Histochemical study of lectin binding in the human fetal minor salivary glands

The emerging synthesis of glycoconjugates containing specific oligosaccharides in developing human fetal labial and lingual salivary glands has been investigated by lectin histochemistry. An avidin-biotin technique was used to study the binding of lectins from Ulex europeus I (UEA-I), Dolichos biflorus (DBA), Glycine maximus (SBA), Helix pomatia (HPA), Arachis hypogaea (PNA) and Triticum vulgare (WGA) to specific sugars on sections of tissue from labial glands, glands of Blandin and Nuhn, glands of von Ebner and the dorsoposterior lingual salivary glands. Incipient synthesis of glycoconjugates in early glands and their presence in the cells and ducts of the later glands was shown. The study also showed a time-related increase in both staining intensity and binding sites of serous acinar cells from all glands and for all lectins used. For mucous cells, peak intensity of staining was reached by the middle phase of development. During later gland development this intensity was maintained in dorsoposterior lingual glands but tended to decline in labial glands. The various lectins showed different degrees of binding but UEA-I lectin generally bound the L-fucose sugar group in all salivary glands at all gestational ages. The results showed that lectins appear to bind to the oligosaccharides on epithelial cell surfaces of fetal salivary glands at all stages of development. The degree of change depends upon the stage of differentiation and maturation of the glands.     

Lectin binding to chronic inflammatory gingival tissue: possible adhesion mechanisms based on lectin-carbohydrate interactions

In this study, we analyzed the expression of different leukocyte surface antigens, of the adhesion molecules ELAM-1 and GMP-140 and binding of various lectins and neoglycoproteins in inflamed gingival tissue. Cell suspensions from collagenase-digested gingiva were analyzed by flow cytometry in a FACScan. The expression of ELAM-1, GMP-140, carbohydrate structures and lectins in gingival specimens was also studied by immunohistochemistry. Gingival tissue of patients with active periodontal disease contained between 5% and 50% CD45⁺ mononuclear cells, consisting mainly of CD19⁺ cells (B lymphocytes). CD62, resembling GMP-140, and ELAM-1 were strongly expressed on endothelial cells of these patients. Control subjects usually contained almost no CD45⁺ cells in their gingiva and no CD62⁺ or ELAM-1-positive endothelial cells could be found in 5 of 6 control persons. Analysis of the glycosylation pattern revealed staining of infiltrating cells by peanut agglutinin (PNA; specificity for galactose), whereas soy bean agglutinin (SBA; specificity for N-acetyl-galactosamine) bound to epithelial cells. An endogenous lactosyl-specific lectin could be detected on endothelial cells by binding of lactosyl-BSA. Ulex europeus I agglutinin (UEA-1, specific for fucose) showed selective staining of endothelial and epithelial cells. Expression of a fucose-binding lectin, demonstrated by binding of fucosylated BSA, could be found on infiltrating cells. The adhesion molecules ELAM-1 and GMP-140 seem to be involved in cell adhesion during chronic inflammation of the gingiva. Interaction of other carbohydrate residues with endogenous lectins might resemble additional adhesion mechanisms inflamed gingiva.     

Histochemical study using lectin and anti-human von Willebrand factor antibody of oral hemangioma

Twenty nine cases of oral hemangiomas composed of capillary hemangioma, cavernous hemangioma and pyogenic granuloma were examined with lectin histochemistry using ulex europaeus agglutinin I, ricinus communis agglutinin I, wheat germ agglutinin, concanavalin A, dolichos biflorus agglutinin, soybean agglutinin and peanut agglutinin, and immunohistochemistry using anti von Willebrand factor antibody in order to understand the nature of each endothelial cell. The following results were obtained; 1. UEA-I that specifically bound alpha-L-fucose was observed mainly in the endothelial cells of capillary hemangioma and hemangioma of granulation tissue type. 2. RCA-I which had specific affinity to beta-D-galactose was so identified in all endothelial cells of all type hemangiomas and normal blood vessels that it was supposed to be a useful marker of the cells. 3. WGA with specific affinity to N-acetyl-glucosamine was also found in various endothelial cells. 4. Con A which had specific affinity to alpha-D-mannose was not observed in various endothelial cells. 5. DBA, SBA and PNA that were specific bound up with N-acetyl-galactosamine were seen in the normal micro-vessels, especially in subepithelial regions. 6. Immunohistochemically, von Willbrand factor was identified chiefly in the endothelial cells of normal blood vessels and cavernous hemangioma, but it was not found in the cells of capillary hemangioma and pyogenic granuloma which had no differentiation in the functional point of view.     

Histochemical studies of obstructive adenitis in human submandibular salivary glands. II. Lectin binding and keratin distribution in the lesions

Lectin-binding profiles and keratin distribution in obstructive adenitis of human submandibular glands (SMGs) are reported and compared those of normal SMGs. Histologically, obstructive changes in the SMGs included acinar atrophy, duct-like structure formation in the early stage, and disappearance of acinar cells and dilation of ductal segments in the later, chronic stage. The following lectins were used: Con A (Glc, Man), PNA(Gal, GalNAc), RCA-I(Gal), DBA(GalNAc), SBA(Gal, Gal-NAc), UEA-I(alpha-L-Fuc) and WGA(GlcNAC, NeuNAc). Lectin staining in atrophic acinar cells was usually reduced except for SBA binding and was irregularly distributed in altered acinar and ductal epithelia. Binding of DBA and UEA-I lectins were particularly intense along the luminar borders of ductal segments in the lesions. Immunohistochemically detectable keratins were characterized by intense staining in atrophic acinar cells and in all the ductal segments, whereas normal acinar cells, either serous or mucous, were negative.     

Histochemical studies of obstructive adenitis in human submandibular salivary glands. II. Lectin binding and keratin distribution in the lesions

Lectin-binding profiles and keratin distribution in obstructive adenitis of human submandibular glands (SMGs) are reported and compared those of normal SMGs. Histologically, obstructive changes in the SMGs included acinar atrophy, duct-like structure formation in the early stage, and disappearance of acinar cells and dilation of ductal segments in the later, chronic stage. The following lectins were used: Con A (Glc. Man), PNA(Gal, GalNAc). RCA1I(Gal), DBA(GalNAc). SBA(Gal, Gal-NAc). UEA-1(α-L-Fuc) and WGA((GlcNAC, NeuNAc). Lectin staining in atrophie acinar cells was usually reduced except for SUA binding and was irregularly distributed in altered acinar and ductal epithelia. Binding of DBA and UEA-1 lectins were particularly intense along the luminar borders of ductal segments in the lesions. Immunohistochemically detectable keratins were characterized by intense staining in atrophie acinar cells and in all the ductal segments, whereas normal acinar cells, either serous or mucous, were negative.     

Splicing determines the glycosylation state of ameloblastin

In developing porcine enamel, the space between enamel rods selectively binds lectins and ameloblastin (Ambn) N-terminal antibodies. We tested the hypothesis that ameloblastin N-terminal cleavage products are glycosylated. Assorted Ambn cleavage products showed positive lectin staining by peanut agglutinin (PNA), Maclura pomifera agglutinin (MPA), and Limulus polyphemus agglutinin (LPA), suggesting the presence of an O-linked glycosylation containing galactose (Gal), N-acetylgalactosamine (GalNAc), and sialic acid. Edman sequencing of the lectin-positive bands gave the Ambn N-terminal sequence: VPAFPRQPGTXGVASLXLE. The blank cycles for Pro(11) and Ser(17) confirmed that these residues are hydroxylated and phosphorylated, respectively. The O-glycosylation site was determined by Edman sequencing of pronase-digested Ambn, which gave HPPPLPXQPS, indicating that Ser(86) is the site of the O-linked glycosylation. This modification is within the 15-amino-acid segment (73-YEYSLPVHPPPLPSQ-87) deleted by splicing in the mRNA encoding the 380-amino-acid Ambn isoform. We conclude that only the N-terminal Ambn products derived from the 395-Ambn isoform are glycosylated.     

Immunofluorescent lectin binding patterns and glycoprotein co-localization in the developing murine molar tooth.

Fluorescein-conjugated lectins were used in conjunction with antibodies to laminin, tenascin and amelogenin to investigate saccharide expression in the developing tooth germ. At the bud stage, peanut agglutinin (PNA) binding demonstrated residues that may be D-galactose-(beta 1----3)DGalNAc, and this staining occurred after the expression of tenascin. Only the cap-stage enamel organ suprabasal cells and the enamel knot stained intensely with Ulex europeus agglutinin-I, but not Lotus tetragonolobus agglutinin, implying the transient presence of blood group H type I oligosaccharides. At the late stages of amelogenesis, enamel synthesis is preceded by en bloc loss of inner enamel basement membrane components. Before this, Bandeiraea (Griffonia) simplicifolia--I (BSL-I) staining was lost from postmitotic ameloblasts, suggesting that a glycosylated species is initially removed. Additionally, PNA was co-localized with amelogenin protein, suggesting that it may express beta-D-galactosyl sequences. These results indicate that the glycosylation patterns of matrix components during odontogenesis may be important as they vary in a manner similar to that of the well-known glycoproteins.     

Lack of effect of hormonal treatment and smoking on cytokeratin expression in buccal mucosa

Differences in cytokeratin expression of clinically normal buccal mucosa were studied in 50 healthy women by indirect immunofluorescence staining with monoclonal antibodies. The subjects were divided into four groups: control group (N= 18), smokers (N=8), oral contraceptive users (N=8) and smokers receiving oral contraceptives (N=16). Our findings indicate that cytokeratin expression in non-cornified stratified epithelium is not influenced by smoking or oestradiol/progesterone treatment. Only cytokeratin No. 19 showed variable patterns of expression but the differences could not be ascribed to smoking or contraceptives. Cytokeratin No. 19 gave a positive reaction in the basal and suprabasal layers in 34 subjects (68%). In 9 (18%) specimens, the staining was positive in the basal cells and showed a positive heterogeneous cytoplasmic reaction in the suprabasal cells. Interestingly, cytokeratin No. 7 was recognized in all epithelial cells except the basal cells. Our results suggest that changes in the serum oestradiol levels do not affect the cytokeratin pattern in buccal mucosa.     

Association of food lectins with human oral epithelial cells in vivo

The association of wheat germ agglutinin (WGA) and peanut agglutinin (PNA) with oral tissues after eating raw wheat-germ or raw peanuts, respectively, was determined. An indirect enzyme-linked immunoassay (ELIA) was used to detect WGA and PNA on epithelial cells. Buccal and tongue epithelial cells, and preparations of salivary sediment exhibited ELIA units of WGA and PNA significantly above background after eating 2--7 g of wheat-germ or 25 g of raw peanuts, respectively. Both lectins were detected also in preparations of salivary bacteria where they persisted for 1--6 h. Samples collected after eating raw wheat-germ and incubated with N-acetylglucosamine, or samples collected after eating raw peanuts and incubated with galactose had reduced ELIA units of lectin present. This suggests that the lectins were not non-specifically adsorbed or present as unbound food particles. Pretreating buccal epithelial cells in vitro with 25 micrograms of WGA increased the numbers of Streptococcus sanguis FC-1 cells which attached. Similarly, statistically significantly higher numbers of Strep. sanguis cells attached to buccal cells which were collected from donors after they had eaten raw wheat-germ compared to buccal cells collected prior to eating.     

Glycosylations in demilunar and central acinar cells of the submandibular salivary gland of ferret investigated by lectin histochemistry

'Resting' submandibular salivary glands obtained post-mortem from mature ferrets of both sexes were examined here. The binding patterns of labelled lectins applied to paraffin sections of tissue slivers fixed in an aldehyde-HgCl2 mixture and the effects of pretreatment procedures on the results were assessed lightmicroscopically. Lectins with affinity for terminal GalNAc residues (DBA, SBA) bound preferentially to demilunar acinar cells which were also strongly reactive with Fuc-directed UEA I. In contrast, lectins with affinity for neuraminic acid (SNA, WGA) bound to central acinar cells where consistent binding of DBA and SNA occurred only after neuraminidase digestion, and variation in the binding of UEA I was seen. The reactivities corresponded with the distribution of secretory granules, but staining in Golgi-like areas occurred in central acinar cells with PNA lectin. The results suggest that glycosylations are more advanced in central than demilunar acinar cells of the ferret submandibular gland. Possibly demilunar and central acinar cells reflect phenotypic changes of a single secretory cell, the 'central' acinar phenotype being influenced by incorporation of neuraminic acid in glycoprotein side chains and by increased Golgi activity.     

Differential lectin-bindings in normal and precancerous epithelium and squamous cell carcinoma of the oral mucosa

In order to establish a useful and objective marker of malignancy of oral mucosa, the binding sites for Ulex europaeus agglutinin I (UEA-I). Bandeiraea simplicifolia agglutinin I (BSA-I) and peanut agglutinin (PNA) were comparatively examined in the surgical materials from the normal, dysplastic and cancerous epithelium of the oral mucosa by a novel lectin-antilectin immunoperoxidase method. Based on the staining patterns of the normal keratinized epithelium, UEA-I was regarded as the marker for the prickle cells, BSA-I for the cells in the upper prickle to the horny layers, and PNA for those in the basal layer. As the degree of dysplasia advanced, all layers of epithelium came to react with UEA-I and PNA, whereas the BSA-I binding was negative. Positive reactions for UEA-I and PNA were seen in most carcinoma cells other than the keratinizing foci stained by BSA-I. The results indicate that a UEA-I-positive reaction in the basal cells, a PNA-positive in the prickle cells and loss of receptor for BSA-I occur in the course of malignant transformation of oral mucosa, and that these lectins may be regarded as useful markers of oral epithelial cytoplasmic differentiation.     

Adhesion of Candida albicans, but not Candida krusei, to salivary statherin and mimicking host molecules

The aim of the present study was to identify salivary molecules affecting adhesion of Candida albicans and Candida krusei to salivary pellicles and epithelial cells. Strains of C. albicans (GDH18, GDH3339, CA1957, ATCC 28366 and ATCC 10321), but not C. krusei (strains ATCC 14243 and Ck9), bound to saliva-coated hydroxyapatite and buccal epithelial cells. Parotid saliva fractions containing statherin, glycosylated proline-rich proteins (PRP) and as yet unidentified components mediated adhesion of strain GDH18; Fuc alpha 1-2Gal beta 1-4Glc partly inhibited the adhesion to those fractions not containing statherin. Pure statherin, but not PRP-1, mediated dose-dependent adhesion of C. albicans strain GDH18 to hydroxyapatite beads. Candida isolates (GDH18, GDH3339 and CA1957) bound somewhat more avidly to statherin/saliva relative to ATCC strains 28366 and 10321, while the opposite was true for adhesion to buccal epithelial cells. Adhesion of C. albicans strain GDH18 to saliva-coated hydroxyapatite and buccal epithelial cells was completely (93%) and partly (43%) blocked by statherin-specific immunoglobulin G (IgG) antibodies, respectively. Control IgG antibodies did not block Candida adhesion. Blockage of Candida adhesion to epithelial cells also occurred with Fuc alpha 1-2Gal beta 1-4Glc (49%) and N-acetylglucosamine (38%), while statherin specific IgG antibodies in combination with Fuc alpha 1-2Gal beta 1-4Glc almost completely eliminated Candida adhesion (79%). In addition, statherin in solution blocked the adhesion of strain GDH18 to epithelial cells by inducing aggregation of Candida cells.     

Expression of cytokeratins in odontogenic jaw cysts: monoclonal antibodies reveal distinct variation between different cyst types

Immunostaining with monoclonal antibodies was used to study and compare the cytokeratin content of odontogenic cysts and normal gingival epithelium. Two monoclonal antibodies, PKK2 and KA1, stained the whole epithelium in all cyst samples. In gingiva, PKK2 gave a suprabasal staining and KA1 reacted with all epithelial cell layers. Antibodies PKK1, KM 4.62 and KS 8.12 gave a heterogeneous staining in follicular and radicular cysts. In keratocysts and in gingiva PKK1 and KM 4.62 reacted mainly with basal cells and KS 8.12 gave a suprabasal staining. Antibodies reacting with the simple epithelial cytokeratin polypeptide No. 18 (PKK3, KS 18.18) recognized in gingiva only solitary cells compatible with Merkel cells. In a case of follicular ameloblastoma a distinct staining of tumor epithelium was revealed with these antibodies. In 2 follicular cysts, but not in other cyst types, a layer of cytokeratin 18-positive cells was revealed. KA5 and KK 8.60 antibodies, reacting exclusively with keratinizing epithelia, including normal gingiva, gave no reaction in radicular cysts, keratocysts and ameloblastoma. Two of the follicular cysts, were negative for PKK3 and KS 18.18, but reacted strongly with KA5 and KK 8.60. The present results show that odontogenic jaw cysts have distinct differences in their cytokeratin content. With the exception of some follicular cysts, they lack signs of keratinizing epithelial differentiation. Only follicular cysts appear to share with some types of ameloblastoma the expression of cytokeratin polypeptide No. 18.     

Expression of cytokeratins in odontogenic jaw cysts: monoclonal antibodies reveal distinct variation between different cyst types

Immunostaining with monoclonal antibodies was used to study and compare the cytokeratin content of odontogenic cysts and normal gingival epithelium. Two monoclonal antibodies, PKK2 and KA1, stained the whole epithelium in all cyst samples. In gingivu, PKK2 gave a suprabasal staining and KA1 reacted with all epithelial cell layers. Antibodies PKK1, KM 4.62 and Ks 8.12 gave a heterogeneous staining in follicular and radicular cysts. In keratocysts and in gingiva PKK1 and K_M 4.62 reacted mainly with basal cells and K_s 8.12 gave a suprabasal staining. Antibodies reacting with the simple epithelial cytokeratin polypeptide No. 18 (PKK3, K_s 18.18) recognized in gingiva only solitary cells compatible with Merkel cells. In a case of follicular ameloblastoma a distinct staining of tumor epithelium was revealed with these antibodies. In 2 follicular cysts, but not in other cyst types, a layer of cytokeratin 18-positive cells was revealed. KA5 and K_k 8.60 antibodies, reacting exclusively with keratinizing epithelia, including normal gingiva, gave no reaction in radicular cysts, keratocysts and ameloblastoma. Two of the follicular cysts, were negative for PKK3 and K_s 18.18, but reacted strongly with KA5 and K_k 8.60. The present results show that odontogenic jaw cysts have distinct differences in their cytokeratin content. With the exception of some follicular cysts, they lack signs of keratinizing epithelial differentiation. Only follicular cysts appear to share with some types of ameloblastoma the expression of cytokeratin polypeptide No. 18.     

细胞角蛋白CK19在口腔粘膜癌变过程中的变化

目的：探讨口腔粘膜癌变过程中CK19的变化。方法：收集正常口腔粘膜、上皮单纯增生、上皮异常增生和口腔鳞癌活检标本共53例，用免疫组化、电泳和Western杂交方法研究上皮中CK19的表达。结果：CK19表达于有上皮异常增生的复层鳞状上层和口腔鳞癌尤其是低分化鳞癌的癌细胞中。随病变程度加重，CK19表达的阳性率，表达强度和占细胞角蛋白构成比显著增加。结论：CK19表达于粘膜上皮的基底上层可作为口腔癌前病变的辅助标志，CK19表达增加是 口腔粘膜病变过程中的早期事件。     

Adhesion of Candida albicans,but not Candida krusei,to salivary statherin and mimicking host molecules

The aim of the present study was to identify salivary molecules affecting adhesion of Candida albicans and Candida krusei to salivary pellicles and epithelial cells. Strains of C. albicans (GDH18, GDH3339, CA1957, ATCC 28366 and ATCC 10321), but not C. krusei (strains ATCC 14243 and Ck9), bound to saliva‐coated hydroxyapatite and buccal epithelial cells. Parotid saliva fractions containing statherin, glycosylated proline‐rich proteins (PRP) and as yet unidentified components mediated adhesion of strain GDH18; Fucα1‐2Galβ1‐4Glc partly inhibited the adhesion to those fractions not containing statherin. Pure statherin, but not PRP‐1, mediated dose‐dependent adhesion of C. albicans strain GDH18 to hydroxyapatite beads. Candida isolates (GDH18, GDH3339 and CA1957) bound somewhat more avidly to statherin/saliva relative to ATCC strains 28366 and 10321, while the opposite was true for adhesion to buccal epithelial cells. Adhesion of C. albicans strain GDH18 to saliva‐coated hydroxyapatite and buccal epithelial cells was completely (93%) and partly (43%) blocked by statherin‐specific immunoglobulin G (IgG) antibodies, respectively. Control IgG antibodies did not block Candida adhesion. Blockage of Candida adhesion to epithelial cells also occurred with Fucα1‐2Galβ1‐4Glc (49%) and N‐acetylglucosamine (38%), while statherin specific IgG antibodies in combination with Fucα1‐2Galβ1‐4Glc almost completely eliminated Candida adhesion (79%). In addition, statherin in solution blocked the adhesion of strain GDH18 to epithelial cells by inducing aggregation of Candida cells.     

Lectin-binding in premalignant lesions during submandibular gland carcinogenesis

Complex carbohydrates in premalignant lesions of mouse submandibular gland tumors were examined by the lectin-peroxidase conjugate method. Peroxidase-conjugated lectins of PNA, RCA-1, DBA, SBA, UEA-1 and WGA were used to detect specific sugar residues of complex carbohydrates in premalignant lesions during experimental carcinogenesis. Marked reduction of PNA and SBA bindings occurred in duct-like structures and cystic lesions which were transformed from granular convoluted tubule cells. Premalignant lesions bound slightly to PNA, RCA-1, DBA, SBA and WGA and manifested increased UEA-1 binding. Squamous metaplastic epithelia of premalignant lesions manifested increased binding to PNA, RCA-1 and SBA as compared to those of duct-like structure and cystic epithelia.     

Lectin-binding in premalignant lesions during submandibular gland carcinogenesis

Complex carbohydrates in premalignant lesions of mouse submandibular gland tumors were examined by the lectin-peroxidase conjugate method. Peroxidase-conjugated lectins of PNA, RCA-1, DBA, SBA, UEA-1 and WGA were used to detect specific sugar residues of complex carbohydrates in premalignant lesions during experimental carcinogenesis. Marked reduction of PNA and SBA bindings occurred in duct-like structures and cystic lesions which were transformed from granular convoluted tubule cells. Premalignant lesions bound slightly to PNA, RCA-1, DBA, SBA and WGA and manifested increased UEA-1 binding. Squamous metaplastic epithelia of premalignant lesions manifested increased binding to PNA, RCA-1 and SBA as compared to those of duct-like structure and cystic epithelia.     

Lectin binding to murine oral mucosa and skin

Carbohydrates on epithelial cell surfaces of oral mucosa and skin from various anatomical regions of C3H mice were demonstrated with fluoresceinated lectins. With an individual lectin, all tissues showed a similar pattern of binding: most lectins showed binding to the cell surfaces of all nucleated cell layers although that to basal cells was often weaker and was occasionally absent. The corneocytes did not typically bind lectins except that the follicular keratin of the tail showed a uniform and intense fluorescence with several lectins. Basement membrane bound all lectins. The results indicate that detectable changes occur in the cell-surface carbohydrate composition as cells differentiate but that cell-surface carbohydrates do not differ markedly from one region to the next. Lectin binding may provide a convenient method of detecting functional changes in normal cells and in cells which have undergone experimental or pathological changes.     

Lectin binding of rat gingival epithelia

Oral gingival epithelium (OGE), oral sulcular epithelium (OSE) and junctional epithelium (JE) were examined histochemically by using different lectins as markers for epithelial differentiation. The staining pattern of gingival epithelia was compared with that of the buccal and palatal epithelia. Binding of WGA had a uniform distribution in all the epithelia examined. A positive reaction was found in all the basal and spinous layers, but not in the cornified layer of the epithelia. BPA binding was seen in the lower spinous layer of OGE, OSE, buccal and palatal epithelia, and in most of the JE. The basal layer and the cells directly attached to the tooth surface at the apical part of JE were nonreactive with BPA. GS-I reacted with the basal and suprabasal layers of each epithelium and with the cells attached to the tooth at the apical part of JE. UEA-I reacted with the upper spinous layer of OGE, OSE and epithelia of hard palate, but not with any of the cells of the JE. Our results agree with previous data suggesting that OGE and OSE exhibit squamous differentiation similar to that of the masticatory epithelium of hard palate. Furthermore, our results suggested that the JE cells undergo differentiation equivalent to that of the suprabasal and lower spinous cells of OGE. The cells along the tooth surface at the apical part of JE, however, form a distinct population of cells with basal nature.