Histochemical alterations of mucin in normal colon, inflammatory bowel disease and colonic adenocarcinoma.

Loss of sialic acid o-acyl substitutions in colonic mucus was studied using specific histochemical techniques in individuals with a variety of large-bowel diseases and in a control population. Changes found included a focal or field (diffuse) loss of side-chain substitutions which were qualitatively similar in all groups studied. The results were tested statistically using a variety of assumptions that field and/or focal loss of o-acyl substitution may be either abnormal or a normal variant. No statistically significant differences in the prevalence of substitutions were detected between normal males and females or between normal individuals aged 0-29 years and 30-80 years. Significant differences were found between ascending and descending colon in both normal individuals and in the non-neoplastic mucosa of patients with cancer. There were also significant differences between the normal descending colon and cases with cancer of the descending colon. These differences seem unlikely to be due to non-specific factors, since for most assumptions there were also differences between colons containing cancer and those from patients with inflammatory bowel disease. In agreement with the work of other investigators, it seems likely that focal loss of o-acetylation results from an acquired gene mutation. It is not clear whether or not this plays a role in carcinogenesis.

Customized implant abutments: technical notes.

Custom-fabricated angulated abutments can be of value when implant angulation or position jeopardizes esthetics and/or function and makes retrievability difficult. A wax pattern is prepared over a castable pattern or gold cylinder. After the casting is completed, a secondary screw channel is created in the angulated abutment to retain the superstructure and the definitive prosthesis. The rationale for the use of customized angulated abutments is described. Technical procedures are presented that allow for modifications of individual cases.

Mechanism of connective tissue techniques. I. The effect of dye concentration and staining time on anionic dye procedures.

Anionic dye connective tissue procedures were performed by staining for 5 min and 24 h with (a) 0.00018 M and 0.0018 M solutions of 28 dyes, and 0.018 M solutions of 21 dyes in saturated picric acid (SPA), and (b) 0.0018 M and 0.018 M solutions of 20 dyes in 1% (w/v) phosphomolybdic acid (PMA). The staining obtained with dyes in SPA was classified as selective (no cytoplasmic staining), moderately selective (traces of cytoplasmic staining) and non-selective (all other staining patterns). The staining of collagen and cytoplasm with dyes in PMA was separately classified on a scale of 1-5 (1 = no staining, 5 = maximum staining). The selectivity of the staining obtained with SPA with solutions of dyes at concentrations of 0.00018 M and 0.0018 M, and both staining times, was correlated (p < 0.001) with an empirical sulphonic acid constant (SAC) defined as the (number of dye sulphonic acid groups/dye molecular weight) x 10(3). Correlation with molecular weight was poor and was significant only when staining was performed with 0.00018 M dye solutions for 24 h. The dyes were divisible into three groups: group 1 (selectivity independent, or almost independent of staining time), group 2 (selective to moderately selective when staining was performed for 5 min), and group 3 (non-selective). The SAC of the group 1 dyes differed significantly from those of the group 2 and 3 dyes. Selectivity was essentially lost at dye concentrations of 0.018 M. The staining with acidic dyes (no amines or substituted amines) in PMA differed significantly (p < 0.001) from that obtained with amphoteric dyes (containing basic substituents).(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical identification of 9-O-acyl sialic acids: studies of bovine submandibular and rat sublingual gland and human colon.

Formalin-fixed tissue specimens containing glycoproteins with side chain O-acylated sialic acids were used to re-examine, compare and evaluate the usefulness of three methods based on the periodic acid-borohydride reduction-saponification-periodic acid-Schiff sequence (PA-Bh-KOH-PAS) for the histochemical identification of 9-O-acyl sialic acids (9-O-AcSA). Method I, modified from Veh et al. (1979), involved a comparison of the staining intensely obtained when both oxidation steps of the PA-Bh-KOH-PAS sequence were carried out with the selective oxidation technique of Volz et al. (1987) with that obtained when the initial oxidation step was carried out with 0.5 M periodic acid for 4 h at room temperature. Methods II and III, modified from Reid et al. (1978), involved an initial PA-Bh step under oxidation conditions that cleaved all the vicinal diols associated with neutral sugars and side chain unsubstituted and 7-O-acyl sialic acids. The Schiff staining obtained following subsequent re-oxidation with either 0.5M (method II) or 1% periodic acid (method III) for 4 h at room temperature (PA-Bh-PAS procedure) identifies 9-O-AcSa.(ABSTRACT TRUNCATED AT 250 WORDS)

A histological study of the Shionogi adenocarcinoma 115 grown in male and female mice.

We have previously demonstrated that growth rate and morphology differ between androgen-responsive Shionogi mouse mammary tumours maintained in male and female mice. Furthermore, we can modulate the growth rate of these tumours in male mice by exposing the mice to psychosocial stressors. In the present study, we were interested in determining if tumours in male mice with a comparable growth rate to that in females, also had a morphology similar to that in females. SC115 tumours were examined using histochemical and immunohistochemical techniques. Tumours in male mice were easily distinguishable from tumours in female mice regardless of growth rate. Tumours maintained in female mice contained osteoid-like regions which stained positive for sialic acid and sulphate moieties. No such regions were observed in any of the tumours from male mice. In addition, although all tumours contained MSA (muscle specific actin)-positive and S100 protein-positive cells, these regions were more extensive in the tumours of female mice. This study suggests that tumour growth rate and morphology are independently regulated by the host environment.

Histochemical classification based upon reaction types and its application to carbohydrate histochemistry.

A method of classification is presented, which divides histochemical visualization reactions into categories based on general reaction types. This scheme is dependent upon the reaction between two elements, the substrate and the probe. The substrate represents a tissue component(s) with one or more reactive groups that can combine directly with the probe. Alternatively, the substrate reactive groups are chemically modified (activation) with a suitable reagent before reaction with the probe. Probes are of three types: those that yield a coloured product, those that result in a colourless product, and those that produce a coloured product only after a further reaction. Methods used in carbohydrate histochemistry are divided into one, two and three probe reactions. Two probe reactions are further subdivided into sequences involving one or two coloured products (one and two dye sequences); three probe reactions into sequences involving one, two or three coloured products (one, two and three dye sequences). This classification permits the rationalization and organization of methods, and provides a framework for the examination of existing methods and the development of new ones.

Histochemical studies of intestinal epithelial goblet cell glycoproteins during the development of the human foetus.

Histochemical studies performed on specimens of intestine from 12 to 37-week human foetuses showed that the epithelial glycoproteins of the goblet cells of the small intestine are non-sulphated sialoglycoproteins containing neutral sugar (hexose, 6-deoxy hexose or N-acetyl hexosamine residues with Periodic acid-Schiff (PAS) reactive vicinal diols), sialic acids without O-acyl substituents, smaller and variable quantities of sialic acids with O-acyl substituents at positions C8 or C9 (or with two or three side chain substituents) and O-acyl sugars (neutral sugars with an ester substituent blocking PAS reactivity). In the lower small intestine glycoproteins containing 8 (or 9)-O-acyl sialic acids are first observed in goblet cells at the tips of the villi. As the foetus matures their quantity increases and they are found in goblet cells located along the length of the villi. Smaller quantities of O-acyl sialic acids and traces of O-acyl sugars occur in the goblet cells of the upper small intestine. The colonic goblet cells contain sulphosialoglycoproteins of two types. The first type, found in the majority of specimens, contains O-sulphate ester, neutral sugar, O-acyl sugars and 8 (or 9)-O-acyl sialic acids. The second type contains O-sulphate ester, neutral sugars, and sialic acids which are either without side chain O-acyl substituents or are a mixture of such acids and 8 (or 9)-O-acyl sialic acids; O-acyl sugars are reduced or absent.(ABSTRACT TRUNCATED AT 250 WORDS)

The histochemical specificity of high iron diamine-alcian blue.

The specificity of the High Iron Diamine-Alcian Blue pH 2.5 (HID-AB 2.5) procedure was examined in tissue sites containing sialogycoproteins alone or differing proportions of sialo- and sulphosialoglycoproteins. Studies with HID in differing final concentrations of hydrochloric acid or sodium chloride confirmed that staining is dependent upon both the pH and the ionic strength of the dye bath and demonstrated a marked heterogeneity in the pKa of the anionic groups of sialosulphoglycoproteins. Use of the sequence High Iron Diamine-Alcian Blue pH 1.0 demonstrated that complete or almost complete staining of O-sulphate esters occurred when HID was prepared in water (final pH 1.3). However, under these conditions HID-AB 2.5 was shown to be non-specific because only black HID staining was observed in sites containing large quantities of sialic acids. This non-specificity was due either to the masking of Alcian Blue staining by HID and/or the black HID staining of anionic groups other than sulphate. These results may account for some of the conflicting data obtained by different groups of investigators who have studied 'transitional mucosa' in human colonic diseases. Caution should be used in drawing conclusions from the use of HID-AB 2.5 without confirmatory evidence from other more specific procedures.

Histochemical changes in cervical mucus-secreting epithelium during the normal menstrual cycle.

Recently developed specific histochemical techniques were applied to sections of human endocervix to investigate possible cyclic changes in stainable intracellular mucin glycoproteins. During the secretory phase, there is a decrease in the sialic acid/O-sulfate ester ratio, a decrease in the neutral sugar/acidic sugar ratio and increased amounts of O-sulfate ester, compared with during the proliferative phase. Endocervical epithelia from prepubertal, pregnant, and postmenopausal females also show decreased sialic acid/O-sulfate ester and neutral sugar/acidic sugar ratios, compared with during the proliferative phase. The authors conclude that proliferative phase mucin is chemically different from secretory phase mucin, and that this difference may be significant in allowing sperm penetration in the periovulatory period.

Histochemical studies of the colonic epithelial glycoproteins of the normal rabbit.

Two general classes of glycoproteins have been identified in the colonic epithelial cells of New Zealand white rabbits. Each is associated with an ultrastructurally distinct secretory cell. The first of these classes is found in cells, termed vesiculated columnar cells, characterized by electron-translucent vesicles, a small rough endoplasmic reticulum-Golgi complex and prominent microvilli. The glycoproteins of the vesiculated cells contain abundant O-sulphate ester, sialic acids with ester substituents at positions C-8 or C-9 (or with two or three side chain substituents) and neutral sugars with vicinal diols whose periodate oxidation is prevented by an O-acyl ester substituent(s). The second class of glycoproteins occurs in goblet cells characterized by electron-dense vesicles, an abundant rough endoplasmic reticulum, a well-developed Golgi apparatus and few, if any, microvilli. Goblet cells along the entire length of the crypts contain neutral sugars with periodate-oxidisable vicinal diols and a ferriferricyanide-reactive component. Cells in the upper halves of the crypts also contain components that are sulphated, Schiff-reactive and acid-fast. In the lower halves of the crypts, the goblet cells contain smaller quantities of the above components plus sialic acids, some of which possibly have an O-acyl substituent located at position C-8 or C-9 (or which have two or three side chain O-acyl substituents). It is suggested that the function of the glycoproteins from the vesiculated columnar cells is protective and that from the goblet cells is lubricative.

Simple procedure for assessing relative quantities of neutral and acidic sugars in mucin glycoproteins: its use in assessing cyclical changes in cervical mucins.

A simple histochemical procedure for assessing relative amounts of neutral and acidic sugars in mucin glycoproteins, and its application in the study of cyclical changes of human cervical mucins, is described. This procedure, the saponification/selective periodate oxidation/borohydride reduction/alcian blue pH 2.5/periodic acid Schiff (KOH/PA*/Bh/Ab 2.5/PAS) method, uses a selective oxidation step to remove the PAS positivity of sialic acid; thus only neutral sugars stain positively with PAS, and acidic sugars (O-sulphate esters and carboxyl groups) stain with alcian blue. This differs from the KOH/Ab/PAS technique which stains sialic acid residues with both alcian blue and PAS. Applying the KOH/PA*/Bh/Ab 2.5/PAS technique to the study of cyclical changes of human cervical mucins, a decreased neutral:acidic sugar ratio in the secretory phase mucins compared with those of the proliferative phase was found. This difference was not seen with KOH/Ab/PAS staining in the same cases. The techniques and reagents used in this procedure can be easily applied in a clinical histopathology laboratory.

A new method for the histochemical demonstration of O-acyl sugars in human colonic epithelial glycoproteins.

A new general method has been developed for the specific histochemical identification of O-acyl sugars in any epithelial glycoprotein. These sugars include hexose, 6-deoxyhexose and N-acetylhexosamine with an ester substituent(s) located on a potential vicinal diol(s). In the procedure reported [the periodic acid-borohydride reduction-saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (PA-Bh-KOH-PA*-Bh-PAS) method] the initial PA-Bh treatment renders vicinal diols located on either sialic acid or neutral sugars PAS unreactive. In the subsequent steps ester substituents are removed from both O-acyl sugars and O-acyl sialic acids by saponification (KOH), sialic acid vicinal diols are selectively removed by the PA*-Bh sequence and O-acyl sugars are stained with the PAS technique. This method has the advantage that the results are obtained with a single section and the results are either positive or negative. Consequently, it is superior to the three indirect methods investigated because it does not require an observer to compare the intensity or the shade of the staining obtained with serial sections. Using the PA-Bh-KOH-PA*-Bh-PAS method we have demonstrated, for the first time, that O-acyl sugars occur in the epithelial goblet cell glycoproteins of adult human colon. The effect of the presence of O-acyl sugars on the interpretation of a number of other methods for the histochemical investigation of glycoproteins is discussed. It is recommended that the results obtained with the PA-Bh-KOH-PA*-Bh-PAS method be evaluated before histochemical procedures for the investigation of neutral sugars and O-acyl sialic acids are selected.

Histochemical studies of epithelial cell glycoproteins in normal rat colon.

Two general classes of glycoproteins have been identified in the colonic epithelial cells of the Sprague Dawley rat. Glycoproteins belonging to the first of these classes contain sialic acids both with and without side chain o-acyl substituents, abundant o-sulphate ester and 'neutral sugars' (hexose, 6-deoxyhexose or N-acetyl hexosamine residues) with oxidisable vicinal diols and are located in the goblet cells of the descending colon and in goblet cells populating the upper halves of the crypts of the ascending colon. In the descending colon, the sulphosialoglycoproteins in the goblet cells in the base of the crypts contain sialic acids without side chain o-acyl substituents. It appears that as these cells migrate up the crypts, there is o-acylation of the side chains of the sialic acids of the glycoproteins and an increase in the quantity of 'neutral sugars' without a corresponding increase in sialic acid. Glycoproteins with similar properties to those of the goblet cells of the upper halves of the crypts of the descending colon, but containing less sulphate, are found in the goblet cells of the upper half of the crypts of the ascending colon. The second general class of glycoproteins contain sialic acids all, or almost all of which, are substituted at position C8 and only relatively small quantities of sulphate. They are located in the mucous cells of the descending colon, the deep crypt secretory cells of the ascending colon and the columnar absorptive cell brush border.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological and histochemical changes in intestinal mucosa in the reserpine-treated rat model of cystic fibrosis.

The present investigation was undertaken to determine whether or not there are histochemical and morphological changes in the intestine of the chronically reserpine-treated rat, an animal model of cystic fibrosis. Male Sprague-Dawley rats were given seven daily intraperitoneal injections of reserpine at dosages of 0.5 (n = 6) or 1.0 mg/kg body weight (n = 6). Control groups consisted of parfed solvent-injected (n = 6), solvent-injected (n = 4), and saline-injected animals (n = 4). Light microscopic histochemical procedures and morphological assessments were performed on sections of "Swiss rolls" of small and large intestine. Chronic reserpine treatment caused an increase in the sulfation of goblet cell mucin in the small intestine without accompanying morphological change; these findings resemble those reported in cystic fibrosis. No qualitative differences in mucin were found in the large intestine but there was an increased number of goblet cells in the surface epithelium and retention of mucus within these cells. Similar although less marked changes were noted in the parfed controls suggesting that those observed in the treated groups may be due, in part, to the reserpine-induced anorexia. The resemblance between the changes in the small intestine of the reserpine-treated rat and those observed in CF patients supports the contention that the chronically reserpine-treated rat is suitable as a model of cystic fibrosis.