Mucosubstances in the normal human oesophageal epithelium

Summary Normal human oesophageal epithelium was investigated with the periodic acid — silver methenamine technique and its variations to demonstrate neutral mucosubstances at the ultrastructural level. The results were compared with the acid phosphotungstic acid method. Neutral mucosubstances were shown in the cell coat and membrane coating granules by both techniques. The silver methods also demonstrated glycogen, the Golgi apparatus and dense bodies. The periodic acid — silver methenamine technique outlined positive material in the intercellular space of the prickle cell layer, but the other silver methods did not.     

Histochemical reactions of gastrointestinal mucosubstances with high iron diamine after prior oxidation and methylation of tissue sections

Summary High iron diamine reactions after the prior methylation and oxidation of tissue sections with performic acid or potassium permanganate (metox-HID or ox-met-HID) in epithelial mucosubstances and in mucosal mast cells were studied in tissue samples from the human gastrointestinal tract and were compared with reactions with high iron diamine without any pretreatment (HID) and high iron diamine with the prior methylation (met-HID). High iron diamine reactions after the prior oxidation (met-ox-HID, ox-met-HID and ox-HID) demonstrated mucosubstances in a way which seemed to operate by the staining of acidic groups evoked by the oxidation of the tissue sections. These acidic groups were not blocked by the methylation. It was supposed that they are sulphonic acids resulting from sulphur groups (sulphydryls or disulphides) in some mucus glycoproteins. Met-ox-HID and ox-met-HID reactions seemed to stain mucosubstances and mast cells in a similar way but differed from the ox-HID reactions with the manner which could be interpretated to be due to the blocking of free sulphate ester groups in reactions of the former. Met-ox-HID (and ox-met-HID) positive mucosubstances were found in the foveolar epithelium of the stomach and in goblet cells of small and large bowel.The study was supported by grants from Sigrid Juselius Foundation and Paulo Foundation, Helsinki, Finland     

Mucosubstances in the normal human oesophageal epithelium. A comparison of periodic acid-silver and phophotungstic acid techniques.

Normal human oesophageal epithelium was investigated with the periodic-acid-silver methenamine technique and its variations to demonstrate neutral mucosubstances at the ultrastruct level. The results were compared with the acid phosphotungstic acid method. Neutral mucosubstances were shown in the cell coat and membrane coating granules by both techniques. The silver methods also demonstrated glycogen, the Golgi apparatus and dense bodies. The periodic acid-silver methenamine technique outlined positive material in the intercellular space of the prickle cell layer, but the other silver methods did not.     

Demonstration of an alkali PAS-effect using periodic acid at low concentration (author's transl)]

It has been shown that an isolated KOH PAS-effect of epithelial mucosubstances in the colonic mucosae of the rat can be demonstrated also without the conventionally used preliminary oxidation/reduction step. The method is based on the use of strongly diluted periodic acid after previous treatment of tissue sections with ethanolic KOH. As the positive reacting material has been proven to be sensitive against treatment with neuraminidase the modified KOH PAS-reaction should be related to the presence of acylated sialic acid residues in mucosubstances of the colonic mucosae of the rat.     

Histochemical reactions of gastrointestinal mucosubstances with high iron diamine after prior oxidation and methylation of tissue sections.

High iron diamine reactions after the prior methylation and oxidation of tissue sections with performic acid or potassium permanganate (metox-HID or ox-met-HID) in epithelial mucosubstances and in mucosal mast cells were studied in tissue samples from the human gastrointestinal tract and were compared with reactions with high iron diamine without any pretreatment (HID) and high iron diamine with the prior methylation (met-HID). High iron diamine reactions after the prior oxidation (met-ox-HID, ox-met-Hid and ox-Hid) demonstrated mucosubstances in a way which seemed to operate by the staining of acidic groups evoked by the oxidation of the tissue sections. These acidic groups were not blocked by the methylation. It was supposed that they are sulphonic acids resulting from sulphur groups (sulphydryls or disulphides) in some mucus glycoproteins. Met-ox-HID and ox-met-HID reactions seemed to stain mucosubstances and mast cells in a similar way but differed from the ox-HID reactions with the manner which could be interpretated to be due to the blocking of free sulphate ester groups in reactions of the former. Met-ox-HID (and ox-met-HID) positive and in goblet cells of small and large bowel.     

Histochemical reactions of gastrointestinal mucosubstances with orcein, high iron diamine and Alcian blue after prior oxidation of tissue sections.

The histochemical orcein reaction (orc) for mucosubstances in tissue samples from the human gastrointestinal tract was compared with PAS, high iron diamine (HID) and Alcian blue reactions at pH 1.0 or 2.5 (AB 1 and AB 2.5). Orc, HID and AB 1 reactions were performed also with prior oxidation of the tissue sections with potassium permanganate or performic acid (ox-orc, ox-HID and ox-AB reactions, respectively). Orc reaction stained mucosubstances similarly to HID and AB 1; only the brush border and goblet cells in the colon were stained. The reactions of the mucosubstances obtained with ox-orc differed from those with PAS, HID, AB 1 or AB 2.5 but were similar to those with ox-HID or ox-AB; the mucosubstances in the brush border and the goblet cells in the colon and small bowel and in the foveolar epithelium of the stomach were strongly stained. Pyloric and cardiac glands were stained faintly with ox-orc but not with ox-HID or ox-AB. Brunner's glands were negative with ox-orc, ox-HID and ox-AB reactions. It was assumed that the orc reaction stains, like HID or AB 1, sulphate groups in epithelial mucosubstances, and that sulphonic acid residues, resulting from oxidation of disulphide groups in the protein core of mucus glycoproteins, are responsible for the ox-orc as well as for the ox-HID and ox-AB reactions.     

Histochemical reactions of gastrointestinal mucosubstances with orcein,high iron diamine and alcian blue after prior oxidation of tissue sections

Summary The histochemical orcein reaction (orc) for mucosubstances in tissue samples from the human gastrointestinal tract was compared with PAS, high iron diamine (HID) and Alcian blue reactions at pH 1.0 or 2.5 (AB 1 and AB 2.5). Orc, HID and AB 1 reactions were performed also with prior oxidation of the tissue sections with potassium permanganate or performic acid (ox-orc, ox-HID and ox-AB reactions, respectively). Orc reaction stained mucosubstances similarly to HID and AB 1; only the brush border and goblet cells in the colon were stained. The reactions of the mucosubstances obtained with ox-orc differed from those with PAS, HID, AB 1 or AB 2.5 but were similar to those with ox-HID or ox-AB; the mucosubstances in the brush border and the goblet cells in the colon and small bowel and in the foveolar epithelium of the stomach were strongly stained. Pyloric and cardiac glands were stained faintly with ox-orc but not with ox-HID or ox-AB. Brunner's glands were negative with ox-orc, ox-HID and ox-AB reactions. It was assumed that the orc reaction stains, like HID or AB 1, sulphate groups in epithelial mucosubstances, and that sulphonic acid residues, resulting from oxidation of disulphide groups in the protein core of mucus glycoproteins, are responsible for the ox-orc as well as for the ox-HID and ox-AB reactions.The study was supported by grants from the Cancer Society of Finland, Foundation of Orion Corporation and from the Paulo's Foundation, Helsinki, Finland     

The Use of Double Staining Techniques for Investigating Bacterial Attachment to Mucopolysaccharide-Coated Epithelial Cells

Double staining techniques were devised to study Escherichia coli attachment to mucopolysaccharide-coated urinary tract epithelial cells. In addition, vital stains were used to distinguish between viable and nonviable epithelial cells. Alcian blue was used to confirm the presence of glycosaminoglycans and periodic acid Schiff was used to distinguish a further group of polysaccharides, proteoglycans, neutral mucosubstances and glycolipids. The staining methods enabled investigations to be carried out concerning the possible importance of mucopolysaccharides in the attachment of bacteria to mucosal surfaces. Staining techniques were also used to investigate whether the presence of a mucopolysaccharide coat is related to epithelial cell viability. The combinations of vital and mucopolysaccharide stains were found to give reproducible results when cell preparations were evaluated by three individuals. Both in vivo and in vitro certain populations of epithelial cells have been found with a large number of bacteria preferentially attached. The double staining techniques described here may help to reveal the nature of these target cells.     

The use of double staining techniques for investigating bacterial attachment to mucopolysaccharide-coated epithelial cells

Double staining techniques were devised to study Escherichia coli attachment to mucopolysaccharide-coated urinary tract epithelial cells. In addition, vital stains were used to distinguish between viable and nonviable epithelial cells. Alcian blue was used to confirm the presence of glycosaminoglycans and periodic acid Schiff was used to distinguish a further group of polysaccharides, proteoglycans, neutral mucosubstances and glycolipids. The staining methods enabled investigations to be carried out concerning the possible importance of mucopolysaccharides in the attachment of bacteria to mucosal surfaces. Staining techniques were also used to investigate whether the presence of a mucopolysaccharide coat is related to epithelial cell viability. The combinations of vital and mucopolysaccharide stains were found to give reproducible results when cell preparations were evaluated by three individuals. Both in vivo and in vitro certain populations of epithelial cells have been found with a large number of bacteria preferentially attached. The double staining techniques described here may help to reveal the nature of these target cells.     

New histochemical techniques for the demonstration of carboxyl groups in mucosubstances

Synopsis The histochemical demonstration of the carboxyl groups of mucosubstances was studied by two methods at the light and electron microscopic levels. Conditions for activating carbohydrate carboxyl groups were elucidated from which a modified carbodiimide reaction procedure was worked out. With this procedure several acid mucosubstances could be demonstrated; some were characterized as sialomucoproteins. The mechanism of the carbodiimide reaction is discussed.A method is also discussed for increasing the electron opacity of Alcian Blue-stained mucosubstances with a sulphide-silver reaction.     

Demonstration eines Alkali PAS-Effektes bei Verwendung von Perjodsäure in niedriger Konzentration

Zusammenfassung Es wird über Untersuchungen berichtet, die zeigen, daß auch ohne Oxidation/Reduktion-Technik ein isolierter KOH PAS-Effekt an epithelialen Mukosubstanzen des Rattendickdarms nachweisbar ist. Die Methode beruht auf der Verwendung stark verdünnter Perjodsäure nach vorausgehender Schnittbehandlung mit äthanolischer KOH. Da sich das mit dieser modifizierten KOH PAS-Reaktion nachweisbare Material als Neuraminidasesensitiv erwies, dürfte die Reaktion auf acylierte Sialinsäurereste in bestimmten Mukosubstanzen des Rattencolon zu beziehen sein.

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Demonstration of an alkali PAS-effect using periodic acid at low concentration

Summary It has been shown that an isolated KOH PAS-effect of epithelial mucosubstances in the colonic mucosae of the rat can be demonstrated also without the conventionally used preliminary oxidation/reduction step. The method is based on the use of strongly diluted periodic acid after previous treatment of tissue sections with ethanolic KOH. As the positive reacting material has been proven to be sensitive against treatment with neuraminidase the modified KOH PAS-reaction should be related to the presence of acylated sialic acid residues in mucosubstances of the colonic mucosae of the rat.

     

Carbohydrate histochemistry of the opossum submandibular and major sublingual glands.

Submandibular and major sublingual salivary glands of the opossum contain histochemically demonstrable neutral mucosubstances, nonsulfated acid musosubstances and sulfomucins. Sialomucins could not be demonstrated conclusively with the methods used in this study. Special serous cells of the opossum submandibular gland contained low concentrations of acidic mucosubstances but no appreciable concentration of neutral mucosubstances was seen. Sulfomucins were not observed in special serous cells. The mucous tubules of the submandibular gland contained high concentrations of neutral mucosubstances. No appreciable acidic mucosubstance was demonstrated in the submandibular gland mucous tubules. Unlike the mucous tubules of the submandibular gland, the major sublingual gland mucous tubules contained high concentrations of both neutral and acidic mucosubstances. The mucous tubules often contained sulfomucin-positive cells interspersed among cells that contained high concentrations of non-sulfated acidic mucosubstance. Marked staining of sulfated acidic mucosubstance was seen only in the major sublingual gland, in both the mucous tubules and in the seromucous demilunes. The seromucous demilunes contained both sulfated and non-sulfated acidic mucosubstances.     

Regional differences in quantities of histochemically detectable mucosubstances in nasal, paranasal, and nasopharyngeal epithelium of the bonnet monkey

Inhaled irritants induce secretory cell hyperplasia in nasal epithelium of animals. To characterize this response histochemically it is first important to know the histochemical character and distribution of epithelial mucosubstance in the normal nasal cavity. An automated image analyzing method was used to detect and quantitate acidic, neutral, and sulfated mucosubstances in the epithelium lining the nasal and paranasal airways of eight bonnet monkeys. Tissue sections 2 micron thick from defined regions of these airways were stained with either alcian blue/periodic acid-Schiff to demonstrate acid and neutral mucosubstances or high iron diamine to demonstrate sulfated mucins. Respiratory epithelium covering maxilloturbinates had the largest volume of stainable mucosubstance per unit surface area of basal lamina, whereas the maxillary sinus epithelium had the least. There was a general anteroposterior increase in the quantity of total epithelial mucosubstance along the septal and lateral walls of the nasal cavity, and there was more acidic than neutral mucosubstance in the posterior nasal airway than in the anterior. Epithelial mucosubstance in the maxillary sinus was predominantly neutral. Therefore, we conclude that there are substantial regional quantitative differences in stainable mucosubstances in the primate nasal epithelium which must be considered when examining nasal mucosa for irritant-induced changes in epithelial mucins.     

Ultrastructural demonstration of cell coat on the cell surfaces of normal human oesophageal epithelium

Synopsis The cell coat in human oesophageal biopsies was studied with Alcian Blue, Ruthenium Red, Safranin O, colloidal iron and the ferrocyanide-osmium tetroxide techniques. Alcianophilic material was found on the cell surface of the basal, prickle cell and functional layers, being most abundant on the superficial cells where it appeared as a continuous coat. In the deeper layers, it tended to have a particulate distribution. Some membrane-coating granules were alcianophilic. Ruthenium Red had a particulate distribution over all cell surfaces. Intercellular debris was also stained. Safranin O produced no staining. Colloidal iron stained the cell coat in a particulate manner. The ferrocyanide-osmium technique showed a uniform filamentous cell coat. The oesophageal epithelial cell coats are, in part, acid mucosubstances which, on the surface cells, may have a protective function.     

Histochemical studies on the mucins of the vertebrate tongues

Summary With a view to augment the understanding of the tongue mucosubstances and their significance in the physiology of taste, tongues of two amphibians were investigated histochemically to determine the distribution and nature of the mucosubstances by employing recent techniques, and the results were considered comparatively with the tongue mucins of other vertebrates and the animal mucosubstances in general. A heterogenous distribution of neutral mucins, sulfomucins and sialomucins in fungiform and filiform papillae, ventral epithelium, lingual glands and connectives tissue could be significantly noted on the basis of which various cell types having specialised mucosubstances were identified in the papillae and the ventral epithelium.The tongue mucosubstances, especially sulfomucins and sialomucins, exhibited inharent heterogenity. Sulfomucins at some sites were hyaluronidas-labile and at other hyaluronidaseresistant, their azurophilia especially at low pH was also different, some being azurophilic and others nonazurophilic. Sialomucins also exhibited such heterogenity, at some sites they were labile to acid hydrolysis and mild methylation but in others they were resistant to the latter, though sialomucins at both sites were sialidase-labile. The mucosubstances localised in the serous glands were highly typical, since they exhibited extraordinary histochemical reactions, they possessed intensely PAS positive reactivity resistant to diastase, hyaluronidase and sialidase, negative to alcian blue both at pH 1 and 2.5, and exhibited no metachromasia at both low and high pH levels, but showed alcianophilia only at high pH levels. Such high pH alcianophilia was sialidase and hyaluronidase resistant but labile to mild methylation. These mucosubstances bear some similarity to those of mammalian parotid.Such heterogenity was reflected at species-specific level, since some interesting speciesspecific differences were observed in mucosubstances of histologically identical cells and tissues of tongues of the two species of amphibians which might assist in the elucidation of phylogenetic importance of mucins.This investigation forms a part of Ph. D. Thesis to be submitted by Mr. M. N. Nalavade to Shivaji University, Kolhapur, under the guidance of Dr. A. T. Varute.     

Histochemistry of mucosubstances in the gallbladder epithelium of the chick embryo

Summary Mucosubstances of epithelial cells of the chick embryo gallbladder were investigated by histochemical methods from days 12–21 of incubation (stages 38–46). On incubation day 14, only neutral mucins were detected; on day 15, neutral and sulfated mucosubstances were observed in the epithelial cells that invaginate the underlying mesenchyme. In the same sites, at day 16 of incubation, neutral, carboxylated and sulfated mucins were seen. From the 17th day of incubation until hatching, neutral, carboxylated and sulfated mucosubstances were present in the surface cells and in the cells lining the epithelial invaginations. During this period, the chemical characteristics of the secretory material are similar to those observed by Yamada and Hoshino (1972) in the fowl.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Studies on the alimentary canal of amphipods: histochemistry of cephalic mucous glands in Talorchestia martensii (Weber) (Crustacea: Amphipoda)

With a view to analyse the chemical nature and the probable functional significance of the cephalic mucous glands the mucosubstances secreted and elaborated by these glands were investigated. All recent and standard histochemical techniques were employed. These reactions revealed that the three groups of glands namely the oesophageal, lateral and maxillipede groups are charged with the task of secreting both acid and neutral mucopolysaccharides. Of these three, maxillipede groups are elaborating most of the neutral mucopolysaccharides and the other two groups are mainly involved in elaborating acid mucosubstances and to a little extent neutral mucosubstances. The acidic nature of the mucosubstances is partly due to hyaluronic acid and partly due to sialic acid. This was confirmed by hyaluronidase and neuraminidase treatment (digestion tests). The glands are also involved in secreting glycoproteins which was evidenced by their positivity to alcian blue/naphthol yellow and Congo red reactions. Entanglement of food and provision of fluid vehicle for lubrication as well as to achieve the desired consistency for digestion may be given as chief functions.     

Histochemistry of mucosubstances in the gallbladder epithelium of the chick embryo

Mucosubstances of epithelial cells of the chick embryo gallbladder were investigated by histochemical methods from days 12-21 of incubation (stages 38-46). On incubation day 14, only neutral mucins were detected; on day 15, neutral and sulfated mucosubstances were observed in the epithelial cells that invaginate the underlying mesenchyme. In the same sites, at day 16 of incubation, neutral, carboxylated and sulfated mucins were seen. From the 17th day of incubation until hatching, neutral, carboxylated and sulfated mucosubstances were present in the surface cells and in the cells lining the epithelial invaginations. During this period, the chemical characteristics of the secretory material are similar to those observed by Yamada and Hoshino (1972) in the fowl.     

Alcianblue/PAS or PAS/alcianblue?

Summary The combined alcian blue (AB)/PAS technic is widely used for the detection and characterization of mucosubstances in tissue sections. Mostly the sequence AB/PAS is used, occasionally also the reserved sequence PAS/AB. The present study shows clearly that the sequence of the combined technic, i.e. AB/PAS or PAS/AB is substantially influencing the results. So it could be demonstrated that by using the combination PAS/AB originally PAS-positive and AB-negative reacting mucosubstances become AB-posltive. This could be caused by periodic acid oxidation followed by addition of hydrogen sulfite to aldehyde group thus providing secondary basophilic resp. AB positive material.