Ulex Europeus Agglutinin Research Articles

MUCIN 1 (MUC1) LEVELS AND GLYCOSYLATION PATTERNS IN SALIVA OF PATIENTS WITH BURNING MOUTH SYNDROME (BMS): A CASE-CONTROL STUDY

<h3>Objectives</h3> Mechanisms underlying burning mouth syndrome (BMS) remain unclear. Evidence of changes in oral mucosal epithelial cells has been found in patients with BMS. This case-control study aimed to investigate whether BMS cases have evidence of impaired mucosal barrier function compared with controls without BMS, focusing on the role of salivary mucins as a first line of defense for oral epithelial cells. <h3>Study Design</h3> A total of 50 women (22 BMS cases and 28 healthy controls) age 50 years or older were included in this study. Participants attended a clinical visit including assessment of BMS symptoms, salivary sample collection, oral examination, swab for candida detection, and DNA collection using the DNA•SAL device. Total protein concentration was measured using the bicinchoninic acid assay. MUC1 protein concentration in saliva samples was quantified by sandwich enzyme-linked immunosorbent assay (Invitrogen). Salivary glycosylation was assessed by protein-normalized dot blot against 3 lectins (Maackia Amurensis Lectin II; WGA: wheat germ agglutinin; UEA: ulex europeus agglutinin (MAL-II), WGA, and UEA). H antigen, the precursor of ABO antigens, secretor status was predicted by performing Sanger DNA sequencing of the FUT2 gene. <h3>Results</h3> The average salivary protein concentration was 1.30 mg/mL in BMS cases vs 0.94 mg/mL in controls (<i>P</i> = .15). Mann-Whitney <i>U</i> test showed a trend toward lower levels of MUC1 in BMS cases (n = 20, sum of ranks = 392.50) compared with controls (n = 24, sum of ranks = 597.50; <i>P</i> = .17). UEA, MALII, and WGA reactivities were each quantitatively lower in cases compared with controls but varied widely (<i>P</i> = NS). The percentage of nonsecretors was 23% among cases vs 38% among controls (<i>P</i> = .28). Genotype-predicted secretors had significantly higher UEA reactivity compared with nonsecretors (<i>P</i> < .001). <h3>Conclusions</h3> Our study overall found trends consistent with higher salivary protein concentration, lower salivary MUC1 levels, and salivary hypoglycosylation in BMS cases compared with controls, although no findings were statistically significant. Study limitations include subject heterogeneity and sample size. As expected, UEA levels were significantly higher in secretors compared with nonsecretors. Other aspects of the oral epithelial barrier, including the quantity of other mucins and glycosylation, remain to be examined.

Can domestication shape Canidae brain morphology? The accessory olfactory bulb of the red fox as a case in point

BackgroundThe accessory olfactory bulb (AOB) is the first integrative center of the vomeronasal system (VNS), and the general macroscopic, microscopic, and neurochemical organizational patterns of the AOB differ fundamentally among species. Therefore, the low degree of differentiation observed for the dog AOB is surprising. As the artificial selection pressure exerted on domestic dogs has been suggested to play a key role in the involution of the dog VNS, a wild canid, such as the fox, represents a useful model for studying the hypothetical effects of domestication on the AOB morphology. MethodsA comprehensive histological, lectin-histochemical, and immunohistochemical study of the fox AOB was performed. Anti-Gαo and anti-Gαi2 antibodies were particularly useful, as they label the transduction cascade of the vomeronasal receptor types 1 (V1R) and 2 (V2R), respectively. Other employed antibodies included those against proteins such as microtubule-associated protein 2 (MAP-2), tubulin, glial fibrillary acidic protein, growth-associated protein 43 (GAP-43), olfactory marker protein (OMP), calbindin, and calretinin. ResultsThe cytoarchitecture of the fox AOB showed a clear lamination, with neatly differentiated layers; a highly developed glomerular layer, rich in periglomerular cells; and large inner cell and granular layers. The immunolabeling of Gαi2, OMP, and GAP-43 delineated the outer layers, whereas Gαo and MAP-2 immunolabeling defined the inner layers. MAP-2 characterized the somas of AOB principal cells and their dendritic trees. Anti-calbindin and anti-calretinin antibodies discriminated neural subpopulations in both the mitral-plexiform layer and the granular cell layer, and the lectin Ulex europeus agglutinin I (UEA-I) showed selectivity for the AOB and the vomeronasal nerves. ConclusionThe fox AOB presents unique characteristics and a higher degree of morphological development compared with the dog AOB. The comparatively complex neural basis for semiochemical information processing in the fox compared with that observed in dogs suggests loss of AOB anatomical complexity during the evolutionary history of dogs and opens a new avenue of research for studying the effects of domestication on brain structures.

Concanavalin agglutinin levels are decreased in peripheral blood of Alzheimer's disease patients.

Alzheimer's disease (AD) seriously threatens patients' lives and causes severe burden to the families. Early prevention and treatment can alleviate the development of the disease; therefore it is important to find new effective and non-traumatic biomarkers for early diagnosis. In this study, peripheral blood samples were collected from 24 AD patients and the same number of age- and gender-matched control subjects. Lectin reactive glycosylation levels including beta-D-galactosyl ricinus communis agglutinin 120 (RCA), peanut agglutinin (PNA), concanavalin agglutinin (Con A), alpha-L-fucosyl ulex europeus agglutinin (UEA), dolichos biflorus agglutinin (DBA), and galanthus nivalis (GNL), in the red blood cells of peripheral blood were examined by western blotting. We found that lectin levels were altered with aging and gender; some lectin levels were different between AD patients and the control subjects. Only Con A levels were significantly decreased in AD patients compared to age-matched control subjects. These results suggest that Con A levels in peripheral blood may be a potent diagnostic marker for AD.

Molecular changes in the cell surface of differentiating epidermal keratinocytes.

The specific binding of lectins to the cell surfaces in particular epidermal strata indicates that oligosaccharide moieties of the glycoconjugates on the cell membrane are altered as the keratinocyte differentiates. The disappearance of binding by the Bandeiraea simplicifolia isolectin I-B4 (BS I-B4) and the appearance of binding by the Ulex europeus agglutinin I (UEA), which occur as the basal cell moves up into the spinous layer of the cutaneous epidermis of the newborn rat, could result from (a) redistribution of existing glycoconjugates in the cell membrane, (b) hydrolysis of galactosyl residues exposing fucosyl binding sites, or (c) addition of fucosyl residues to pre-existing oligosaccharides with specificity for the galactoside. Isolation of glycoproteins from the cell membrane using lectin-affinity column chromatography on Sepharose-4B showed a decrease in the amount of protein which bound to the BS I-B4 and an increase in the protein with affinity for the UEA as the ratio of spinous to basal cells increased in the population of cells from which the glycoproteins were obtained. These data indicate the existence of two different classes of glycoproteins on the surfaces of cells in the lower epidermis and argue against a redistribution of glycoconjugates as the explanation for the selective binding of specific lectins observed in tissue sections. Exposure of sections of skin to alpha-galactosidase completely eliminated the binding of fluorescein isothiocyanate-labeled (FITC) BS I-B4 in the lower epidermis but did not result in additional binding of UEA. Exposure to fucosidase eliminated binding by UEA and increased the binding of BS I-B4 in the spinous layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate profiling of fungal cell wall surface glycoconjugates ofAspergillusspecies in brain and lung tissues using lectin histochemistry

The aim of this study was to evaluate, through lectin histochemistry, the expression of N-acetyl-D-glucosamine, L-fucose, D-galactose and glucose/mannose on the cell wall surfaces of Aspergillus species in histopathological specimens of brain (n = 1) and lung (n = 6) tissues obtained during autopsy of patients diagnosed postmortem as having had invasive aspergillosis. Concanavalin A (Con A), wheat germ agglutinin (WGA), Ulex europeus agglutinin I (UEA-I) and peanut agglutinin (PNA), all conjugated with horseradish peroxidase, were employed. Lectin-binding was visualized using 3,3-diaminobendizine (DAB) and hydrogen peroxide in phosphate buffer solution (PBS). We observed expression of N-acetyl-D-glucosamine and methyl-α-D-mannoside on the cell wall surfaces of all evaluated Aspergillus species, while the expression of L-fucose and D-galactose demonstrated inter and intra-specific variations. The results obtained from this study indicate that the use of WGA and Con A lectins permits visualization of Aspergillus structures such as hyphae, conidial heads and conidia in histopathological specimens of brain and lung tissues.

Carbohydrate profiling of fungal cell wall surface glycoconjugates of Trichophyton tonsurans and other keratinophilic filamentous fungi using lectins

Various researchers have concluded that lectins are useful reagents for the study of fungal cell wall surface glycoconjugates. In this study, we evaluated the expression of N-acetyl-D-glucosamine, L-fucose, D-galactose and glucose/mannose on the cell wall surface of Trichophyton tonsurans and other keratinophilic filamentous fungi, using a simple lectin-binding protocol. The fungal cultures used were isolated from soils obtained from public parks by the hair-bait technique. The lectin assays used concanavalin A (Con A), wheat germ agglutinin (WGA), Ulex europeus agglutinin I (UEA-I) and peanut agglutinin (PNA), all conjugated with horseradish peroxidase. Adhesive tape was placed sticky-side down over the fungal colony, gently pressed and then removed. The fungal-tape samples were incubated with the lectin for 1 h at 4 °C. Lectin binding was visualised using 3,3-diaminobendizine (DAB) and hydrogen peroxidase. There was a high expression of N-acetyl-D-glucosamine on the cell wall surface of all fungi species tested, whereas the expression of L-fucose, D-galactose and glucose/mannose demonstrated inter-specific variations. The lectin-binding assay presented in this article eliminates many of the laborious steps involved in other protocols. The amount and quality of the mycelium and spores immobilised by the adhesive tapes were suitable for obtaining the carbohydrate profile in glycoconjugates of the cell wall surface of filamentous fungi.

Inhibition of Glycogen Synthase Kinase 3β (GSK3β) Decreases Inflammatory Responses in Brain Endothelial Cells

Immune mediators and leukocyte engagement of brain microvascular endothelial cells (BMVECs) contribute to blood-brain barrier impairment during neuroinflammation. Glycogen synthase kinase 3beta (GSK3beta) was recently identified as a potent regulator of immune responses in in vitro systems and animal models. However, the role of GSK3beta in regulation of immune endothelial functions remains undetermined. Here we evaluated the effect of GSK3beta inhibition on the regulation of inflammatory responses in BMVECs. A focused PCR gene array of 84 genes was performed to identify the cytokine and chemokine gene expression profile in tumor necrosis factor (TNF) alpha-stimulated BMVECs after GSK3beta inactivation by specific inhibitors. Fifteen of 39 genes induced by TNFalpha stimulation were down-regulated after GSK3beta inhibition. Genes known to contribute to neuroinflammation that were most negatively affected by GSK3beta inactivation included IP-10/CXCL10, MCP-1/CCL2, IL-8/CXCL8, RANTES/CCL5, and Groalpha/CXCL1. GSK3beta suppression resulted in diminished secretion of these proinflammatory mediators by inflamed BMVECs detected by ELISA. GSK3beta inhibition in BMVECs reduced adhesion molecule expression as well as monocyte adhesion to and migration across cytokine stimulated BMVEC monolayers. Interactions of monocytes with TNFalpha-activated BMVECs led to barrier disruption, and GSK3beta suppression in the endothelium restored barrier integrity. GSK3beta inhibition in vivo substantially decreased leukocyte adhesion to brain endothelium under inflammatory conditions. In summary, inhibition of GSK3beta emerges as an important target for stabilization of the blood-brain barrier in neuroinflammation.

CD133+Endothelial Progenitor Cells as a Potential Cell Source for a Bioartificial Glomerulus

Development of a bioartificial glomerulus, a hemofilter in which the inner surface of hollow fibers is endothelialized, requires expandable, nonimmunogenic, antithrombogenic, and highly permeable endothelial cells. We used human umbilical cord blood CD133(+) endothelial progenitor cells (EPCs) to evaluate the feasibility of application of EPCs for bioartificial glomerulus. Numbers of adhered CD133(+) EPCs (adhered EPCs) was approximately 25 to 30 times as great in the expansion culture group as in the non-expansion group. Adhered EPCs had endothelial cell features, including the expression of CD31, Kinase domain region, von Willebrand factor, vascular endothelial-cadherin, positive for Ulex europeus agglutinin I staining, and up-take of acetylated low-density lipoprotein. Adhered EPCs secreted 6-keto-prostaglandin F(1alpha) identically to that secreted by human umbilical vein endothelial cells (HUVECs). The cells also expressed messenger RNA for phospholipase A(2), cyclooxygenase (COX)-1, COX-2, prostaglandin I(2) synthase, tissue plasminogen activator, and thrombomodulin (TM). TM protein in adhered EPCs properly activated protein C. Scanning electron microscopy revealed the suppression of platelet adhesion and aggregation on the surface of cell monolayer. Adhered EPCs treated with 50 microg/mL of cytochalasin B induced a larger diameter and a greater number of fenestrae, subsequently producing significantly more ultrafiltration than the non-treated cell. These results suggest that CD133(+) EPCs would potentially be applicable in bioartificial glomerulus.

Lectins binding during alloxan-induced diabetes in rat soleus muscle

Membrane structural changes of soleus muscle of alloxan-diabetic rats were detected with a panel of six biotinylated lectins. Samples of muscles were obtained from normal and diabetic rats. The biotinylated lectins in staining were detected by avidin-peroxidase complex. Lectin stainning of soleus muscle cryostat sections from alloxan-diabetic rats were compared with similar cryostat sections from normal rats. All lectins were bounded to soleus muscle cell membranes of normal and diabetic rats except the Ulex europeus agglutinin I (UEA-I). UEA-I was not stained for cell membranes of soleus muscles of normal and diabetic rats. But intercellular areas of normal and diabetic soleus muscle were stained for UEA-I. Griffonia simplicifolia I (GS-I) lectin was bounded more strongly to cell membrane of diabetic rats than other lectins. Not only cell membranes but also cytoplasmic miyofibrills of diabetic muscle cells were stained by Griffonia simplicifolia (GS1) lectin. It was concluded that cell membranes of soleus muscle of diabetic rats were differently stained for lectins than those of normal rats. Our data can supplement on the use of lectins as probes for changes in carbohydrate-containing constituents of the alloxan-diabetic muscle cell membrane information obtained from normal and diabetic

UEA I-bearing nanoparticles for brain delivery following intranasal administration

Surface engineering of nanoparticles with lectins opened a novel pathway to improve the brain uptake of agents loaded by biodegradable PEG-PLA nanoparticles following intranasal administration. Ulex europeus agglutinin I (UEA I), specifically binding to l-fucose, which is largely located in the olfactory epithelium, was selected as a promising targeting ligand and conjugated onto the PEG-PLA nanoparticles surface with an optimized protocol relying on maleimide-mediated covalent binding technique. The in vivo results in rats suggested that UEA I modification at the nanoparticles surface facilitated the absorption of a fluorescent marker—6-coumarin associated with the nanoparticles into the brain following intranasal administration with significant increase in the area under the concentration–time curve (about 1.7 times) in different brain tissues compared with that of coumarin incorporated in the unmodified ones. UEA I-conjugation also elevated the brain-targeting efficiency of nanoparticles. Inhibition experiment of specific sugar suggested that the interactions between the nasal mucosa and the lectinised nanoparticles were due to the immobilization of carbohydrate-binding pockets on the surface of the nanoparticles. Distribution profiles of UEA I-modified nanoparticles indicated their higher affinity to the olfactory mucosa than to the respiratory one. Therefore, the UEA I-modified nanoparticles might serve as potential carriers for brain drug delivery, especially for mental therapeutics with multiple biological effects.

Vasculogenic Mimicry: How Convincing, How Novel, and How Significant?

In a recent publication, Maniotis et al 1 report that blood vessels of malignant eye tumors known as uveal melanomas are formed by tumor cells instead of endothelial cells. The authors use the term vasculogenic mimicry to describe this phenomenon and consider it a novel concept in the biology of tumor vascularization. The paper has received widespread attention and apparent validation through two commentaries, one published along with the paper in The American Journal of Pathology 2 and another published concurrently in Science. 3 Despite the paper’s impact the evidence is, in our view, unconvincing. The problems are, however, not easily detected by readers unfamiliar with the background or pitfalls of this specialized topic. Although it is intriguing and worthy of further study, the evidence presented in Maniotis et al for a functionally significant contribution of tumor cell-lined blood vessels to vascularization and blood flow in uveal melanomas is neither persuasive nor novel. The purpose of this commentary is to examine the evidence for vasculogenic mimicry and the reasons for our assessment. (Note: This commentary does not address the in vitro or microarray data presented by Maniotis et al, because the interpretation of these results is dependent on the histological, immunohistochemical, and electron microscopic evidence that is the focus of our remarks. Also, this commentary does not question the validity of the relationship between the periodic acid-Schiff (PAS) staining pattern of uveal melanomas and clinical outcome, as reported by Folberg et al in several publications. 4,5 This correlation may be clinically useful even if the PAS staining pattern does not faithfully represent the microvascular architecture. Neither does our commentary question the usefulness of microvascular density as a prognostic factor for survival in uveal melanomas. 6,7 Indeed, PAS staining pattern and microvascular density may offer complementary indices of the lethality of these tumors. 6-9 )

A histopathological study of the effects of low-power laser irradiation on wound healing of exposed dental pulp tissues in dogs, with special reference to lectins and collagens

This study investigated the effects of low-power laser irradiation on exposed pulp tissue in dogs. Gallium-aluminium-arsenide diode laser (300 mW) irradiation was applied to the exposed surface of the pulp, and histopathological changes were observed at 1, 3, and 7 wk after the operation. In addition, the lectin (concanavalin A, Dolichos biflorus agglutinin, peanut agglutinin, Ricinus communis agglutinin I, soybean agglutinin, Ulex europeus agglutinin I, and wheat germ agglutinin) binding pattern and distribution of collagens (type I, III, and V) were examined to determine the histochemical and immunohistochemical nature of wound healing. The fibrous matrix formation and the continuing changes in the dentin bridge formation of the irradiation group were observed earlier (1 wk after the operation) than in the nonirradiation control group. Lectin histochemistry and collagen immunohistochemistry showed that concanavalin A, peanut agglutinin, wheat germ agglutinin, and collagens (types I, III, and V) were distributed in the fibrous matrix and dentin bridge. The expression of these lectins and collagens occurred earlier in the laser irradiation group than in the control group. These results suggest that laser irradiation accelerates wound healing of the pulp and the expression of the lectins and collagens. Furthermore, D-glucose-, D-mannose-, N-acetyl-D-galactosamine-, and N-acetyl-neuraminic acid-binding sugars and type I, III, and V collagens play an important role in the healing of pulp wounds.

Lectin histochemistry of dorsal skin of Wistar-derived hypotrichotic WBN/Ila-Ht rats.

A lectin histochemical study was carried out on the dorsal skin of Wistar-derived hypotrichotic WBN/Ila-Ht rats (HtRs) and Wistar rats (WRs) at 3, 7 and 24 weeks of age to clarify the lectinhistochemical characteristics of the skin during their development. The lectins examined were Concanavalia ensiformis (Con A), Dolichos biflorus agglutinin (DBA), Griffonia simpliciolia (GS-I), Helix pomatia agglutinin (HPA), Arachis hypogaea (PNA), Glycine maximus agglutinin (SBA), Ulex europeus agglutinin (UEA-I) and Triticum vulgaris agglutinin (WGA). None of the nucleated cell layers of the epidermis had DBA-binding sites, but they were all stained intensely with HPA and weakly with Con A irrespective of the strain and age of the rats. As to the other 5 lectins, the intensity of binding activity was generally weaker in HtRs than in WRs and at 3 weeks of age than at 7 or 24 weeks of age, respectively. Among them, UEA-I mainly bound to the spinous cell layer but not to the basal cell layer, suggesting that alpha-L-fucose would be expressed on the cell surface according to the differentiation of keratinocytes. In addition, GS-I, HPA and UEA-I bound to the hair follicle epithelium and many lectins stained sebaceous gland epithelial cells. In conclusion, except for the binding intensity of some lectins, there were no specific differences between HtRs and Wrs in the lectinhistochemical characteristics of the dorsal skin epidermis. The present data on the rat skin would be useful from the viewpoint of comparative lectinhistochemistry.

Selective expression of adhesion molecules on human brain microvascular endothelial cells

Human microvascular endothelial cells were isolated from children's brain and examined for their morphological characteristics and upregulation of cell adhesion molecules in response to TNF α. Our human brain microvascular endothelial cells (HBMEC) were positive for factor VIII-Rag, carbonic anhydrase IV, Ulex Europeus Agglutinin I, took up fluorescently labeled acetylated low density lipoprotein and expressed gamma glutamyl transpeptidase, demonstrating their brain endothelial cell characteristics. Upon treatment with TNF α, VCAM and ICAM but little ELAM was expressed on HBMEC, while VCAM, ICAM and ELAM were clearly evident on HUVEC. This selective expression of cell adhesion molecules was also demonstrated by in situ stimulation of brain tissues. In conclusion, microvascular endothelial cells from childrens brains display selective expression of cell adhesion molecules, which differ from macrovascular endothelial cells. This may have consequences for leukocyte trafficking into the central nervous system.

Ulex Europeus Agglutinin (UEA-I) Lectin Binding in Breast Carcinoma and its Relationship to Prognostic Factors

In this study, lectin binding was compared with pathological prognostic factors and clinical follow-up details. Formalin-fixed, paraffin-embedded tissues from 43 cases of breast carcinoma were studied for binding with Ulex Europeus Agglutinin (UEA-I) lectin. Staining results were compared with tumor size, histologic and nuclear grade, lymph node status (number, capsular and pericapsular invasion), blood and lymphatic vessel invasion, ER and PR status, clinical stage and the patients' short-term follow-up details. Analysis of staining with UEA-I showed a significant relationship with blood vessel invasion (P < 0.01) and lymphatic vessel invasion (P < 0.05). Furthermore, PR showed a significant inverse correlation with lectin binding (P < 0.05). Staining with UEA-I related significantly with axilliary lymph node metastases (P < 0.05). UEA-I was positive in four (66.6%) out of six cases with distant organ mestastasis. This study confirms that, in breast cancer, lectin binding to the cancer cells can be a reliable indicator for axilliary metastases, and the need for additional therapeutic interventions.

Endothelin-1-like immunoreactivity in human atherosclerotic coronary tissue: a detailed analysis of the cellular distribution of endothelin-1.

Endothelin (ET) is a very potent vasoconstrictor peptide, which was originally reported to be produced by endothelial cells and to act locally in a paracrine fashion to regulate vascular tone. Recent studies have demonstrated that endothelin-1 (ET-1) not only is produced by endothelial cells, but is also present in non-endothelial cells of atherosclerotic lesions. The present study was therefore designed to characterize the cell type and distribution of ET-expressing cells in different areas of human atherosclerotic coronary plaques, obtained by directional atherectomy of 30 patients. In addition, ET-1 messenger RNA (mRNA) distribution was studied in human atherosclerotic plaque tissue by in situ hybridization (ISH). The strongest ET-1-like immunoreactivity (ET-1-IR) was present in all cell-rich areas of 27 plaques. In fibrotic areas of 27 tissue samples, ET-1-IR was found in 44 per cent (12/27). ET expression was most prevalent in foamy macrophages (MPs, HAM 56-positive) and myofibroblasts (MFBs, alpha-actin-positive) in the vicinity of necrotic areas with signs of previous intraplaque haemorrhage. By contrast, ET-1-IR was weak and inconsistently found in MPs (11/27; 40 per cent) and MFBs (12/27; 44 per cent) in fibrous areas. Luminal endothelial cells (Ulex europeus agglutinin reaction-positive, UEA) exhibited strong ET-1-IR, whereas endothelial cells of intraplaque microvessels demonstrated inconsistent staining for ET-1. ISH revealed that ET mRNA is produced locally in intimal MPs showing strong ET-1-IR. These findings demonstrate that ET-1 is produced by human MPs, the principal inflammatory cell type in atherosclerosis, suggesting a role for ET-1 in the chronic inflammation associated with complicated atherosclerosis.

Carbohydrate distribution in the living utricular macula of the guinea pig detected by lectins

Carbohydrate distribution in the fresh utricular macula of the guinea pig was analysed using lectins such as Concanavalin A (ConA), Dolichos biflorus agglutinin (DBA), peanut agglutinin (PNA), soybean agglutinin (SBA), Ulex europeus agglutinin (UEA-I) and wheat germ agglutinin (WGA) by means of confocal laser scanning microscopy. The ciliary bundle was strongly reactive to ConA, PNA, SBA and WGA but not to DBA and UEA-I, showing that the ciliary bundle has abundant d-galactose (Gal), N-acetyl- d-glucosamine (GlcNac), d-mannose (Man) and sialic acid(s) (Sia) but not detectable amounts of l-fucose (Fuc) and terminal N-acetyl- d-galactosamine (GalNAc). Similar patterns of lectin bindings with moderate-to-weak intensities were observed on the non-cilial apical surface, on the surface of the otoconia and in the gelatinous layer of the otoconial membrane. On the contrary, the globular substance, a precursor of the otoconia, was scarcely reactive to any lectin examined, implying that it lacks glycoconjugates on its surface. Previous histochemical studies reported that the otoconial membrane possesses a much higher affinity for lectins than does the sensory epithelium (including the cilia) in the vestibular organ. This discrepancy suggests that factors in the preparation process may affect the otoconial membrane or the surface coat of the cilia to change their lectin affinity. Meanwhile, sialidase treatment augmented the affinity of the ciliary bundle for DBA and PNA, indicating that sialylated GalNAc and Gal are present on the vestibular ciliary bundle.

A method for the isolation and serial propagation of keratinocytes, endothelial cells, and fibroblasts from a single punch biopsy of human skin.

When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment to Ulex europeus agglutinin I-coated paramagnetic beads. To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS). Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin.(ABSTRACT TRUNCATED AT 250 WORDS)

Ulex europeus agglutinin I binding pattern during chemical carcinogenesis in the rat gastrointestinal tract

Fluorescein isothiocyanate-Ulex europeus agglutinin I stain (UEA1) was postulated as a prominent histochemical marker for premalignant mucosa in dimethylhydrazine (DMH)-treated animals. UEA1 (evaluated by two scanning methods) and high iron diamine Alcian blue (HIDAB) stain were used in attempt to detect premalignant colonic mucosa in this animal model. The authors also examined the influence of the duodenal medium on colonic segments transposed to the upper gastrointestinal tract. Rats were placed into three groups: those with interposed intestine, those receiving the sham operation, and controls. Half of the animals received DMH, and surviving rats were killed at 2, 4, and 8 months. The authors found no differences in tumor development in the transposed and nontransposed colons of animals treated with DMH. Several transposed segments of animals without carcinogen induction showed dysplastic areas. These findings suggest a trophic role of certain duodenal factors in the epithelial kinetics of the transposed colons. The authors did not find HIDAB stain useful in the identification of premalignant colonic mucosa. The quantitative evaluation method of UEA1 binding was more reliable. Fifteen percent of all the colon specimens of animals without chemical induction were stained with UEA1 with this form of evaluation. Positive staining of the interposed colon samples was the most important factor for these findings. In this animal model, UEA1 staining is a potentially useful marker of premalignant mucosa, particularly when the nontransposed distal colon of animals treated with DMH is considered.

Epithelioid Hemangioma of Bone

Epithelioid hemangiomas are benign vascular tumors that usually occur in the skin and subcutis. They have been infrequently recognized in bone. Because of their unusual cytologic appearance and growth patterns, they are commonly confused with malignant tumors. We report a series of 12 epithelioid hemangiomas of bone occurring in adult patients, including five males and seven females whose ages at presentation ranged from 24 to 74 years, with a mean of 46 years. Five tumors were associated with involvement of the adjacent soft tissue. A single patient had multifocal bone disease. The most common presenting symptom was localized pain. Treatment of the patients varied widely; however, none of the tumors behaved aggressively. In 11 cases, adequate tissue was available for immunohistochemical analysis, which revealed positive staining for the epithelial markers cytokeratin and epithelial membrane antigen in nine cases. All 11 tumors stained for factor VIII-related antigen and Ulex europeus agglutinin. We believe that many of the vascular tumors of bone that have been reported as low-grade malignant hemangioendotheliomas probably represent examples of epithelioid hemangiomas. We recommend that the criteria for diagnosing vascular tumors of bone conform to those used for morphologically similar tumors that arise in the soft tissues.