at 0.75 Gy, while in the otic vesicles it appeared at 0.50 Gy. A dose-effect relationship was established for the DBA lectin affinity of the vascular endothelium. In comparison to DBA, SBA and PNA displayed more extensive staining after irradiation. The reactivity of these lectins appeared especially pronounced on the blood vessels within the central nervous system and in the luminal surface of the ependymal cells. It is of interest that maximal binding for PNA was observed at 0.25 Gy and for SBA at 0.50 Gy at the junctions between neuroepithelial cells.     

Quantitative analysis of heterogeneity of estrogen binding in human breast tumor specimens

A quantitative assessment was made of the heterogeneity of estrogen binding among histologically distinct regions and among nuclei within histologically uniform regions in ten human breast tumors. An autoradiographic method was used that employed sections from fixed tissues and required exposure times of one to two weeks. Grain counting of autoradiograms probably indicated both type I and type II estrogen binding. There were significant decreases in estrogen binding in all specimens after competition with excess diethylstilbestrol, suggesting that a component of binding was specific for estrogen. Significant differences in amount and distribution (nuclear vs cytoplasmic) of estrogen binding were seen not only among tissues with different histologies but also within histologically identical regions of a tissue. In histologically uniform regions, wide ranges of values for nuclear estrogen binding were seen. This method allows for the rapid evaluation of estrogen-binding profiles of tumor specimens and has potential for analyzing the process of neoplastic transformation in mammary epithelia as it affects, or is affected by estrogen binding.     

Cellular localization of the galactose-binding lectin from human serum

An SL2 lectin was isolated from human serum and characterized previously; cellular localization of the lectin was studied using polyclonal rabbit antibodies. According to cytofluorimetry, anti-SL2 antibodies bound only to lymphocytes and monocytes but not to other blood cells. Antibodies bound to Jurkat T cell lymphoma but did not interact with IM-9 cells of B cell origin. Moreover, the Jurkat cells bound oligosaccharides having the highest affinity to SL2 (GalNAcalpha_and Fucalpha1-2Gal), and this interaction was inhibited by anti-SL2 antibodies. Lysis of the Jurkat cells with subsequent electrophoresis and Western blotting indicates that anti-SL2 antibodies recognized a 14-kD protein.     

Selective binding of lectins to normal and neoplastic urothelium in rat and mouse bladder carcinogenesis models

Bladder cancer adjuvant intravesical therapy could be optimized by more selective targeting of neoplastic tissue via specific binding of lectins to plasma membrane carbohydrates. Our aim was to establish rat and mouse models of bladder carcinogenesis to investigate in vivo and ex vivo binding of selected lectins to the luminal surface of normal and neoplastic urothelium. Male rats and mice were treated with 0.05 % N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water and used for ex vivo and in vivo lectin binding experiments. Urinary bladder samples were also used for paraffin embedding, scanning electron microscopy and immunofluorescence labelling of uroplakins. During carcinogenesis, the structure of the urinary bladder luminal surface changed from microridges to microvilli and ropy ridges and the expression of urothelial-specific glycoproteins uroplakins was decreased. Ex vivo and in vivo lectin binding experiments gave comparable results. Jacalin (lectin from Artocarpus integrifolia) exhibited the highest selectivity for neoplastic compared to normal urothelium of rats and mice. The binding of lectin from Amaranthus caudatus decreased in rat model and increased in mouse carcinogenesis model, indicating interspecies variations of plasma membrane glycosylation. Lectin from Datura stramonium showed higher affinity for neoplastic urothelium compared to the normal in rat and mouse model. The BBN-induced animal models of bladder carcinogenesis offer a promising approach for lectin binding experiments and further lectin-mediated targeted drug delivery research. Moreover, in vivo lectin binding experiments are comparable to ex vivo experiments, which should be considered when planning and optimizing future research.     

The lectin binding pattern of normal and pathologically altered synovial tissue

Light-microscopical lectin-binding studies were carried out in healthy and pathologically altered synovial tissue (osteoarthrosis, rheumatoid arthritis (RA)). Seven lectins were studied: Con A, DBA, PNA, RCA, SBA, UEA-I, and WGA. Con A and WGA mark all lining cells and the majority of subintimal synovial cells. RCA and SBA stain only a portion of lining cells, regardless of the basic pathology. The lectin PNA reacts only with RA and arthrotic material, and is thus suitable for the diagnosis of inflammatory changes in synovial tissue. UEA-1 is a consistent marker for capillary endothelium and large vessels.     

Gram staining and lectin binding properties of Myxosporea and Sporozoea

The staining method developed by Christian Gram was introduced as a simple and highly selective tool for demonstrating myxosporean and coccidian sporogonic stages. When using standard blood staining procedures for those enigmatic parasites it is sometimes difficult to distinguish them from fish host tissue. They clearly exhibit a partial Gram-positive reaction in histological sections, but staining is variable in air dried fish organ imprints. To visualize the Gram-negative background of different host tissue components in histological sections, the conventional safranin counterstain of the Gram protocol may be modified as follows: after application of 2% crystal violet (basic violet 3) and Lugol's solution, sections are stained with 0.1% nuclear fast red-5% aluminum sulfate and 0.35% aniline blue (acid blue 22) dissolved in saturated aqueous picric acid. Replacement of the Gram-specific dye crystal violet with 2% malachite green gave similar results in organ imprints containing myxospores or coccidia, but only in sections containing myxosporea. Staining for 1 min with an aqueous solution of 0.5% malachite green and followed 1 min washing was sufficient for rapidly demonstrating the parasite spores in organ imprints of both myxosores and oocysts. With regard to the role of acid mucopolysaccharides and other carbohydrates in the Gram reaction of spores, alcian blue 8GX staining was compared to the binding of FITC-labeled WGA, GS I and GS II. Each lectin was applied at 20 μl/ml PBS, HEPES for 1 hr. Whereas WGA yielded a nonspecific pattern like the alcian blue staining, GS II resulted in a pattern similar to the Gram staining results. This binding was weak in untreated specimens, but was significantly enhanced when digested first within trypsin overnight in a humid chamber at 37 °C. The binding of GS II to both myxosporidian and coccidian spores suggests that they are both composed of polymers containing N-acetyl-D-glucosamine residues. Furthermore, the results suggest that this hexosamine plays a key role in the Gram reaction.     

Glycoconjugates of the normal human colorectum: a lectin histochemical study

Summary Previous studies of the normal human colorectum by lectin histochemistry have used a mixture of tissues, including those derived from colons harbouring neoplasia and inflammatory bowel diseases. In the current investigation, tissues from patients without either of these conditions have been examined with a wide panel of lectins, encompassing specificities directed against both N- and O-linked sequences, using an avidin peroxidase revealing system and evaluated with a semiquantitative scoring method. The results of binding of these lectins have been compared with those seen in the resection margins of (at least 5 cm away from) colorectal carcinomas. Consistent regional variations were noted between right- and left-sided colonie tissues, with more diverse glycan structures and a greater sialyl content in the distal colon. There was evidence of graduation of formation of oligosaccharide chains in developing crypts, possibly related to the maturation and expression of glycosyl transferases responsible for the incorporation of mannose residues of N-linked oligosaccharides and of N-acetylgalactosamine and N-acetylglucosamine. Comparison with previous reports has revealed some variations, possibly related to tissue fixation and processing and to lectin concentrations employed, which raises the question of standardization of methodologies in lectin histochemical investigations.     

A comparison of lectin binding in rat and human peripheral nerve

Eleven different fluorescein- or peroxidase-conjugated lectins with different sugar-binding affinities were employed to analyze and compare glycoconjugates of rat and human peripheral nerves at the light microscopic level. A majority of lectins showed a distinct binding pattern in different structures of the nerve. Lectin binding was similar but not identical in rat and human nerves. Limulus polyhemus agglutinin did not stain any structures in rat or human nerves. In both species, all other lectins bound to the perineurium. Perineurial staining was intense with Canavalia ensiformis (Con A), Triticum vulgaris (WGA), Maclura pomifera (MPA); moderate with Glycine max (SBA), Griffonia simplicifolia-I (GS-I) and GS-II; weak with Ulex europaeus (UEA), Dolichos biflorus (DBA), and Ricinus communis (RCA). In the endoneurium of both species, ConA staining was intense, MPA and WGA moderate, SBA, GS-II, PNA, and RCA weak, and UEA and DBA absent. Interestingly, GS-I stained rat but not human endoneurium. Most lectins bound to blood vessels. GS-I bound to rat but not human, whereas UEA bound to human but not rat vessels. The results show that lectins can be used to reveal heterogeneity in sugar residues of glycoconjugates within neural and vascular components of nerves. They may therefore be potentially useful in detecting changes in glycoconjugates during nerve degeneration and subsequent regeneration after trauma or in pathological states.