Lectin and proteoglycan histochemistry of Merkel cell carcinomas.

Changes in carbohydrate residue expression and in proteoglycan distribution occur during different stages of tumor development and progression. However, few data concerning carbohydrate residue analysis as performed by lectin histochemistry and proteoglycan distribution of Merkel cell carcinoma, a rare malignant tumor of the skin, have been reported. Hence, lectin- and proteoglycan immunohistochemistry was performed on paraffin wax material of 9 cases of Merkel cell carcinomas characterized by cytokeratin and neurofilament immunohistochemistry. The lectin binding pattern of tumor cells varied between lectins with different sugar binding specificities, while within a given nominal sugar specificity intensities were remarkably similar between tumors from different patients. The most intensive reaction was observed using Con A (mannose/glucose-specific) followed by LCA with the same specificity and the N-Acetyl glucosamine-specific lectins (WGA, UDA, CMA), while no fucose binding sites were detected (UEA-I). In addition, N-Acetyl galactosamine residues were only occasionally detected. The lectin binding pattern of Merkel cell carcinoma cells indicated that predominantly N-linked glycans and not O-linked glycans, typical for mucins of most epithelia, were present. Hence these tumor cells were relatively undifferentiated and resembled stem cells more closely than differentiated epithelia. The tumor stroma was especially evaluated in this study and showed a lectin reaction, which was intermediate between the tumor cells and extra-tumoral stroma. For example, the reactions of N-Acetyl galactosamine-specific lectins were intensive in the extra-tumoral stroma but nearly negative in tumor cells, while the lectin reaction of the intra-tumoral stroma was similar to the cellular reaction. These results indicated an influence of tumor cells on the stromal constituents. Antibodies against chondroitin type glycosaminoglycans reacted with the tumor stroma and the pericellular substance around the tumor cells most intensely in - and around the major tumor septae which, in general, were well vascularized. The most intensive immunoreactivity was detected using the chondroitin-6-sulfate antibody. The cellular and membrane-associated reaction for heparan sulfate was less intensive in comparison to epidermal cells. In conclusion the pattern of lectin-binding sites, the high chondroitin(sulfate) specific reactivity and the relatively low intensity of heparan sulfate immunohistochemistry indicate a low degree of differentiation and high malignity of the tumors, which is consistent with the clinical behavior of Merkel cell carcinomas.

Lectin and proteoglycan histochemistry of feline pacinian corpuscles.

We studied carbohydrate residues of glycoproteins and proteoglycans (PGs) in peritoneal Pacinian corpuscles of five adult cats. Terminal monosaccharides of glycoproteins and related polysaccharides were identified by lectin histochemistry and the PGs and glycosaminoglycans (GAGs) by specific antibodies. The most intensive lectin staining reactions indicated an abundance of glycoconjugates with terminal mannose (Man) or sialic acid residues, but no complex-type oligosaccharides were detected within the corpuscles. Terminal fucose (Fuc) and galactose (Gal) residues typical for O-linked mucin-type glycoproteins generally associated with high water binding capacity were also absent. Antibodies against unsulfated chondroitin (C-0-S), chondroitin-4-sulfate (C-4-S), and decorin showed positive reactions in the interfibrillar spaces between the lamellae, around collagen fibers, and around the lamellae of the perineural capsule, especially in the outer parts known to contain Type II collagen. Biglycan showed a preference for the innermost part of the perineural capsule (intermediate layer), known to contain Type V collagen. Collagen V and biglycan are both linked to growth processes. Hyaluronic acid (HA), chondroitin-6-sulfate (C-6-S) chains, and a chondroitin sulfate proteoglycan (CSPG) were co-localized in the terminal glia. The study of carbohydrates with high water binding capacity may contribute to our understanding of the high viscoelasticity of Pacinian corpuscles.

Proteoglycan distribution pattern during aging in the nematode Caenorhabditis elegans: an ultrastructural histochemical study.

Glycosaminoglycans are important constituents of the extracellular matrix of vertebrates, where distinct changes in their distribution pattern occur during aging. However, little is known about their changes in the nematode Caenorhabditis elegans, which ages extremely rapidly compared to mammals. The presence of glycosaminoglycans was analysed in cross-sections of all organs of the nematode, in three different age groups (60, 144, 228 h), using the electron-dense dye Cuprolinic Blue in conjunction with the critical electrolyte concentration method and specific glycosaminoglycan degrading enzymes. The nematodes (strain DH 26) were grown at 25.5 degrees C. The results indicate the presence of an organ-specific distribution pattern. Chondroitin-4-sulphate and/or chondroitin-6-sulphate are present in the epicuticula. Chondroitin-4-sulphate and/or chondroitin-6-sulphate and dermatan sulphate are detected in the mesocuticula. If stained by conventional methods the mesocuticula shows an empty fissure, which is filled by chondroitin sulphates and dermatan sulphate as shown by Cuprolinic Blue staining and enzymes. Heparan sulphate is found in the terminal web of intestinal cells while dermatan sulphate is revealed in the central cores of microvilli. An unknown polyanion staining at high electrolyte concentrations is observed in the gonads. Age-related changes do not impair the composition of the glycosaminoglycan fraction. In conclusion an unexpected highly differentiated pattern of glycosaminoglycans with high stability during aging exists.

Lectin binding pattern and proteoglycan distribution in human eccrine sweat glands.

The distribution pattern of glycoconjugates in human eccrine sweat glands has been studied by the binding of newly discovered lectins and by antibodies against a chondroitin sulphate proteoglycan and chondroitin sulphate glycosaminoglycans. Mannose-specific lectins labelled large intracellular granules, part of which could be extended cisternae of the endoplasmic reticulum or Golgi apparatus. In contrast, lectins specific for terminal mannose/glucose residues predominantly labelled basement membranes and the glycocalyx. Lectins recognizing terminal N-acetylgalactosamine groups left most parts of the glands unstained, but stained some dark cells intensely. These last cells were also intensively labelled by N-acetylglucosamine-specific and by fucose-specific lectins. Sialic acid residues were preferentially located in luminal borders of secretory coils. No terminal galactose residues were detected. All antibodies against chondroitin glycoconjugates stained large granules similar to those revealed by the mannose-specific lectins in the secretory cells. The basement membrane is only stained by the proteoglycan antibody and the chondroitin-6-sulphate antibody. Thus, a complex composition of glycoconjugates exists not only in matrix elements but also in the cells of eccrine glands of the human skin. A possible secretion of glycoconjugates is discussed.

Age-related histochemical staining patterns of glycosaminoglycans in cell nuclei of different regions of the rat brain: a pilot study.

The brains of 6 rats aged 12 months (adult) and 6 rats aged 24 months (aged) were embedded in paraffin following steady state perfusion with fixation solution. Glycosaminoglycans (GAGs) were demonstrated by histochemical methods using the Alcian blue CEC method in combination with the Feulgen reaction and testis hyaluronidase. Cell nuclei revealed different patterns of GAGs in different layers of the brain cortex and in different cell types. In neuronal cell nuclei of layer 2, no GAGs are found and this may be the case also in certain types of pyramidal cells. There was a reduction of the blue staining components of the chromatin network by hyaluronidase, and also a reduction of the electronmicroscopic contrast by this enzyme in pilot study using a specimen of one animal. The enzyme effects were found to be more marked or even exclusively present in the group of aged animals. Thus, the contents of chrondroitin sulfates or hyluronate which are substrates of the enzyme may be increased either relatively or absolutely in cell nuclei of aged brains whereas GAGs resistant to the enzyme may be reduced in activity. Since GAGs are known to influence DNA activity, the variations demonstrated may be assumed to be of significance for the aging process in postmitotic cells.

Changes of the glycosaminoglycan metabolism in human lung fibroblasts and porcine aortic endothelium cells influenced by the number of subcultures in vitro and by lipids in the medium.

In vitro aging fibroblasts and porcine aortic endothelium cells were treated with a commercial soy-bean lipid emulsion and incubated in 35S-sulfate or 3H-glucosamine respectively. The glycosaminoglycans (GAGs) were characterized by enzyme digestion following proteolysis. Uronic acid concentrations were determined in fibroblast cultures. The GAG content is lower in the last possible passage than in the foregoing ones. Lipid treatment leads to an increase, especially in early passages. The incorporation of radiolabeled precursors decreases with increasing in vitro age in both cell types. By lipid treatment the heparansulfate (HS) radioactivity of fibroblasts is increased insignificantly, while the GAG radioactivity of endothelium cells and the chondroitinsulfate (CS) radioactivity of fibroblasts are decreased. The in vitro age-related reduction of the precursor incorporation is more pronounced in CS as compared to HS, whereby the HS radioactivity shows a percentual increase, especially following lipid treatment. The effect of lipids shows changes related to in vitro aging. The role of cellular GAG metabolism and lipid loads in arteriosclerosis is discussed. Even in the arterial wall different cell types may react differentially.

[Histotopochemical quantification of glycosaminoglycans in melanomas and the surrounding epidermis]. / Histotopochemische Quantifizierung von Glykosaminoglykanen in Melanomen und umgebender Epidermis.

15 cases of human malignant melanoma were studied and classified into 5 superficial speading (SSM), 5 nodular melanomas (NM), and 5 melanoma metastasis (Met). The tissue was fixed with formaldehyde and cetylpyridium chloride (CPC). Glycosaminoglycans (GAG) or proteoglycans respectively were characterized by Alcian blue staining following the method of critical electrolyte concentration (CEC) (Scott, Dorling 1965) and by testes hyaluronidase. The staining intensities were quantified by a Leitz MPV photometer microscope in basement membranes (BM) and tumor septa. Tumor septa, which may be looked on as correlates of epithelial BM material, show increased straining intensities as compared to the normal BM (nBM) around the tumor. It is concluded from the sensitivity to testes hyaluronidase and the straining pattern that these are caused by increased straining of GAG of the type of chondroitin sulfates and possibly of dermatan sulfate while unsulfated GAG are rather decreased. The GAG pattern in BM in SSM shows characteristics of tumor septa and of nBM as well. The staining of the tumors shows higher intensities than that of all structures in the normal skin. It is concluded that increasing malignancy is accompanied by increasing changes in GAG which can be quantified by the method used topohistochemically discerning the healthy tissue from malignant structures.

[Age-related changes in glycosaminoglycans in the cell nuclei of the rat liver--biochemical and histochemical pilot studies]. / Alternsabhängige Veränderungen der Glycosaminoglycane in Zellkernen der Rattenleber--biochemische und histochemische Pilotstudien.

Cell nuclei of the livers of 12 male Wistar rats aged 24 months, and 16 male Wistar rats aged 4 months were isolated. 0.4 microCi [3H]-glucosamine/kg body weight were administered intraperitoneally. The livers of four animals were used for each biochemical analysis. The tissue was degraded by proteolysis, the glycosaminoglycans (GAGs) were isolated and the GAG types were determined by enzyme digestion, nitrite degradation and radiometry. The amount of GAGs was photometrically determined in single samples of pooled material of each of the two age groups. In paraffin sections (from other male Wistar rats of a different age), the influence of enzymes on specific GAG staining was observed. Most of the radioactivity was found in heparan sulfate (HS) and a lower content in chondroitin sulfates (CSs). In HS the incorporation increased with increasing age. The amount of HS showed no age-related differences. The results indicate an age-related activation of the HS metabolism. The GAG pattern and the age-related changes of the GAG types in total tissue (earlier results) are different from those in the nuclei. By histochemical methods, we observed a small but distinct effect of heparitinase in the cell nuclei.

Embryotoxic effects of thalidomide-derivatives in the non-human primate Callithrix jacchus. I. Effects of 3-(1,3-dihydro-1-oxo-2H-isoindol-2-yl)-2,6-dioxopiperidine (EM12) on skeletal development.

The response of pregnant marmosets (Callithrix jacchus) to the thalidomide derivative EM 12 was evaluated. EM 12 was selected for these studies because it is more active than thalidomide and is much more stable for hydrolysis. Skeletal gross structural abnormalities were observed when EM 12 was given to marmosets for 3-7 days during the period between days 49 and 60 post ovulation. Using the treatment schedule finally adapted in our laboratory, i.e. treatment during days 51-57 post ovulation, doses of 5 (or 10) mg EM 12/kg body wt induced the typical limb abnormalities known from man with an 80-100% certainty. In some animals we could observe the typical pattern of abnormalities even with doses as low as 1 mg EM 12/kg body wt. Abnormalities of the skeleton induced during this sensitive period are described. None of these (except some bifurcations of ribs) were seen in any of the ten litters (23 fetuses) serving as controls during the period of the study.

Embryotoxic effects of thalidomide derivatives in the non-human primate Callithrix jacchus. II. Elucidation of the susceptible period and of the variability of embryonic stages.

Data are presented on the normal intra- and inter-litter variability of the embryonic stages in the common marmoset (Callithrix jacchus) on day 56 of pregnancy. The embryology of the marmoset is described with respect to the susceptible period for the teratogenic action of thalidomide.

Sulfate incorporation into glycosaminoglycans of human lung fibroblasts as influenced by the number of subcultures and glycosaminoglycan polysulfate.

The influence of a glycosaminoglycan polysulfate (GAGPS) on the glycosaminoglycan (GAG) metabolism was investigated in relation to in vitro ageing of cultured human lung fibroblasts (Flow 2002). The incorporation of 35S-sulfate was determined following proteolytic digestion and specific degradation methods for the individual GAGs. In untreated cultures the pericellular matrix and medium showed a decrease of chondroitin sulfate (CS) 35S-radioactivity of the higher passage numbers, while the pericellular heparan sulfate (HS) showed increasing values. In GAGPS-treated cultures the decrease of 35S-sulfate incorporation into CS and dermatan sulfate of the cells and medium as well as into the pericellular CS was negatively correlated to the passage numbers, since GAGPS preferentially increased the radioactivity in cultures of low passage numbers. The HS values, however, were changed in the same direction as observed in untreated controls. Thus, age-related changes in the GAG metabolism become visible when exogenous GAGPS is used as a stimulus.

[Age-related changes in cellular activity in organ and tissue cultures of the human cornea]. / Altersabhängige Veränderungen der zellulären Aktivität in Organ- und Gewebekulturen der menschlichen Cornea.

Human autopsy eyes and material of eyes enucleated by various causes were used to prepare explants of the whole chamber angle and isolated stroma corneae for organ and tissue cultures. The age-related changes in chamber angle explants are obscured by interactions of the different cell types. In 75 human stroma explants aged from 5 weeks to 93 years the latent period for cell activation (i.e. the time needed for the activation of cells revealed by histological methods) shows its lowest values in cases under 20 years of age. The latent period for outgrowth of cells is not significantly related to age. The long-term reorganization of the tissue structure in the explants is disturbed in higher age.

[Age dependence of the early reaction in glycosaminoglycan metabolism of the rat liver on damage following a single dose of thioacetamide]. / Die Altersabhängigkeit der frühen Reaktion im Glycosaminoglycan-Stoffwechsel der Rattenleber auf eine einzeitige Thioacetamidschädigung.

Wistar rats aged 7 or 22 months resp. were treated with 50 mg/kg of thioacetamide (TAA) i.p. The TAA-treated animals as well as control groups of the same ages and containing the same number of animals were injected i.p. with 3H-glucosamine (0.2 microCi/g body weight) 2 hours before sacrifice. Previously to sacrifice, the livers had been perfused in narcosis with NaCl (0.9%). Following degradation of the tissue by pronase, specimen of the substances precipitable by ethanol were digested with chondroitinases, streptomyces-hyaluronidase, keratanase, and nitrite. The quantities of substances were estimated photometrically using Alcian blue; radiometry was performed following precipitation with cetylpyridiniumchloride. In aging animals the amount of dermatan-sulphate (DS) and of the total acidic glycosaminoglycans (a.GAG) is increased as compared to the younger animals, although there ist no age related difference in the incorporation of glucosamine. The incorporation per microgram a.GAG (spec. act.) is decreased. Following TAA the amount of DS is increased in both age groups, whereas the total amount of GAG is increased only in the younger animals. The incorporation per mg dry weight is also increased in all GAG types of both age groups which is also the case with the spec. act. (with the exception of HS in the younger animals). The increase of the incorporation per mg dry weight in DS is significantly lower in the older TAA-animals as compared to the younger ones. HS shows the same tendency, but there is no significance. The age related increase of hydroxyproline parallels that of DS.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related changes in the composition of proteins in the trabecular meshwork of the human eye.

The composition of the trabecular meshwork proteins of human eyes ranging in age from 36 days to 84 years was examined by polyacrylamide gel electrophoresis and amino acid analysis. Proteins of different molecular weights could be extracted from the tissue with acetic acid. Although their electrophoretic patterns became less distinct with increasing age, proteins of molecular weights ranging from 50 000 to 69 000 always prevailed. The amino acid compositions of the acetic acid-insoluble trabecular meshwork residues revealed the prevalence of collagenous proteins. The peptide maps produced by treatment with cyanogen bromide indicate that most of the fragments solubilized from the trabecular meshwork of younger eyes are derived from type I collagen. Beyond 40 years of age, the trabecular meshwork was resistant to cyanogen bromide and pepsin digestion. A rough estimate of the distribution of collagen types in the trabecular meshwork was based on 3-hydroxyproline/4-hydroxyproline ratios, indicating an age-related increase of type I collagen from about 55 to 70 per cent, and of type IV collagen from about 2 to 5 per cent of the total protein present. During ageing, some of the protein-bound methionine is oxidized to methionine sulfoxide, reaching about 35 per cent of the total methionine content at the age of 20 years and, with a slower rate of oxidation, a mean value of 40 per cent at 80 years of age. Electron-microscopic analysis of specimens remaining undissolved after cyanogen bromide cleavage and pepsin treatment no longer revealed regular collagenous fibrils but rather elastic-like fibers surrounded by wide sheaths consisting of fine fibrils with a regular cross-banding periodicity of 40-50 nm. In addition, clusters of so-called curly (lattice) collagen were found. The amino acid composition of this insoluble material suggests that altered collagen-like molecules prevail among the proteins of the residues.

Changes in cell cultures of bovine corneal endothelium cells as related to donor age and number of passages in vitro.

Aging changes of the acidic glycosaminoglycans (aGAG) were investigated in bovine corneal endothelium cells grown in vitro, using histochemical methods and the determination of hexosamine and uronic acid. The time interval between trypsination and confluency of the monolayer and the cell counts at different time intervals of the same passage number served as parameters for the proliferation capacity of the cultures. A cell line, derived from the eye of a 1,5 year old cattle showed a low proliferation capacity in the first 9 passages, which increased until the 15th passage, to decrease thereafter until the 29th passage. In cells from eyes aged 11 and 12 years respectively the initial proliferation capacity was very low to reach the same values as in cultures of the young eye at the 10th passage but remained constant thereafter. Chromosome counts showed an increased number of hyperploids in late passages of cells from the aging eye with counts around 4n-8n increased to 17% as compared to 5% in the cells derived from the young eye. The cell densities were increased in cultures of high passage numbers. Histochemically the aGAG were shown in the cell nucleus, and the cytoplasm as well as in the intercellular space. In the nuclei of cells from aging eyes and in late passage cells of the young eye they showed an increased resistance against resting hyaluronidase. The hexosamine and uronic acid values were high in early passages of cultures from the young eye and decreased in late passages of the young eye as well as in early passages of cells from the aging eyes. The changes in the aGAG as well as the changes in the cell proliferation are similar comparing early passage cells of aging eyes to late passage cells of the young eyes. But the aGAG changes are more pronounced in cells of eyes aged in vivo. In the eye the proliferation of the corneal endothelium cells is inhibited. As such these cells provide a useful model to comparing in vivo-aging changes of resting cells to changes of the same cells proliferating in vitro.

Synthesis and composition of glycosaminoglycans by cultured human trabecular meshwork cells.

A human trabecular meshwork cell line with a limited number of population doublings was established in monolayer culture. All cultures produced hyaluronic acid, heparan sulfate, chondroitin sulfate, and dermatan sulfate. Following [14C]-glucosamine incorporation into proliferating (phase II) cultures, 70%--80% of the medium glycosaminoglycan label was found in hyaluronic acid and 8%--14% in heparin sulfate. About 60% of the cell-bound activity was associated with hyaluronic acid and 30% with heparan sulfate. Long-term cultivation under nondividing ("senescent") conditions resulted in a change of the pattern of synthesized and excreted (medium) [14C]-glucosamine-labeled glycosaminoglycans with a relative decrease of hyaluronic acid and a relative increase of heparan sulfate.