High Iron Diamine-alcian Blue 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.

High-risk individuals for gastric cancer would be missed for surveillance without subtyping of intestinal metaplasia.

The use of Operative Link on Gastritis Assessment (OLGA) and Operative Link on Gastritis Assessment based on Intestinal Metaplasia (OLGIM) staging system is recommended for identifying subjects at risk for developing gastric cancer; usually high-risk lesions are considered only as stages III and IV. Accumulating evidence suggests that incomplete intestinal metaplasia (IM) is important in the development of gastric cancer.Our aim has been to identify the prevalence of incomplete IM in patients with low-risk OLGA/OLGIM stages among a high-risk general population. Healthy adult volunteers aged 40-64years were invited to undergo upper endoscopy within a regional GISTAR pilot study in Kazakhstan (n = 166). Gastric lesions were staged according to OLGA/OLGIM staging system. High iron diamine-alcian blue (HID-AB) was used for subtyping IM. IM prevalence overall was 45.8%. Incomplete IM was present in 52.6% (type II in 30.3% and type III in 22.3%), whereas complete IM was found in 47.4% individuals. The prevalence of OLGIM I and II stage were 39.8 and 4.8%, respectively, whereas OLGIM III was observed in 1.2%. The prevalence of incomplete IM in patients stratified to OLGIM I was 54.5% (type II in 31.8% and type III in 22.7%). High prevalence of incomplete IM was detected not only in subjects with extensive IM, but in those stratified as at the OLGIM I stage. Without IM subtyping, patients with high risk of gastric cancer development would be missed for surveillance.

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.

Chemoradiotherapy-induced Changes in Mucinous Components in Rectal Cancer Tissue: Evaluation on High Iron Diamine-alcian Blue and Mucin 1 Staining.

The standard treatment for rectal cancer is neoadjuvant chemoradiotherapy (nCRT) followed by surgery. Mucinous carcinoma responds poorly to nCRT. In some patients, mucin lakes (MLs) are induced by nCRT. Identifying whether MLs are induced or originally present would be of great importance. We studied 20 patients with MLs (CRT-MC group) among 205 patients who received nCRT. Among 88 patients who did not receive nCRT, we studied 9 patients with mucinous carcinoma (non-CRT-MC group) and 18 patients with MLs in differentiated adenocarcinoma (non-CRT-AC group). Tumors were stained with high iron diamine-Alcian blue (HID-AB) and MUC1 staining. Rate of AB>HID staining of cancer cells was significantly higher in the CRT-MC group than in non-CRT-MC group (p=0.0004). Rate of MUC1 staining in MLs was significantly higher in the CRT-MC group (p=0.0254). nCRT can induce qualitative changes in mucinous components, however, other methods are required to distinguish induced components from originally existing components.

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.

The distribution of mucous secreting cells in the gastrointestinal tracts of three small rodents from Saudi Arabia: Acomys dimidiatus, Meriones rex and Meriones libycus

The proportion of mucin phenotypes (which form the protective biofilm of the gastrointestinal tract) differs between intestinal regions. This study examines the distribution of mucin secreting cells in the gastrointestinal tracts of the Eastern spiny mouse (Acomys dimidiatus), King jird (Meriones rex) and Libyan jird (Meriones libycus), which inhabit the dry and hot deserts of Saudi Arabia. Intestinal tract samples were processed to wax and tissue sections stained with Alcian Blue-Periodic Acid Schiff (AB-PAS) and High Iron Diamine-Alcian Blue (HID-AB) in order to determine different mucin phenotypes by quantitative analysis. Mixed mucin secreting cells (combined neutral and acid) was the predominant mucin secreting cell type observed throughout the gastrointestinal tract in all species. Acid mucin secreting goblet cells were mainly located in the colon. A. dimidiatus presented with significantly more total sialo than sulfomucin secreting cells while the opposite was true for both Meriones species. The distribution of mucin secreting cells is therefore similar to previously reported results for small mammals not living under arid conditions.

Tissue content of sulfomucins and sialomucins in the colonic mucosa, without fecal stream, undergoing daily intervention with sucralfate.

To measure the content of acidic mucin, sialomucin, and sulfomucins in the colonic mucosa without fecal stream submit to intervention with sucralfate (SCF). Thirty-six rats were submitted to a right colostomy and a distal mucous fistula and divided into two groups according to sacrifice to be performed two or four weeks. Each group was divided into three subgroups according daily application of enemas containing saline, SCF at 1.0 g/kg/day or 2.0 g/kg/day. Colitis was diagnosed by histological analysis. Acid mucins were determined with the Alcian-Blue and sulfomucin and sialomucin by high iron diamine-alcian blue (HID-AB) techniques. The mucins were quantified by computer-assisted image analysis. Mann-Whitney and ANOVA tests were used to analyze the results establishing the level of significance of 5% for both (p<0.05). SCF enemas decreased the inflammation score and was related to the concentration used and time of the intervention. SCF at both concentrations increased the content of acid mucin, which was related to the concentration used and to the improvement in the inflammatory score. There was an increase in the content of sulfomucins and sialomucins in SCF groups. SCF increased sulfomucins from 2 weeks of intervention, which was not related to the dose or time of application. The increase in sialomucin content was related to the time and dose used in the intervention. Sucralfate increased the content of acidic mucins, primarily at the expense of sialomucin, which was affected by the dose and time of intervention.

Expression of Das-1, Ki67 and sulfuric proteins in gastric cardia adenocarcinoma and intestinal metaplasia lesions

The aim of this study was to characterize histo-chemical patterns and Das-1 and Ki67 protein expression in gastric cardia adenocarcinoma (GCA) and intestinal metaplasia (IM) lesions adjacent to GCA. Histochemical techniques, including Alcian blue/periodic acid-Schiff (AB/PAS), high iron diamine/Alcian blue (HID/AB) and avidin-biotin-peroxidase complex (ABC) immunohistochemistry were applied to GCA and IM samples from patients (n=200) in Linzhou, Henan, China, a high incidence area for GCA and esophageal squamous cell carcinoma (SCC). The detection rate of IM lesions in resected tissues adjacent to GCA was 32.5% (65/200). GCA and IM lesions presented a high frequency of Das-1 and Ki67-positive staining with statistical significance (P<0.01). The expression of sulfuric proteins did not show co-expression with Das-1 and Ki67 in GCA and surrounding IM lesions (P>0.05) from the same GCA patient. The high frequency of co-expression of Das-1 and Ki67 in GCA and adjacent IM lesions indicates that IM adjacent to GCA may undergo similar molecular changes to GCA, which may be one of the mechanisms for malignant transformation of IM in the population studied.

Quantification by computerized morphometry of tissue levels of sulfomucins and sialomucins in diversion colitis in rats

To quantify the intensity of sulfomucin and sialomucin expression in the colon mucosa, by means of computer-assisted image processing, comparing segments with and without fecal stream and correlating with the duration of fecal transit exclusion. Forty-five Wistar rats were subjected to diversion of the fecal stream in the left colon by means of constructing a proximal colostomy and distal mucosal fistula. They were distributed randomly into three experimental groups of 15 animals, of which 10 were subjected to colon diversion (experimental subgroup) and five were only subjected to laparotomy, without colon diversion (control subgroup). The three experimental groups were formed according to the sacrifice date, which was to be performed six weeks after the surgical procedure (Group A), 12 weeks (Group B) and 18 weeks (Group C). The sulfomucin and sialomucin expression in the colon mucosa was evaluated using the histochemical technique of high iron diamine-alcian blue (HID-AB). The tissue expression was quantified for each animal, in the segments with and without fecal stream, at a location where there were four complete contiguous crypts in two random fields, with the aid of the computer-assisted image analysis software. The final value was taken to be the mean reading from the two fields selected, in the segments with and without fecal stream. To compare the expressions of the two mucin subtypes in the segments with and without fecal stream, the paired Student t test was used. To analyze variance according to duration of exclusion, ANOVA with the Newman-Keuls post-test was used, setting the significance level at 5% (p<0.05). There were significant reductions in tissue sulfomucin and sialomucin content in the colon without fecal stream, independent of the duration of exclusion considered. There was increased tissue sulfomucin content and decreased tissue sialomucin in the segments without fecal stream, with increasing duration of exclusion. Diversion of the fecal transit decreased the tissue sulfomucin and sialomucin content in the segments without fecal stream. Notwithstanding the reduction in the levels of both subtypes of acid mucin in the segments without fecal stream, there was increased tissue sulfomucin content and decreased tissue sialomucin with increasing duration of intestinal diversion.

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.

Intestinal metaplasia subtype and gastric cancer risk

is mostwidely used and consists of complete and incomplete types. Thecomplete type (type I) is characterized by the presence of absorp-tive cells, Paneth cells, and goblet cells secreting sialomucins andcorrespondstothesmallintestinephenotype.Theincompletetype,which encompasses types II and III, is characterized by the pres-ence of columnar and goblet cells secreting sialomucins and/orsulfomucins: neural and acid sialomucins in type II and sulfomu-cins in type III. Several studies have shown that type I has noincreased risk of developing carcinoma, whereas type III is asso-ciated with an increased risk of malignant transformation.

Effects of repeated silver nanoparticles exposure on the histological structure and mucins of nasal respiratory mucosa in rats

To investigate the effects of repeated silver nanoparticle exposure on the nasal septum respiratory mucosa, 6-week-old SD rats were exposed to silver nanoparticles at concentrations of fresh air control, low-dose (1.73 × 10 4/cm, 0.5 μg/m 3), middle-dose (1.27 × 10 5/cm 3, 3.5 μg/m 3) and high-dose (1.32 × 10 6 particles/cm 3, 61 μg/m 3) in an inhalation chamber for 6 h per day, 5 times a week for 28 days. The animals were sacrificed after the 28 days of exposure period. Histochemical staining, including periodic acid Schiff (PAS), alcian blue (AB) pH 2.5, and high iron diamine-alcian blue (HID-AB) pH 2.5, was used to evaluate changes in the mucosubstance properties of the goblet cells in the respiratory epithelium. In a histopathological study, the nasal cavity and lungs from the exposed groups exhibited no remarkable changes compared to the control group. However, a slight increase in the neutral mucins was noted for all the silver nanoparticle-exposed groups when compared to the control group, although without statistical significance. Nonetheless, the size and number of goblet cells containing neutral mucins increased significantly in the groups exposed to silver nanoparticle at middle- and high-dose ( P < 0.05). While the densities of the stained mucosubstances showed no difference among the exposed groups, the amount of neutral mucins did tend to increase slightly, although acid mucins including sulfomucins and sialomucins showed no change in any of the exposed groups. Therefore, the present results did indicate that silver nanoparticles have an influence on the neutral mucins in the respiratory mucosa, yet without toxicological significance.

Clinic-pathological characteristics of colonic carcinoma in relation to localization and histologic type

A significant increase in colonic carcinoma has been analyzed in numerous clinico-pathological studies and genetics models. The aim of this study was to determinate the differences in clinico-pathological parameters of colonic carcinoma regarding localization and histological type. The study enrolled 124 patients who had undergone surgery for colorectal carcinoma at the Clinic of Surgery in the town of Nis within 2005. Paraffin sections were stained with Hematoxilin-eosin (H&E), High iron diamine-Alcian blue (HID-AB) (pH-2.5) and Alcian blue-Preiodic arid Shiff (AB-PAS) (pH-2.5) methods. From pathological report we used data regarding: localization (right or left colon), histological type, histological grade, and parameters which determinate the tumor stage. The total number of patients with right-sided colonic carcinoma was 40 (32.36%) and 84 (67.74%) with left-sided colonic carcinoma. Histopathologically, 96 (77.42%) adenocarcinomas and 28 (22.58%) mucinous adenocarcinomas were verified. There were no statistically significant differences between the right-sided and left-sided colonic carcinoma regarding sex, age, histological grade and tumor stage (p > 0.05). Mucinous adenocarcinomas was statistically significantly more frequent in right-sided colon (35.00%) than in left-sided colon (16.67%) (p < 0.05). There were no statistically significant differences between adenocarcinomas and mucinous adenocarcinomas regarding sex and disease stage. In younger patients the percentage of mucinous adenocarcinomas (28.57%) compared to non-mucinous ones (11.46%) was significantlly higher (p < 0.05) than in older patients. Mucinous adenocarcinomas had statistically significantly more frequently poor differentiation in comparision to adenocarcinomas (46.43% versus 9.37%, p < 0.001). According to the presented results it can be concluded that the lower grade of differentiation of the colon adenocarcinoma and mucinous secretion are significantly often present in younger patients.

Expression of COX-2 in Different Subtypes of Gastric Intestinal Metaplasia and Gastric Carcinoma by Tissue Microarray

Objective: To study the expression of cyclooxygenase-2 (COX-2) protein in different subtypes of intestinal metaplasia (IM) and gastric carcinoma, evaluate the possibility of COX-2 forecasting the risk of malignant potential of IM, and the relationship between COX-2 expression and gastric carcinogenesis. Methods: Forty cases of chronic atrophic gastritis (CAG) with IM, 40 cases of gastric carcinoma and corresponding paracancerous tissues were selected to construct a tissue microarray. High iron diamine/alcian blue (HID/AB) staining and Hematoxylin and Eosin (HE) staining was used to classify IMand gastric carcinoma, and the expression of COX-2 protein detected in different subtypes of IM and gastric cancer by using immunohistochemistry. Results: The positive expression rate of COX-2 was 45.65%, 59.38% and 77.27% in IM foci in CAG, IM foci in paracancerous tissues, and intestinal-type gastric carcinoma, respectively, significantly higher than in diffuse-type gastric cancer (16.67%)(P<0.05, 0.005 and 0.005, respectively), and the expression intensity of COX-2 protein showed a increased tendency gradually in the sequence of IM foci in CAG→IM foci in paracancerous tissues→intestinal-type gastric carcinoma (P<0.005). The positive expression rate of COX-2 protein in type III IM was significantly higher than in type I and type II IM (P<0.005 and 0.05, respectively), and the expression intensity also showed a increased tendency gradually from type I to type III IM (P<0.005). Conclusion: The expression level of COX-2 was increased gradually along with the increase of the risk of malignancy of IM, and its expression level may be a useful index to forecast the risk of malignant potential of IM. COX-2 expression was associated with intestinal-type gastric carcinoma, but it might also have some role in the carcinogenesis of diffuse-type gastric carcinoma.

Mucin‐depleted foci have β‐catenin gene mutations, altered expression of its protein, and are dose‐ and time‐dependent in the colon of 1,2‐dimethylhydrazine‐treated rats

Mucin-depleted foci (MDF) are purported preneoplastic lesions that can be easily visualized in the unsectioned colon of carcinogen-treated rats stained with high-iron diamine alcian blue (HID-AB). In F344 rats treated twice with 150 mg/kg of 1,2-dimethylhydrazine (DMH) and sacrificed after 5, 9, 13 and 28 weeks, MDF increased over time from 5 to 13 weeks, whereas they decreased at 28 weeks, when tumors appear. MDF multiplicity (crypts/MDF) linearly increased with time. Increasing doses of DMH (100, 150 and 200 mg/kg x 2 times) caused a dose-related increase in MDF. Mutations in Ctnnb1 gene codifying for beta-catenin were identified with PCR amplification and direct sequencing in 6/15 tumors (40%), 7/28 MDF (25%) and 2/27 (7%) aberrant crypt foci (ACF) identified in HID-AB-stained colon. All mutations in tumors and MDF caused amino acid substitution, while one mutation in ACF was silent. Beta-catenin detected at membrane level by immunohistochemistry was markedly reduced in MDF and tumors and, to a lesser extent, in ACF identified with HID-AB. By contrast, nuclear localization of beta-catenin was significantly increased in MDF and tumors, while no variation was observed in ACF. Beta-catenin cytoplasmic expression was also significantly increased in MDF and tumors but to a lesser extent in ACF. In conclusion, MDF are induced dose-dependently by DMH, increase in size with time, have mutations in the beta-catenin gene and marked alterations in beta-catenin cellular localization. Since all these phenomena are considered specific steps for colon tumorigenesis, these results further support the hypothesis that MDF are cancer precursors and can be proposed as endpoints in short-term carcinogenesis experiments.

Distinction between short-segment Barrett’s esophageal and cardiac intestinal metaplasia

To investigate the roles of mucin histochemistry, cytokeratin 7/20 (CK7/20) immunoreactivity, clinical characteristics and endoscopy to distinguish short-segment Barrett's esophageal (SSBE) from cardiac intestinal metaplasia (CIM). High iron diamine/Alcian blue (HID/AB) mucin-histochemical staining and immunohistochemical staining were used to classify intestinal metaplasia (IM) and to determine CK7/20 immunoreactivity pattern in SSBE and CIM, respectively, and these results were compared with endoscopical diagnosis and the positive rate of gastroesophageal reflux disease (GERD) symptoms and H pylori infection. Long-segment Barrett's esophageal and IM of gastric antrum were designed as control. The prevalence of type III IM was significantly higher in SSBE than in CIM (63.33% vs 23.08%, P< 0.005). The CK7/20 immunoreactivity in SSBE showed mainly Barrett's pattern (76.66%), and the GERD symptoms in most cases which showed Barrett's pattern were positive, whereas H pylori infection was negative. However, the CK7/20 immunoreactivity in CIM was gastric pattern preponderantly (61.54%), but there were 23.08% cases that showed Barrett's pattern. H pylori infection in all cases which showed gastric pattern was significantly higher than those which showed Barrett's pattern (63.83% vs 19.30%, P< 0.005), whereas the GERD symptoms in gastric pattern were significantly lower than that in Barrett's pattern (21.28% vs 85.96%, P< 0.005). Distinction of SSBE from CIM should not be based on a single method; however, the combination of clinical characteristics, histology, mucin histochemistry, CK7/20 immunoreactivity, and endoscopic biopsy should be applied. Type III IM, presence of GERD symptoms, and Barrett's CK7/20 immunoreactivity pattern may support the diagnosis of SSBE, whereas non-type III IM, positive H pylori infection, and gastric CK7/20 immunoreactivity pattern may imply CIM.

Gastric carcinogenesis by duodenal reflux through gut regenerative cell lineage.

To elucidate the histogenesis of gastric stump cancer, we performed an operation in rats to make all duodenal contents flow back into the glandular stomach. The subjects were 41 rats, and sequential morphological changes of the duodenogastric stoma and the incidence of stump cancers were studied. Serial sections around the stoma were studied with mucin stains such as paradoxical concanavalin A (Con A), galactose oxidase Schiff (GOS), and high-iron diamine-Alcian blue (HID-AB). An immunohistochemical study on cell proliferation with bromodeoxyuridine (BrdU) was also done. At week 30, pyloric gland type cells positive for Con A first appeared at the base of the intestinal crypts and the fundic glands adjacent to the anastomosis. These glands became large with time, resulting in formation of cystically dilated glands. These gland cells were partially stained with GOS, and then they retained a proliferative activity. These changes seemed to resemble "gastritis cystica profunda" in human remnant stomachs. At 50 and 80 weeks, adenocarcinomas were observed in 4 of 10 rats (40.0%) and in 16 of 21 rats (76.2%), respectively. We have noted that the early change of cystic proliferation of mucous glands resembled the so-called "ulcer associated cell lineage (UACL)" described by others, but our characteristic finding was not only pyloric but also foveolar metaplasia. This pyloric-foveolar metaplasia subsequently led to development of glands with intestinal-type goblet cells, which looked like incomplete intestinal metaplasia. This sequence was different from UACL, and very recently, we proposed a concept of "gut regenerative cell lineage (GRCL); from pyloric-foveolar to with goblet cell metaplasia in regeneration," common to all parts of the gut, and the stump cancer appeared to arise from GRCL.

Is subtyping of intestinal metaplasia in the upper gastrointestinal tract a worthwhile exercise? An evaluation of current mucin histochemical stains

Intestinal metaplasia is a premalignant condition that occurs in the upper gastrointestinal tract and can be subdivided into three types (I, II and III). Previous studies suggest that type III carries the highest cancer risk. The high iron diamine/alcian blue (HID/AB) technique traditionally has been used to identify this subtype; however, the technique uses reagents that are toxic and potentially carcinogenic. Therefore, in this study we evaluate various alternative histochemical techniques. Our results indicated that the only suitable alternative is Gomori’s aldehyde fuchsin/AB technique. The study also revealed that subtyping of intestinal metaplasia is a subjective procedure, open to varying interpretation. Consequently, we suggest that previous work linking cancer risk to metaplasia subtypes should be viewed with some circumspection.

Cell Kinetic Study on Histogenesis of Barrett's Esophagus Using Rat Reflux Model

Background: To elucidate the histogenesis of Barrett's esophagus and esophageal adenocarcinoma, we designed a duodeno-gastric reflux model in which normal stomach function and normal nutritional status are retained. Methods: Male Wistar rats were used in the experiment. The esophago-gastric junction was side-to-side anastomosed to a loop of jejunum about 3 r cm distal to Treitz's ligament. The animals were not exposed to any known carcinogens during the experiment. Sequential morphological changes were studied for up to 50 weeks after surgery. Serial sections were made and stained with hematoxylin and eosin (H&E). In addition, immunohistochemical staining for bromodeoxyuridine (BrdU) was performed along with histochemical staining for mucins using paradoxical concanavalin A (ConA), galactose oxidase Schiff (GOS), and high-iron diamine-alcian blue (HID-AB). Results: Severe esophagitis with squamous cell hyperplasia was noted in all animals after surgery. At week 20 after surgery, glandular metaplastic cells positive for ConA first appeared within the basal cell layer of esophageal squamous cell epithelium, and then GOS-positive cells and HID-AB goblet cells appeared. This is a characteristic of the specialized columnar epithelium of Barrett's esophagus. We detected esophageal adenocarcinomas in 1 out of 8 subjects at week 40 and in 3 out of 8 subjects at week 50 after surgery. Conclusions: Reflux of duodenal contents causes specialized columnar epithelium of Barrett's esophagus and esophageal adenocarcinoma. As part of the sequence of events leading to the development of Barrett's esophagus, pyloric-foveolar metaplasia was observed followed by the appearance of intestinal goblet cells. The pyloric-foveolar metaplasia appears to be associated with chronic mucosal damage and regeneration. This multiplastic cell lineage is referred to as 'gut-regenerative cell lineage' (GRCL).