High Iron Diamine-alcian Blue Staining Research Articles

DOP35 High-fat diet reduces gut microbiota-derived metabolite indole-acetic acid and aggravates colitis

Abstract Background It is widely accepted that high-fat diet (HFD) has been considered as one of the risk factors of inflammatory bowel disease(IBD), while the mechanism is little known. We aimed to examined whether dietary high fat promotes colonic inflammation through modulating gut microbial tryptophan metabolites. Methods The C57BL/6 female mice were fed with chow diet (12kcal% fat) or HFD (60kcal% fat) for 2 weeks. Fresh fecal samples were collected before administration of 2% DSS. LC-MS/MS analysis was performed to detect fecal microbial tryptophan metabolites. Next, 8-week-old mice were administrated 3-indoleacetic acid (IAA) for 7 days prior to 2.5% DSS treatment. The severity of colitis was assessed and the colonic sections were collected. RNA sequencing was performed to analyze the differential genes in intestinal tissue of mice fed with IAA or not. Real-time PCR and Western blotting were performed to detect the transcription and protein expression of mucin sulfation-related genes. High iron diamine-alcian blue (HID-AB) staining of colon tissues and LS174T cells was used to detect the abundance of sulfated mucin. Additionally, the Chromatin immunoprecipitation (ChIP) sequencing is performed to confirm the binding of aryl hydrocarbon receptor(AHR) to 3’-phosphoadenosine 5’-phosphosulfate (PAPS) synthase 2 (PAPSS2). Results Short-term HFD increased the susceptibility to colitis of mice. Mice fed with HFD have impaired tryptophan metabolism with remarkable lower level of IAA. IAA supplementation resulted in ameliorative colitis symptoms. Mechanistically, the transcriptome sequencing detected that IAA supplementation enriched the the cytosolic sulfonation pathway and significantly increased the expression of PAPSS2 of colon tissue, a rate-limiting enzyme on the mucin sulfation pathway. Next, in vivo and in vitro experiments verified IAA increased the expression of PAPSS2 and its downstream genes and promoted the mucin sulfation in golgi apparatus of colon through AHR. ChIP sequencing and ChIP-PCR verified IAA can enhance the binding of AHR to the promoter region of PAPSS2 , which further promoted the expression of PAPS transporter 2 (PAPST2 or Slc35b3) and ultimately promotes the mucin sulfation. Conclusion HFD altered the gut microbial tryptophan metabolites and disrupt the protective effect of IAA on the intestinal barrier. IAA could relief colitis through AHR-PAPSS2-PAPST2-mucin sulfation axis, which may present a potential approach for precise prevention and treatment of IBD.

Quantitative Analysis of Mucin Expression Using Combined Alcian Blue-Periodic Acid Schiff (AB-PAS) Stain and Combined High Iron Diamine-Alcian Blue (HID-AB) Stain and the Correlation With Histomorphological Score in Chronic Calculous Cholecystitis.

Introduction Chronic calculous cholecystitis is the most common and important contributing factor for cholecystectomy across the country, with a prevalence of 2-29%. Cholesterol supersaturated bile plays a major role in stone formation. It is very essential to identify the pathogenesis of stone formation in order to prevent its formation. This study is aimed to evaluate histomorphological features of chronic calculous cholecystitis and to quantitatively evaluate alteration in mucin expression using the combined Alcian blue-periodic acid Schiff (AB-PAS) stain and the combined high iron diamine Alcian blue (HID-AB) stain, to correlate with each other and also with biochemical features of gall stones. Methods A cross-sectional study of 64 chronic calculous cholecystitis were taken for histomorphological assessment and grading using Hematoxylin and Eosin (H&E) stainand Masson's trichrome stains.Expression of the type of mucins wasanalyzed using histochemical stains by a standardized scoring system. Results A significant positive correlation was observed between an increase in grades of inflammation and fibrosis with an increase in the quantity of sialomucin and neutral mucin in the deep layers of epithelium, and a significant negative correlation was observed between an increase in grades with a decrease in acidic mucin and sulfomucin of both superficial and deep epithelium except sulfomucin in fibrosis. No significant correlation was obtained with muscle thickness, adipose tissue deposition, and epithelial hyperplasia. A higher frequency of mixed-type stones was associated with severe inflammation. Conclusion Inflammation and fibrosis were strongly correlated with quantitative alteration and reversal of mucin composition in chronic cholecystitis; hence we conclude that these features play a significant role in the pathogenesis of stone formation. Using Combined AB(2.5pH)-PAS stain and Combined HID-AB(2.5 pH) stain to detect mucin hypersecretion and composition of altered mucin is relatively accurate and cost-effective rather than performing costly immunohistochemical (IHC) markers.

Comparison of Mucin Patterns in Colonic Pathologies by Histochemistry

Introduction: High Iron Diamine Alcian blue (HID-AB) is a well-established technique for demonstrating colonic acid mucins namely sulphomucins (brown- black) and sialomucins (blue). In certain colonic pathologies expression of sulphomucin, the predominant mucin in normal colonic epithelium is altered. Objective: To compare the changes in the pattern of mucin staining in, adenocarcinoma (AC), normal colonic epithelium, solitary rectal ulcer (SRU), transitional zone mucosa (TZ) (i.e.: normal non dysplastic mucosa adjacent to adenocarcinoma) and in ulcerative colitis (UC). Method: AC-21, normal colonic epithelium-30, SRU-23, TZ-20, UC-29 biopsies were stained with the HID-AB stain. Two investigators assessed the percentages of mucin staining by counting the number of cells staining for sulphomucin and sialomucin in an area of colonic epithelium measuring 1mm in length. The differences in these 5 categories were assessed for statistical significance using the one-way ANOVA test and a Post Hoc Comparison of the mean values was carried out to establish which groups were different. Results: ACs showed no sulphomucin or sialomucin staining while normal colonic epithelium showed nearly 100% sulphomucin staining. The percentage mean value for sulphomucin staining was AC-12.47%, normal colonic epithelium - 96.7%, SRU-35.7%, TZ-48.98%, and UC –75.43%. Hence the differences between these categories reached a level of statistical significance except between TZ and SRU. Conclusion: The patterns of mucin staining observed with HID/AB staining included relatively similar changes in mucin staining in the TZ and SRU, the loss acid mucin staining in AC and the relatively mild degree of sulphomucin loss in UC. Though there are significant differences in mucin staining between some of these colonic pathologies practical problems were encountered in using HID/AB as a diagnostic tool.

The abundance of Akkermansia muciniphila and its relationship with sulphated colonic mucins in health and ulcerative colitis

Akkermansia muciniphila utilises colonic mucin as its substrate. Abundance is reduced in ulcerative colitis (UC), as is the relative proportion of sulphated mucin in the mucus gel layer (MGL). It is unknown if these phenomena are related, however reduced sulphated mucins could contribute to reduced abundance, owing to a lack of substrate. The aim of this study was to quantify A. muciniphila within the MGL and to relate these findings with markers of inflammation and the relative proportion of sulphomucin present. Colonic biopsies and mucus brushings were obtained from 20 patients with active UC (AC), 14 with quiescent UC (QUC) and 20 healthy controls (HC). A. muciniphila abundance was determined by RT-PCR. High iron diamine alcian-blue staining was performed for histological analysis. Patients with AC had reduced abundance of A. muciniphila compared to HC and QUC. A positive association was found between A. muciniphila abundance and higher percentage of sulphated mucin (ρ 0.546, p = 0.000). Lower abundances of A. muciniphila correlated with higher inflammatory scores (ρ = 0.294 (p = 0.001)). This study confirms an inverse relationship between A. muciniphila and inflammation and a positive association between A. muciniphila abundance and percentage of sulfated mucin in the MGL.

Expression of GCRG213p, LINE-1 endonuclease variant, significantly different in gastric complete and incomplete intestinal metaplasia

BackgroundIntestinal metaplasia (IM) of the gastric mucosa is classified as complete (Type I) and incomplete IM (Type II and III) subtypes, which showed significantly different risk for developing to gastric adenocarcinoma (GAC). GCRG213, a variant of L1-endonuclease (L1-EN), first identified in our lab, was upregulated in GAC tissue. However, the relationship between GCRG213 and IM subtypes is not clear. Our study explored the association of GCRG213 protein (GCRG213p) with IM subtypes.MethodsGastric cancer and/or para-tumor tissue samples were collected from 123 patients who underwent gastrectomy for intestinal type gastric adenocarcinoma. The subtypes of IM were characterized with Alcian blue-periodic acid-Schiff and High Iron Diamine-Alcian blue staining methods. Immunohistochemistry of GCRG213p was performed, and its expression in gastric adenocarcinoma and para-tumor tissue including dysplasia, IM, and normal mucosa were analyzed.ResultsGCRG213p was expressed in 48.94% IM, 57.14% dysplasia and 55.32% GAC, respectively. GCRG213p expression was higher in well and moderately differentiated adenocarcinoma (P = 0.037). In IM glands, GCRG213p expressed mainly in the cytoplasm of absorptive enterocytes with defined brush borders, but not in goblet cells. The expression of GCRG213p in type I IM (90.00%) was significantly higher than that in type II (36.36%) and type III (25.00%) (P < 0.001). In normal gastric mucosa, GCRG213p was exclusively positive in the cytoplasm of gastric chief cells.ConclusionsThe expression of GCRG213p in complete IM was significantly higher than in incomplete IM, which implies that GCRG213p may play a role on the developing of IM to adenocarcinoma. GCRG213p was exclusively expressed in chief cells, suggesting that it might be involved in cell differentiation from the chief cells to IM.

Effect of Different Doses of Herbal Cake-partitioned Moxibustion on Histopathological Changes of Colon Tissue in Ulcerative Colitis Rats

To observe the effect of different-doses of herbal cake-partitioned moxibustion (Moxi) on histopathological changes of the damaged colon tissue in rats with ulcerative colitis (UC), so as to select an optimal dosage of Moxi in the treatment of UC. Sixty-three male Sprague Dawley (SD) rats were randomized into 7 groups: normal control, model, medication, and 1, 2, 3 and 4 moxa-cone Moxi (n＝9 rats per group). The UC model was developed by subcutaneous injection of emulsion (1 mL) containing colon mucosa-prepared protein suspension and complete Freund's adjuvant into the toes, groin and back. On the 38th day, enema of 3% formalin and the aforementioned emulsion was used. Herbal-cake (composed of monkshood, cinnamon, etc.) partitioned Moxi with 1 or 2 moxa-cones (about 5 min/cone) was applied to bilateral "Tianshu" (ST 25) once daily or once every other day. The rat's general conditions (diet, movement, response ability, stool, and body weight) were observed, and histopathological changes (adhesion, ulcer formation and inflammation) of colon tissues were examined after hematoxylin-eosin (HE) staining, and scored (histopathological score). Gross score was given according to the severity of adhesion, ulcer formation and inflammation of colonic tissues under stereo microscope. The average optical density (AOD) values of colonic mucins were detected after periodic acid-schiff (PAS) staining, and those of the sulfated mucus content detected after high iron dia-mine-alcian blue (HID-AB) staining. Compared with the normal group, rats in the model group presented loose stool, or with pus and blood, and slowly increased body weight (P<0.01), obvious congestion of colon with ulcer spots or continuous superficial ulcer regions which had irregular glandular cavities, swelling and serious inflammatory infiltration in lamina propria and submucosa, and increased score of colon tissue damage (P<0.01). PAS and HID-AB staining showed a marked decrease of AOD values of colonic mucins and sulfated mucus in the model group relevant to the normal control group (P<0.01), suggesting a reduction of mucus secretion of intestinal glands. Following the intervention, rats in the Moxi groups presented an increase of the body weight, formed feces, and an improvement of the damaged colon tissues as mucosal healing and inflammatory reduction, and a marked decrease of the damage score relevant to the model rats. No significant differences were found in the gross scores among the medication, 1, 2, 3 and 4 moxa-cone groups (P>0.05). The histopathological scores were significantly lower in the 1 and 4 moxa-cone Moxi groups than in the medication group (P<0.05, P<0.01); and significantly lower in the 1, 3, 4 moxa-cone Moxi groups than in the 2 moxa-cone Moxi group (P<0.05, P<0.01). PAS staining showed a significant increase of the AOD values of colonic mucins in the 1, 2, 3 and 4 moxa-cone and medication groups relevant to the model group (P<0.01); and the AOD values of colonic mucins in the 1, 3, 4 moxa-cone Moxi groups were significantly increased than that in the 2 moxa-cone Moxi group (P<0.05, P<0.01). HID-AB staining showed that the AOD values of sulfated mucus content were significantly higher in the 2 and 4 moxa-cone Moxi groups than in the 3 moxa-cone Moxi group (P<0.01). The two-level two-factor factorial analysis showed an interaction existed between the moxa-cone number and Moxi frequency in reducing the gross score and histopathological score and in facilitating colonic mucin and sulfated mucus secretion. The histopathological score of the 4 moxa-cone Moxi group was significantly lower than that of the 2 moxa-cone Moxi group (P<0.05), and the sulfated mucus content was significantly higher in the 4 moxa-cone group than in the 3 moxa-cone group (P<0.01). The effect of Moxi given on alternate days was superior to that of daily Moxi in improving colonic histological damage. Herbal cake-partitioned moxibustion at ST 25 can promote repair of the damaged colonic tissue and secretion of mucin in UC rats. The number of moxa cones and intervention frequency affect the efficacy of Moxi in improving histopathological changes. The Moxi intervention on alternate days and with 2 moxa-cones every time is recommended.

Development of a total colectomy and ileorectal anastomosis rat model to evaluate colonic metaplasia

BackgroundUlcerative colitis is an idiopathic inflammatory condition of the colon that may require surgical intervention including proctocolectomy and either ileal pouch-anal anastomosis or in the pediatric population, low ileorectal anastomosis (IRA). Often, subsequent physiologic alteration (or colonic metaplasia) occurs in the anastomosed small bowel that includes changes in mucin content, villous blunting, and increased expression of WNT5A, a marker of colonic crypt regeneration. We developed a rat low IRA model to assess and study the development of colonic metaplasia. Materials and methodsWe subjected male Sprague–Dawley rats (n = 17) to total colectomy and low IRA surgery and evaluated healing periodically by endoscopic evaluation. The ileum upstream of the anastomosis was assessed by hematoxylin and eosin staining, and the mucin content was measured by high iron diamine-Alcian blue staining. Wnt5a transcripts were quantified by reverse transcription and quantitative polymerase chain reaction at the 8-wk study end point. ResultsAlthough no gross endoscopic evidence of inflammation was seen throughout the course of the study, colonic metaplasia in the small bowel was detected in 7 out of 10 (70%) rats at the study end point. In rats with colonic metaplasia, enhanced expression of Wnt5a was evident at the study end point compared to levels in the terminal ileum at the time of surgery. ConclusionsWithin 4-8 wk, the majority of rats subjected to IRA developed colonic metaplasia defined by villous blunting, changes in mucin content, and increased expression of Wnt5a. This model provides a method to study small bowel colonic metaplasia.

Mucin-Related Molecular Responses of Bronchial Epithelial Cells in Rats Infected with the Nematode Nippostrongylus brasiliensis.

Although mucins are essential for the protection of internal epithelial surfaces, molecular responses involving mucin production and secretion in response to various infectious agents in the airway have not been fully elucidated. The present study analysed airway goblet cell mucins in rats infected with the nematode Nippostrongylus brasiliensis, which migrates to the lungs shortly after infection. Goblet cell hyperplasia occurred in the bronchial epithelium 3–10 days after infection. The high iron diamine-alcian blue staining combined with neuraminidase treatment showed that sialomucin is the major mucin in hyperplastic goblet cells. Immunohistochemical studies demonstrated that goblet cell mucins were immunoreactive with both the major airway mucin core peptide, Muc5AC, and the major intestinal mucin core peptide Muc2. Reverse transcription real-time PCR studies demonstrated upregulation of gene transcription levels of Muc5AC, Muc2, the sialyltransferase St3gal4, and the resistin-like molecule beta (Retnlb) in the lungs. These results showed that nematode infection induces airway epithelial responses characterised by the production of sialomucin with Muc5AC and Muc2 core peptides. These mucins, as well as Retnlb, might have important roles in the protection of mucosa from migrating nematodes in the airway.

Distribution and severity of spontaneous lesions in the neuroepithelium and Bowman’s glands in mouse olfactory mucosa: age-related progression

Age-related changes were examined in the distribution and severity of spontaneous lesions in the neuroepithelium and Bowman's glands in mouse olfactory mucosa. The olfactory mucosa of female ICR mice at postnatal ages from 10 days to 16 months were investigated histologically by hematoxylin and eosin staining, high-iron diamine-Alcian blue (HID-AB) staining, and immunohistochemistry for olfactory marker protein (OMP), betaIII tubulin (betaIIIT), and Ki67. The lesions in the neuroepithelium and Bowman's glands were quantitatively assessed by morphometric analyses of sections stained with anti-OMP antibody or HID-AB. The first appearance of neuroepithelial abnormality was observed in the dorsomedial portion of the olfactory mucosa in 5-month-old mice. The distribution and severity of lesions progressed with increasing age. In mildly affected epithelium in which OMP-positive olfactory receptor neurons (ORNs) were present but in smaller amounts, the numbers of betaIIIT-positive and Ki67-positive neuroepithelial cells tended to be increased, indicating that neurogenesis was upregulated in these areas. In contrast, severely affected epithelium in which OMP-positive ORNs were virtually absent showed high variability in the numbers of betaIIIT- and Ki67-positive cells among the areas examined, probably reflecting differences in the capacity of the basal cells remaining in the affected area to generate new neuronal cells. Histological analysis with HID-AB revealed that spontaneous lesions in Bowman's glands also occurred in aged mouse olfactory mucosa. Lesions in the neuroepithelium and underlying Bowman's glands tended to be spatially co-localized, suggesting a close association between pathogeneses in these two structures. Moreover, lesions in Bowman's glands were associated with changes in the biochemical composition of mucus on the olfactory mucosa. This information should prove useful in improving the understanding of the pathogenetic mechanisms underlying age-related changes in the peripheral olfactory system.

Indistinguishable cellular changes in gastric mucosa between Helicobacter pylori infected asymptomatic tribal and duodenal ulcer patients.

To investigate the changing pattern of different histological parameters occurring in the stomach tissue of Helicobacter pylori (H pylori) infected tribal populations and duodenal ulcer patients among ethnic Bengalis and correlation of the genotypes of H pylori with different histological parameters. One hundred and twelve adult individuals were enrolled into this study between 2002 and 2004. Among them, 72 had clinical features of duodenal ulcer (DU) from ethnic Bengali population and 40 were asymptomatic ethnic tribals. Endoscopic gastric biopsy samples were processed for histology, genotyping and rapid urease test. Histologically, haematoxylin and eosin staining was applied to assess the pathomorphological changes and a modified Giemsa staining was used for better detection of H pylori. For intestinal metaplasia, special stainings, i.e. Alcian blue periodic acid-Schiff and high iron diamine-Alcian blue staining, were performed. PCR was performed on bacterial DNA to characterize the presence or absence of virulence-associated genes, like cagA, and distribution of different alleles of vacA and iceA. Intraglandular neutrophil infiltration, a hallmark of activity of gastritis, was present in 34 (94%) of tribals (TRs) and 42 (84%) of DU individuals infected with H pylori. Lymphoid follicles and aggregates, which are important landmarks in H pylori infection, were positive amongst 15 (41%) of TRs and 20 (40%) of DU subjects. Atrophic changes were observed in 60% and 27.7%, respectively, among DU cases and tribals (P > 0.003). Metaplastic changes were detected in low numbers in both groups. Moderate to severe density distribution of H pylori in the gastric mucosa was 63% among TRs, whereas it was 62% in DU subjects. There were no significant differences in the distribution of virulence-associated genes like cagA, vacA and iceA of H pylori strains carried by these two populations. Our study showed almost similar distribution of inflammatory cells among asymptomatic tribals and DU Bengali patients. Interestingly, the tribal population are free from any clinical symptoms despite evidence of active histologic gastritis and infection with H pylori strains carrying similar virulence markers as of strains isolated from patients with DU. There was an increased cellular response, especially in terms of neutrophil infiltration, but much lower risk of developing atrophy and metaplastic changes among the tribal population.

Effects of Tea Catechins on the Gastrointestinal Mucosa in Rats

Although tea catechins are known to exert a potent antiulcer effect on the alimentary tract, there is scant information concerning their effects on normal mucus cell functions. Using original anti-mucin monoclonal antibodies, we studied the influences of long-term administration of catechins on the quantity and quality of mucin in rat gastrointestinal mucosa. Administration of 0.5% tea catechins significantly increased the mucin content of the ileum, but not the stomach. An enzyme-linked immunosorbent assay (ELISA) showed no remarkable qualitative changes in gastric mucin, but a selective increase and decrease in sulfo- and sialomucins, respectively, in the ileum of rats administered catechins. The ELISA results were consistent with both the immunohistochemical findings and the high-iron diamine-alcian blue staining pattern. These findings indicate that tea catechins modulate ileal mucin metabolism in the ileal mucosa, suggesting that further studies focusing on the ileal epithelium will assist in further elucidation of the mechanism of catechin effects.

Expression of small intestinal and colonic phenotypes in complete intestinal metaplasia of the human stomach

The incomplete intestinal metaplasia (IM) that is reported to be a risk factor for gastric carcinogenesis in man usually features sulfomucin production and thus is considered of colonic type. To cast light on the underlying mechanisms, we here examined the proportions of colonic and small intestinal phenotypes in IM by immunohistochemistry and real-time reverse transcription-polymerase chain reaction at the single isolated gland level. Carbonic anhydrase 1 (CA1) is a specific marker of colonic epithelial cells, whereas sucrase is specific to absorptive cells of the small intestine. Totals of 139 (23.5%) and 452 (76.5%) IM glands were judged to be CA1 positive and CA1 negative, respectively, in resected pyloric mucosa from cancer patients. The average score for MUC5AC in CA1-positive IMs was significantly lower than in CA1-negative counterpart tissue (P<0.0001), whereas the opposite was the case for sucrase (P<0.0001). High iron diamine-Alcian blue staining revealed CA1 expression to coincide with type I complete IM. The expression of CA1 mRNA strongly correlated with that of sucrase-isomaltase, and inversely with that of MUC5AC in isolated IM glands. In conclusion, CA1 could be colocalized with small intestinal proteins such as sucrase, but only rarely with the gastric mucin, MUC5AC. Its expression warrants further study, with the focus on stimulation and/or suppression mechanisms by gastric and intestinal transcription factors.

Type III intestinal metaplasia and carcinoma express an identical carbohydrate-epitope revealed by a novel monoclonal antibody (2D11).

A monoclonal antibody (2D11, IgG2b) obtained by immunizing mice with a mucin fraction of the human gastric mucosa reacted specifically to intestinal metaplasia of human gastric mucosa and fetal intestinal mucosa but not to normal adult gastric, small intestinal or colonic mucosa in immunohistochemical staining. The results of Western blotting indicated that 2D11 recognized the high molecular weight glycoprotein(s) (mucin) of the stomach. Treatment of the antigens with sodium periodate abolished their reactivity to 2D11, and digestion of the antigens with beta-galactosidase reduced their reactivity to 2D11. Digestion of the antigens with pronase had no effect, however, suggesting that 2D11 recognizes the oligosugar moiety but not the peptide moiety of the antigens. Further immunohistochemical investigation showed that the reactivity of 2D11 was restricted to the Type IotaIotaIota intestinal metaplasia that is identified by a characteristic staining pattern with the high iron diamine-Alcian blue stain. 2D11 also reacted in high frequency to adenocarcinomas of the stomach (66.7%), pancreas (66.7%) and gallbladder (50.0%), but in low frequency to those in lung (8.3%) and colon (11.1%). It is of interest that 2D11 reacted to very restricted regions of the gastric adenocarcinomas. All monoclonal antibodies to mucin polypeptides (MUC1, 2, 3, 5AC and 6) examined stained intestinal metaplasia and carcinomas in a different pattern from 2D11 in immunohistochemistry. These facts indicate that Type IotaIotaIota intestinal metaplasia and carcinomas express carbohydrate chains identical to those expressed in the fetal intestinal mucosa, suggesting that both of them are closely related to fetal intestinal mucosa.

Cytokeratin immunoreactivity of intestinal metaplasia at normal oesophagogastric junction indicates its aetiology

BACKGROUND AND AIMSCytokeratin (CK) 7 and 20 patterns are specific for long and short segments of Barrett's oesophagus but their use has not been assessed in intestinal metaplasia arising in...

Observations of High Iron Diamine-Alcian Blue Stain in Uterine Cervical Glandular Lesions

We studied epithelial glycoproteins in uterine cervical lesions, including glandular lesions, and investigated whether a more accurate diagnosis could be obtained using high iron diamine-alcian blue (HID-AB) stain and immunostaining with carcinoembryonic antigen (CEA). In addition, we examined the usefulness of preoperative diagnosis using biopsy specimens stained with HID-AB and CEA. Normal endocervical glands showed a predominance of sulfomucin, while adenocarcinoma predominantly showed sialomucin. CEA was highly detected in adenocarcinoma, but not in normal endocervix or in glandular dysplasia, suggesting that this stain has high specificity. The staining patterns of biopsy specimens and hysterectomy specimens were similar. Therefore HID-AB stain and CEA stain may be useful as a supplementary means of diagnosing uterine cervical lesions.

The expulsion of Echinostoma trivolvis from C3H mice: differences in glycoconjugates in mouse versus hamster small intestinal mucosa during infection.

Mucosal glycoconjugates were examined in C3H mice and in hamster small intestines infected with Echinostoma trivolvis and in uninfected rodents, using periodic-acid Schiff (PAS) and high-iron diamine-alcian blue (HID-AB) staining and three different fluorescein-conjugated lectins: Triticum vulgaris agglutinin (WGA), Helix pomatia agglutinin (HPA) and Griffonia simplicifolia agglutinin (GSA-II). Lectin-labelling by electron microscopy was also undertaken with WGA and HPA lectin-gold probes. HID-AB stain demonstrated that the most mature goblet cells of the mouse villi contain sulfomucins, whereas those of hamsters contain sialomucins. The expression of lectin-binding sites and the intensity of the lectin binding in the small intestines were changed by echinostome infection. Specific differences in the reaction to mucin glycoproteins were clearly observed between the mouse and hamster intestines infected with E. trivolvis; lectin-binding to hyperplastic goblet cells and crypts in the infected mice increased, while no marked increase in the number of goblet cells and reaction to the glycoconjugates were observed in the infected hamsters. These findings indicate that the expression of terminal N-acetyl-D-galactosamine, sialic acid and N-acetyl-D-glucosamine increased in mucins secreted from hyperplastic goblet cells associated with E. trivolvis infection in mice. No marked increase in these glycoconjugates occurred in hamster infections. These findings reflect clear differences in infectivity of E. trivolvis in C3H mice versus hamsters.

Histochemical, lectin-histochemical and morphometrical characteristics of intestinal goblet cells of germfree and conventional mice.

Histochemical, lectin-histochemical and morphometrical studies were carried out on intestinal goblet cells of 8-week-old germfree (GF) and conventional (CV) mice of the BALB/c strain. Except for the reactivity of cecal goblet cells to Dolichos biflorus agglutinin (DBA) and Ulex europeus-I agglutinin (UEA-I), there was no difference between GF and CV mice in histochemical and lectin-histochemical properties. In the cecal mucosa, DBA stained the goblet cells strongly in CV mice but not in GF mice and UEA-I stained the goblet cells strongly in the lower part of crypts in CV mice but only faintly in GF mice. These findings suggest that terminal residues of cecal goblet cell mucin were different in GF and CV mice. Morphometrically, cecal goblet cells were fewer in number and smaller in size in GF mice than in CV mice. In addition, high iron diamine-alcian blue staining made a very clear border between the cecum and colon, because cecal goblet cells were exclusively positive for sulfomucin and colonic goblet cells were predominantly positive for sialomucin.

Qualitative study of sialomucins changes during N-methyl-N-nitrosourea-induced colonic carcinogenesis in mice

The sequential histological and sialomucins histochemical changes in colonic epithelium during the N-methyl-N-nitrosourea (MNU) induced colonic carcinogenesis were studied in female ICR mice. The sialomucin histochemical changes were assessed by use of high iron diamine-alcian blue staining (HID-AB) at pH 2.5. In the untreated controls, the epithelial cells in the distal colon are characterized by a predominance of sulfomucins, and the cells in the proximal site stain for abundant sialomucins, with occasional sulfomucins in the base of the crypt. The earlier changes detected at the 4th week are focal areas of hyperplasia in which sulfomucins still predominant as in controls. As the experimental progress, the number of lesions with severe dysplasla increase, with carcinoma insitu and invasive carcinoma appearing from the 10th week. The areas of mild or moderate dysplasia show marked changes in the composition of mucin, characterized by the progressively increased of staining of sialomucins and accompanied in general by a decrease of sulfomucins, similar to those changes described in transitional mucosa (TM) adjacent to colorectal cancer in the human. It is suggested the morphological malignant transformation of colonic epithelium were paralleled qualitatively enhanced slalomucin staining in concert with reduced sulfomucin staining of epithelial cells, providing further evidence that the mucin changes in TM characterized by an increase in sialomucins might reflect early primary premalignant transformation.

Long-term follow-up study of patients with gastric adenomas with malignant transformation. An immunohistochemical and histochemical analysis.

Little is known about the changes in the immunohistochemical and histochemical characteristics that take place during the malignant transformation of gastric adenoma (GA). Nine patients with GA who developed carcinoma (Group A) and 33 who did not (Group B) during an equivalent follow-up period (mean, 76.4 months; range, 38-166 months) were studied. Tissue sections from these patients were stained for p53, c-erbB-2, bcl-2, carbohydrate antigen 19-9 (CA 19-9), carcinoembryonic antigen (CEA), and proliferating cell nuclear antigen (PCNA), and by high iron diamine-alcian blue staining. The gastric or intestinal phenotypes of the GA cells were evaluated histochemically by paradoxical concanavalin A and galactose oxidase-Schiff reactions. In Group A, the following were more frequent; severe dysplasia (77.8% vs. 0%, P < 0.001), villous structures (66.7% vs. 9.1%, P < 0.05), sulfomucin secretion (88.9% vs. 39.4%, P < 0.05) and mixed gastric-intestinal phenotype (77.8% vs. 9.1%, P < 0.001). The development of carcinoma in Group A positively correlated with an increase in p53 expression and PCNA-labeling index. None of the adenomas was positive for c-erbB-2, but four of nine carcinomas showed positive reactions. Mucin histochemistry of GA may be of some value in assessing the potential for subsequent carcinoma. Immunoexpression of c-erbB-2, bcl-2, CA 19-9, and CEA have only limited value. In contrast to p53, the immunoexpression of c-erbB-2 is a late event in the malignant transformation of GA.

Ulex europeus agglutinin I binding pattern during chemical carcinogenesis in the rat gastrointestinal tract

Fluorescein isothiocyanate-Ulex europeus agglutinin I stain (UEA1) was postulated as a prominent histochemical marker for premalignant mucosa in dimethylhydrazine (DMH)-treated animals. UEA1 (evaluated by two scanning methods) and high iron diamine Alcian blue (HIDAB) stain were used in attempt to detect premalignant colonic mucosa in this animal model. The authors also examined the influence of the duodenal medium on colonic segments transposed to the upper gastrointestinal tract. Rats were placed into three groups: those with interposed intestine, those receiving the sham operation, and controls. Half of the animals received DMH, and surviving rats were killed at 2, 4, and 8 months. The authors found no differences in tumor development in the transposed and nontransposed colons of animals treated with DMH. Several transposed segments of animals without carcinogen induction showed dysplastic areas. These findings suggest a trophic role of certain duodenal factors in the epithelial kinetics of the transposed colons. The authors did not find HIDAB stain useful in the identification of premalignant colonic mucosa. The quantitative evaluation method of UEA1 binding was more reliable. Fifteen percent of all the colon specimens of animals without chemical induction were stained with UEA1 with this form of evaluation. Positive staining of the interposed colon samples was the most important factor for these findings. In this animal model, UEA1 staining is a potentially useful marker of premalignant mucosa, particularly when the nontransposed distal colon of animals treated with DMH is considered.