Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands.

The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.

Agminated histiocytomas in a 23-year-old patient.

A 23-year-old patient presented with a group of 17 reddish-brown papules and nodules on the left shoulder lasting for 4 years. The histopathologic examination after a punch biopsy was repetitively consistent with the diagnosis of fibrous histiocytoma (FH). We use the term agminate histiocytomas for the first time to stress the presence of grouped lesions in one skin segment. Intralesional corticosteroids and cryotherapy were partially successful in this patient.

Functional consequences of the glycophenotype of squamous epithelia--practical employment.

Squamous epithelia represent a morphologically and differentiation-dependent stratified tissue. The stem cells are located in the bulge region of hair follicles or in the basal layer of interfollicular epidermis and in the limbus of the cornea. This article summarizes the data about the glycobiological aspects of squamous epithelia cell differentiation under physiological as well as pathological conditions in relation to the function of this epithelial tissue. The entries about the LC, Merkel cells and melanocytes are also mentioned. The employment of the described data in the diagnostics of carcinomas derived from this type of epithelium as well as in the cell therapy of skin defects are shown.

Dendritic cells and their role in skin-induced immune responses.

This artide gives a brief review on dendritic cells (DC) with regard to their origin, life cyde and functions. The regulation of immune responses by DC functioning as antigen-presenting cells is discussed. Special attention is given to epidermal DC, e.g. Langerhans cells. The perspectives of DC-based therapy are also mentioned.

Expression of galectin-3-reactive ligands in squamous cancer and normal epithelial cells as a marker of differentiation.

The definition of biological markers for oropharynx and larynx cancer is essential to predict their clinical behavior. Since cellular glycans play an important role in biological information transfer, we have employed an endogenous lectin, galectin-3, to examine in primary squamous carcinomas, lymph node metastases, and physiological squamous epithelia whether glycans recognized by this lectin are altered in relation to the state of differentiation. The expression of galectin-3 was concomitantly evaluated by immunohistochemistry using the A1D6 monoclonal antibody. In addition, other antibodies were used for the detection of cytokeratins and desmosomal proteins (desmoplakin-1 and desmoglein). The results show the expression of galectin-3-reactive ligands in moderately/highly differentiated carcinomas only in areas exhibiting a high level of keratinization. Except for one patient out of 14, metastatic cells in lymph nodes expressed no accessible binding sites for galectin-3. No galectin-3-reactivity was detected in the basal cell layer of all studied normal epithelia (which contains the proliferating cells). The suprabasal layers were positive in epidermis and epithelium of tongue and cornea and negative in epithelium of palatine tonsil. The tumor cells expressed galectin-3 with an intensity positively correlated with tumor differentiation. The position of galectin-3-reactive sites colocalized with the two tested desmosomal proteins. However, presence of these proteins was also detected in areas of tumor and suprabasal layers of tonsil epithelium where no binding reactivity for galectin-3 was found. The present study showed that expression of galectin-3-reactive glycoligands is differentiation-dependent in normal as well as malignant squamous cells. Colocalization of galectin-3-reactive sites with desmosomal proteins (desmoplakin-1 and desmoglein) suggests an association of the galectin-3 ligand(s) with the cell surface, pointing to a potential participation of galectin-3 in mediation of intercellular contacts in these tumor types.

Postmitotic basal cells in squamous cell epithelia are identified with Dolichos biflorus agglutinin--functional consequences.

Dolichos biflorus agglutinin (DBA) is a plant lectin specifically recognizing alpha-N-acetylgalactosamine. Controversial reports regarding the binding of DBA to the epidermis have been published. Using a double labeling procedure at the single-cell level, we studied the expression of DBA-reactive binding sites in conjunction with markers of cell proliferation and differentiation in normal human epidermis, cornea, and malignant tumors as well as in cultured keratinocytes. The results characterize the cells recognized by DBA as postmitotic early differentiating cells, identifiable by their lack of expression of the proliferation marker (Ki-67). The Golgi complex of a limited number of cultured keratinocytes was recognized by DBA and some of these cells show the accumulation of beta1 integrin chain in the Golgi complex. This process seems to be important for the migration of postmitotic cells from the basal to the suprabasal layers.

Biological properties of copolymer of 2-hydroxyethyl methacrylate with sulfopropyl methacrylate.

Interaction of organism with non-toxic implanted polymers depends on the physicochemical properties of the implant surface, which influence the adsorption of bioactive proteins and subsequently adhesion and growth of cells. The synthetic hydrogels are known as poorly adhesive surfaces. In this study we demonstrated the adsorption of albumin, fibrinogen, fibronectin, basic fibroblast growth factor, heparin-binding epidermal growth factor-like growth factor and epidermal growth factor to poly(2-hydroxyethyl methacrylate) (pHEMA) and copolymer of 2-hydroxyethyl methacrylate (HEMA) and potassium salt of 3-sulfopropyl methacrylate (SPMAK). The adhesion and growth of 3T3 cells and human keratinocytes on surface of these polymers was tested without and with pretreatment of polymers with heparin-binding epidermal growth factor-like growth factor. The adhesion of mixture of human granulocytes and monocytes to these surfaces was also tested. The strips of both polymers were subcutaneously and intracerebrally implanted into the rat and the extent of foreign body reaction and brain biocompatibility was evaluated. The results showed the extensive adsorption of basic fibroblast growth factor and heparin-binding epidermal growth factor-like growth factor to copolymer containing SPMAK. However the adhesion (and growth) of cells to this type of copolymers was very low. Preadsorption of human plasma to pHEMA clearly stimulated the leukocyte adhesion in contrary to copolymer containing SPMAK. The extent of foreign-body reaction was significantly higher against the pHEMA compared to tested copolymer p(HEMA-co-SPMAK). In conclusion, the tested copolymer was a poorly adhesive substrate that is only poorly recognized by the non-specific immunity, although the adsorption of basic growth factors to this substrate is highly significant. Both polymers were well tolerated by the brain tissue. The phenotype of surrounding neurons was more close to the control neurons in the brain tissue surrounding the p(HEMA-co-SPMAK) implants.

Cells of porcine epidermis and corneal epithelium are not recognized by human natural anti-alpha-galactoside IgG.

Human natural antibodies against Galalphal,3Gal-R are mainly responsible for hyperacute rejection of xenografts transplanted to the human host. In addition to the anti-alpha-Gal activity, human serum also contains anti-beta-Gal IgG fractions. Employing biotinylated IgG subfractions with anti-alpha- and anti-beta-Gal activity purified from human natural IgG, we have studied expression of reactive epitopes in porcine and human skin, porcine cultured keratinocytes and porcine and human cornea, porcine liver and human lacrimal gland, tear fluid and capillaries. No reactivity of porcine and human epidermis as well as anterior corneal epithelium was observed for human anti-alpha-Gal IgG. Serving as positive control, porcine capillaries gave the expected signal with the anti-alpha-Gal antibody. The anti-beta-Gal subfraction recognized cell nuclei in the epidermis of both these species. The pig liver cells interacted with antibodies against alpha- and beta-galactosides like cells of the human lacrimal gland. alpha-galactoside-reactive glycoproteins were also detected in the human tear fluid. The carbohydrate specificity of the reaction was ascertained by using melibiose as competitive sugar for alpha-galactoside-mediated binding. These results reveal the presentation of Galalpha1,3Gal in epithelial cells of human lacrimal gland, its biosynthetic origin being unclear. With respect to a potential clinical perspective, the given results facilitate consideration of the use of porcine epidermal cells in engineering of non-permanent wound covers to improve treatment.

Human epidermal Langerhans cells are selectively recognized by galectin-3 but not by galectin-1.

Langerhans cells are dendritic antigen-presenting cells residing predominantly in the epidermis. Since endogenous galactoside-binding lectins with the jelly-roll motif (galectins) are known to trigger cellular responses, including mediator release, we investigated by lectin histochemistry the cells' capacity to bind two common members of this family, i.e. galectin-1 and -3. Actually, surrounding keratinocytes express a high level of galectin-3, and these cells can be considered as donors of this lectin to Langerhans cells. Employing biotinylated galectin-1 and -3, and concomitantly an antibody against CD1a as a second marker, to visualize the position of Langerhans cells in the human epidermis, the expression of galectin-3-reactive glycoligands in contrast to the lack of binding of galectin-1 was observed. Although the functional consequences of this selectivity are unclear, these results reveal an example for differential cellular reactivity towards two related endogenous lectins.

Synthetic hydrogel capacity to induce formation of foreign-body giant multinucleate cells differs in vivo and in vitro.

The granulomatous reaction accompanied with MGC formation represents the most striking feature of the non-favourable biological tolerance of implanted devices. We compared MGC formation in the course of the granulomatous reaction in vitro and in vivo employing three types of hydrogels whose biocompatibility had been well studied earlier. The efficiency of the in vitro assay for the granulomatous reaction, including MGC formation, was verified employing the nematode Nippostrongylus brasiliensis, a well-known inductor of MGC formation in vitro. The in vitro results demonstrated a very low level of MGC formation in reaction against all three types of hydrogels without polymer-specific differences in comparison with the nematode experiment characterized by a high extent of MGC formation. On the other hand, the extent of MGC formation was implant type-specific in vivo: pHEMA-co-DMAEMA > pHEMA > pHEMA-co-NaMA. These results indicate that in the in vitro assay it was not possible to discriminate among the types of polymers used in the experiment in comparison with the animal experiment. They also indicate potential differences between granuloma formation induced by parasites and by foreign bodies.

Coexpression of binding sites for A(B) histo-blood group trisaccharides with galectin-3 and Lag antigen in human Langerhans cells.

Galectin-3 is an immunomodulatory protein with binding capacity for various glycoconjugates including IgE. It has been shown to be produced by epidermal keratinocytes and is present on the surfaces of skin Langerhans cells (LC). Therefore, it may have a role in the pathogenesis of various skin diseases, such as atopic dermatitis. To study the expression of galectin-3 in LC, we used, in addition to specific antibodies, a panel of synthetic, carrier-immobilized, specific oligosaccharides of the A- and B-histo-blood group, which are recognized by this lectin. In the mean time, Birbeck granules were visualized with an anti-Lag antibody. The double labeling experiments showed a remarkable colocalization of signals for Lag antigen (Birbeck granules) and galectin-3, as well as the binding sites for A- and B-histo-blood group trisaccharides. The specificity of the oligosaccharide binding was demonstrated by the lack of binding by Le(c), Le(d) (H blood group antigen), and sLe(x), which are not recognized by galectin-3. These results suggest that galectin-3 is present in Birbeck granules, where it retains reactivity for its glycoligands.

Simultaneous detection of endogenous lectins and their binding capacity at the single-cell level--a technical note.

Endogenous lectins are proteins/glycoproteins which selectively recognize distinct saccharide ligands and are different from immunoglobulins and carbohydrate-utilizing enzymes. Expression of these molecules can be detected immunohistochemically using nonblocking monoclonal antibodies (A1D6: anti-galectin-3, MR-15-2-2: anti-175 kD mannose receptor). Alternatively, biotinylated (neo)glycoconjugates which are recognized by a studied lectin can be employed as convenient probes to demonstrate specific binding of sugar epitopes by carbohydrate recognition domains (CRD) of endogenous lectins. In this study, we describe a new procedure for immunocytochemical visualization of the expression of endogenous lectins and glycochemical visualization of the reactivity of carbohydrate recognition domain(s), performed simultaneously at the single-cell level.