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Supplementary Data from Locked Nucleic Acid In situ Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

Supplementary Figure 3 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 12 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 4 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Methods, Figure Legends 1-13 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 9 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Table 3 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 8 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Tables S1-S2, Figures 1-7 from Frequent Loss of Brm Expression in Gastric Cancer Correlates with Histologic Features and Differentiation State

The mammalian SWI/SNF chromatin remodeling complex, an essential epigenetic regulator, contains either a single Brm or BRG1 molecule as its catalytic subunit. We observed frequent loss of Brm expression but not of BRG1 in human gastric cancer cell lines. Treatment with histone deacetylase inhibitor rescued Brm expression, indicating epigenetic regulation of this gene, and an RNA interference–based colony formation assay revealed antioncogenic properties of Brm. Brm immunostaining of 89 primary gastric cancers showed an obvious reduction in 60 cases (67%) and a severe decrease in 37 cases (42%). Loss of Brm is frequent in the major gastric cancer types (well- or moderately-differentiated tubular adenocarcinoma and poorly-differentiated adenocarcinoma) and positively correlates with the undifferentiated state. Among the minor gastric cancer types, Brm expression persists in signet-ring cell carcinoma and mucinous adenocarcinoma, but a marked decrease is observed in papillary adenocarcinoma. Intestinal metaplasia never shows decreased expression, indicating that Brm is a valid marker of gastric oncogenesis. In contrast, BRG1 is retained in most cases; a concomitant loss of BRG1 and Brm is rare in gastric cancer, contrary to other malignancies. We further show that Brm is required for villin expression, a definitive marker of intestinal metaplasia and differentiation. Via regulating such genes important for gut differentiation, Brm should play significant roles in determining the histologic features of gastric malignancy. [Cancer Res 2007;67(22):10727–35]

Supplementary Figure 2 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 5 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 7 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 6 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Table 1 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Table 2 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

The chromatin remodeling complex SWI/SNF is an important epigenetic regulator that includes one Brm or BRG1 molecule as catalytic subunit. Brm and BRG1 do not function identically, so this complex can regulate gene expression either positively or negatively, depending on the promoter to which it is recruited. Notably, Brm attenuation due to posttranscription suppression occurs often in human tumor cells, in which this event contributes to their oncogenic potential. Here, we report that the 3′-untranslated region of Brm mRNA has two sites that are efficiently targeted by the microRNAs miR-199a-5p and -3p, revealing a novel mechanism for modulation of Brm-type SWI/SNF activity. Computational mapping of the putative promoter region of miR-199a-2 (miPPR-199a-2) has defined it as the major contributing genetic locus for miR-199a-5p and-3p production in these tumor cell lines. We validated this predicted region by direct promoter analysis to confirm that Egr1 is a strong positive regulator of the miR-199a-2 gene. Importantly, we also showed that Egr1, miR-199a-5p, and miR-199a-3p are expressed at high levels in Brm-deficient tumor cell lines but only marginally in Brm-expressing tumor cells. Finally, we also obtained evidence that Brm negatively regulates Egr1. Together, our results reveal that miR-199a and Brm form a double-negative feedback loop through Egr1, leading to the generation of these two distinct cell types during carcinogenesis. This mechanism may offer a partial explanation for why miR-199a-5p and -3p have been reported to be either upregulated or downregulated in a variety of tumors. Cancer Res; 71(5); 1680–9. ©2010 AACR.

Nodular lymphoid hyperplasia (NLH) of the human colon has been associated with multiple diseases and symptoms. Causes include food allergies, infections, inflammatory bowel disease, and immunodeficiency, and gastrectomy is not usually considered to be the etiology. Nine rats two weeks after total gastrectomy and 12 control rats were sacrificed and submitted for histological examination. In the gastrectomy group, we found lymphoid hyperplasia throughout the entire colon mucosa. The cross-sectional area of lymphoid follicles was increased to be five-fold larger than that in the rats in the control group (sham surgery). Lymphoid follicles were classified into primary and secondary follicles according to the presence/absence of germinal centers; the gastrectomy group had a significantly larger number of secondary follicles. When T cell and B cell classification of lymphocytes was performed, there was no difference between gastrectomy and control groups at T:B = 40:60. When the lymphoid follicles were classified, the proportion of T lymphocytes increased in the secondary follicle (T:B = 40:60) compared with in the primary follicle (T:B = 20:80). Gastrectomy significantly activated lymphocytic intestinal immunity by altering the intestinal environment, causing colonic NLH. Gastrectomy in rats is a good animal model for the study of NLH in colorectal diseases.

Ovarian serous cystadenofibroma is a relatively rare subtype of serous cystadenoma classified as ovarian benign epithelial tumor. We report a rare case of ovarian serous cystadenofibroma with scattered lesions in pelvic cavity, like malignant disseminations. The patient was 22 years old, gravida 0, para 0. In the laparoscopic surgery, numerous hard yellowish-white solid masses of various sizes were present in the bilateral ovaries. Grossly similar masses were scattered in the fimbria of the fallopian tubes, peritoneum, and great omentum. Because the intraoperative rapid histological diagnosis was benign tumor, surgery was completed for only tumor excision. Postoperative histopathological diagnosis is serous cystadenofibroma. Similar pathological findings were noted in the scattered lesions in the peritoneum and great omentum. No malignant or borderline malignant finding was observed. Because of a benign disease, careful treatment taking fertility preservation into consideration is necessary, especially for young patients.

Ceramide synthase 6 (CERS6) promotes lung cancer metastasis by stimulating cancer cell migration. To examine the underlying mechanisms, we performed luciferase analysis of the CERS6 promoter region and identified the Y‐box as a cis‐acting element. As a parallel analysis of database records for 149 non–small‐cell lung cancer (NSCLC) cancer patients, we screened for trans‐acting factors with an expression level showing a correlation with CERS6 expression. Among the candidates noted, silencing of either CCAAT enhancer‐binding protein γ (CEBPγ) or Y‐box binding protein 1 (YBX1) reduced the CERS6 expression level. Following knockdown, CEBPγ and YBX1 were found to be independently associated with reductions in ceramide‐dependent lamellipodia formation as well as migration activity, while only CEBPγ may have induced CERS6 expression through specific binding to the Y‐box. The mRNA expression levels of CERS6, CEBPγ, and YBX1 were positively correlated with adenocarcinoma invasiveness. YBX1 expression was observed in all 20 examined clinical lung cancer specimens, while 6 of those showed a staining pattern similar to that of CERS6. The present findings suggest promotion of lung cancer migration by possible involvement of the transcription factors CEBPγ and YBX1., Schematic illustration showing CEBPγ functions for cancer cell migration.

Background: The definitive diagnosis of eosinophilic gastrointestinal disorders (EGID) relies on gastrointestinal endoscopy and histopathology, especially for the determination of the total number of infiltrating eosinophils per high-power field. The objective of this study was to clarify the difference in eosinophil counts by hematoxylin and eosin (H&E), Giemsa, and direct fast scarlet (DFS) staining using pathological tissue from patients with EGID. Methods: This retrospective study included 28 patients with eosinophilic gastroenteritis (EGE) and 10 with eosinophilic esophagitis (EoE) between January 2016 and June 2019. The judges included three pathologists and one clinical laboratory technician; we also included an outsider, who was not a judge, to set a specific site and set a site for measuring the eosinophil count using H&E, Giemsa, and DFS staining at the same site. The data were analyzed using a linear mixed-effect model. In addition, the difference in color value for each staining method was analyzed using the color value of the Commission International de l’Eclairage (CIE) luminance-a-b model (L a*b*). A certain color difference (ΔE) was obtained from the L a*b* value of the eosinophilic granules, with the difference from the other L a*b* values (ΔL, Δa, Δb), and the total color difference was calculated by ΔE.Results: Compared with the H&E stain, the Giemsa and DFS stains had higher counts, with DFS having the highest count (β = 35.151, 95%CI: 27.220 - 43.083, p < 0.001). There was a difference among all the raters (p < 0.001); however, the closer they were to being an expert, the smaller was the difference in the mean values for Giemsa and DFS stains. Additionally, the ΔE of the DFS stain was higher than that of the Giemsa stain in the plasma cells, erythrocytes, and stroma.Conclusions: The number of counted eosinophils was higher with Giemsa and DFS staining than with H&E staining. Eosinophil counting is reliable for any staining method and, although there were differences between the raters, relying on experts and expert assistants can reduce these differences. Therefore, the use of Giemsa and DFS staining with H&E staining may be useful for counting eosinophil.

The enzyme-labeled antigen method is an immunohistochemical technique detecting plasma cells producing specific antibodies in tissue sections. The probe is an antigen labeled with an enzyme or biotin. This immunohistochemical technique is appliable to frozen sections of paraformaldehyde (PFA)-fixed tissues, but it has been difficult to apply it to formalin-fixed, paraffin-embedded (FFPE) sections. In the current study, factors inactivating the antibody reactivity during the process of preparing FFPE sections were investigated. Lymph nodes of rats immunized with horseradish peroxidase (HRP) or a mixture of keyhole limpet hemocyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activity) or biotinylated proteins in 4% PFA-fixed frozen sections, significantly decreased in unbuffered 10% formalin-fixed frozen sections. The positive cells were further decreased by paraffin embedding following formalin fixation. In paraffin-embedded sections fixed in precipitating fixatives such as ethanol and acetone and those prepared with the AMeX method, the antigen-binding reactivity of antibodies was preserved. Fixation in periodate-lysine-paraformaldehyde and Zamboni solution also kept the antigen-binding reactivity in paraffin to some extent. In conclusion, formalin fixation and paraffin embedding were major causes inactivating antibodies. Precipitating fixatives could retain the antigen-binding reactivity of antibodies in paraffin-embedded sections.

Diagnosis of gastric adenocarcinoma using small biopsy samples is occasionally difficult. Various markers have been employed for improving the diagnostic accuracy, but there remains room for improvement. A total of 129 endoscopically biopsied samples were studied, consisting of 104 intramucosal tubular adenocarcinomas, 24 non-cancerous lesions and one cancer sample originally suspected of non-cancer but revised as cancer after immunostaining. We evaluated the association between histopathology and immunohistochemical expression of MUC1, HER2, p53, CEA, E-cadherin, β-catenin and claudin-18. Regarding β-catenin and claudin-18, not only membranous expression (β-catenin(M) and claudin-18(M)) but also nuclear expression (β-catenin(N) and claudin-18(N)) were analyzed. When subtyped with mucin core protein expression, the gastric-type cancers dominantly expressed claudin-18(M), while claudin-18(N) was significantly encountered in intestinal- and mixed-types. Expression of MUC1 (P = 0.0010), HER2 (P = 0.0173), p53 (P = 0.0002), CEA (P = 0.0019) and claudin-18(N) (P < 0.0001) revealed significant correlation with gastric cancers. Negative correlation of claudin-18(M) (P = 0.0125) was also noted. MUC1 and p53 were negative in non-cancer lesions. The non-cancer group exceptionally expressed HER2 and β-catenin(N). Membranous expression of E-cadherin was consistent in both groups. Logistic regression analysis showed that MUC1 (P = 0.0086), p53 (P = 0.0031), claudin-18(M) (P = 0.0158) and claudin-18(N) (P = 0.0190) were independently associated with gastric cancers. Nuclear expression of claudin-18 should be the novel diagnostic marker for gastric cancer.

Pit-1, the pituitary-specific transcriptional factor, has been known as a gene that regulates the functional differentiation of the anterior pituitary gland, especially in GH, PRL, and TSH production. Our immunohistochemical studies were performed to elucidate the functional roles of the pit-1 product in human pituitary adenomas. Eighty-six pituitary adenomas consisted of 51 GH-producing adenomas, 18 nonfunctioning (NF) adenomas, 1 1 PRL adenomas, and 6 TSH adenomas. Indirect immunoperoxidase method was performed using antibodies against GH, PRL, ACTH, the α subunit (SU), FSH β-SU, LH β-SU, TSH β-SU, and pit-1 product. Pit-1 product was expressed in 38 patients in the nuclei of the adenoma cells. The frequency of positive pit-1 product was significantly high in the patients with TSH and GH adenomas compared with those who had NF adenomas. These results suggest a role for pit-1 in functional differentiation of the human pituitary adenomas, especially TSH and GH adenomas.

Intestinal metaplasia induced by ectopic expression of caudal-type homeobox (CDX)2 and/or CDX1 (CDX) is frequently observed around gastric cancer (GC). Abnormal expression of CDX is also observed in GC and suggests that inappropriate gastrointestinal differentiation plays essential roles in gastric tumorigenesis, but their roles on tumorigenesis remain unelucidated. Publicly available databases show that GC patients with higher CDX expression have significantly better clinical outcomes. We introduced CDX2 and CDX1 genes separately into GC-originated MKN7 and TMK1 cells deficient in CDX. Marked suppression of cell growth and dramatic morphological change into spindle-shaped flat form were observed along with induction of intestinal marker genes. G0-G1 growth arrest was accompanied by changed expression of cell cycle-related genes but not with apoptosis or senescence. Microarray analyses additionally showed decreased expression of gastric marker genes and increased expression of stemness-associated genes. Hierarchical clustering of 111 GC tissues and 21 non-cancerous gastric tissues by selected 18 signature genes based on our transcriptome analyses clearly categorized the 132 tissues into non-cancer, "CDX signature"-positive GC, and "CDX signature"-negative GC. Gene set enrichment analysis indicated that "CDX signature"-positive GC has lower malignant features. Immunohistochemistry of 89 GC specimens showed that 50.6% were CDX2-deficient, 66.3% were CDX1-deficient, and 44.9% were concomitant CDX2/CDX1-deficient, suggesting that potentially targetable GC cases by induced intestinal differentiation are quite common. In conclusion, exogenous expression of CDX2/CDX1 can lead to efficient growth inhibition of CDX-deficient GC cells. It is based on rapidly induced intestinal differentiation, which may be a future therapeutic strategy.

Gastric cancer (GC) is rich in many different histological types, but how the histological pattern is defined remains to be proved. The relation between GC histological types and the expression of nectin1, which is one of the cell adhesion molecules that composes adherens junction, has not been reported. According to a publicly available database of 406 GC patients, the median overall survival of Nectin1 high expression patients was 55.4 months and that of low expression patients was 25.6 months (P = 0.0246). Using surgically or endoscopically resected GC samples, nectin1 expression was analyzed by immunohistochemistry. Nectin1 expressed at adherens junction in all the normal epithelial cells. However, nectin1 expressed not at adherens junction but at apical membrane in epithelial cells in intestinal metaplasia. The expression pattern of nectin1 in intestinal type GC resembled to intestinal metaplasia. In order to analyze the difference in nectin1 expression between GC histological types, a total of 116 intestinal type GC and 33 diffuse type GC. The expression of necitin1 in diffuse type GC (3.0%) was remarkably decreased compared to that in intestinal type GC (65.5%) (P < 0.0001). In conclusion, this is the first report showing an association between nectin1 expression and histological subtypes of GC.

The mechanism behind the pathogenesis and carcinogenesis of these neoplasms is not fully understood. The objective of this study was to identify genetic markers and pathways specific to precancerous duodenal adenomas and early stage adenocarcinomas through gene expression analysis. Gene expression profiling was performed in 4 pairs of duodenal adenoma/adenocarcinomas and corresponding matched normal tissue. Genes with consistent expression differences were identified and confirmed in 7 independent pairs. Gene set enrichment analysis (GSEA) was performed to characterize gene expression profiles of duodenal adenoma/adenocarcinomas, together with immunohistochemical staining of candidate oncogenic genes. 626 probes consistently demonstrated over a twofold expression difference between tumor–normal pairs. Reverse transcriptase polymerase chain reaction of genes with the most prominent difference in expression between tumors and normal mucosa (KLK7, KLK6, CEMIP, MMP7, KRT17, LGR5, G6PC, S100G, APOA1) validated the results of gene expression analysis. GSEA demonstrated a strong association between duodenal adenoma/adenocarcinomas with colorectal adenomas (p

Sporadic nonampullary duodenal epithelial tumors (NADETs) are uncommon, and thus their clinicopathological features have not been fully assessed. In this study, we have analyzed a series of early sporadic NADETs, focusing on various immunohistological features. We conducted a multicenter retrospective analysis of 68 patients with endoscopically resected sporadic NADETs. Associations between immunohistological features and clinicopathological features were statistically analyzed. The 68 patients consisted of 46 men (68%) and 22 women (32%) with a mean age of 60.7 ± 12.2 years (range 37–85 years). The 68 tumors were composed of 39 adenomas (57%) and 29 early-stage adenocarcinomas (43%). Duodenal adenocarcinomas were larger in size than adenomas and had papillary architecture in their pathological diagnosis with statistical significance. Duodenal adenocarcinomas also demonstrated a significantly higher expression of gastric markers (MUC5AC and MUC6) and a higher MIB-1 index. Duodenal adenomas were contrastively apt to express intestinal markers (MUC2, CDX1 and CDX2). Of the 68 cases analyzed, there were only 3 tumors positive for p53 staining, all of which were adenocarcinoma. When 7 submucosal invasive cancers and 21 intramucosal cancers were compared, submucosal invasion was positively associated with expression of MUC5AC. Also, submucosal invasion showed strong association with double-positivity of MUC5AC and MUC6. Our results indicate that immunohistochemical evaluation is useful for predicting malignant potential of NADETs, especially focusing on the expression of gastrointestinal markers.

We report the case of a 66 year-old woman with chronic atrial fibrillation, hypertrophic cardiomyopathy (HCM), and spinocerebellar atrophy (SCA). Her mother and first-born son had died of heart disease at the ages of 65 and 16 years, respectively. Four of her 8 siblings had died suddenly of unknown cause or of heart disease, and 2 others of cerebral infarction by the 7th decade. Genetic testing revealed that she had a novel mutation (c. 482C > A, p. Ala161Asp) in the troponin I gene (TNNI3), and no abnormality of the GAA repeat in the frataxin gene. Her older brother with SCA but without HCM was also analyzed, with no abnormality noted in either gene. The Ala161Asp mutation in TNNI3 was implicated in the pathogenesis of her HCM, though an association between HCM and SCA was not revealed.

Neutrophil extracellular traps (NETs) represent an extracellular, spider's web-like structure resulting from cell death of neutrophils. NETs play an important role in innate immunity against microbial infection, but their roles in human pathological processes remain largely unknown. NETs and fibrin meshwork both showing fibrillar structures are observed at the site of fibrinopurulent inflammation, as described in our sister paper [Acta Histochem. Cytochem. 49; 109-116, 2016]. In the present study, immunoelectron microscopic study was performed for visualizing NETs and fibrin fibrils (thick fibrils in our tongue) in formalin-fixed, paraffin-embedded sections of autopsied lung tissue of legionnaire's pneumonia. Lactoferrin and fibrinogen gamma chain were utilized as markers of NETs and fibrin, respectively. Analysis of immuno-scanning electron microscopy indicated that NETs constructed thin fibrils and granular materials were attached onto the NETs fibrils. The smooth-surfaced fibrin fibrils were much thicker than the NETs fibrils. Pre-embedding immunoelectron microscopy demonstrated that lactoferrin immunoreactivities were visible as dots on the fibrils, whereas fibrinogen gamma chain immunoreactivities were homogeneously observed throughout the fibrils. Usefulness of immunoelectron microscopic analysis of NETs and fibrin fibrils should be emphasized.

Japanese apricot (Prunus mume; ume) is a traditional food in Japan that has been shown to have various beneficial health effects. There is some evidence to suggest that ume is also effective against allergic disease. Here, we conducted a cross-sectional epidemiological pilot study to examine the association between ume intake frequency and allergic symptoms including rhinitis in 563 adults (288 men and 275 women) who resided in Wakayama, Japan. After adjusting for age, present illness and medication, women with high ume intake had significantly lower odds ratio (OR) for the presence of symptoms of allergy [OR: 0.49 with 95% confidence interval (CI): 0.25–0.97]. Therefore, we investigated the anti-allergic effect of ume on passive cutaneous anaphylaxis (PCA) reaction in immunoglobulin E (IgE)-sensitized mice. The animal study demonstrated that oral administration of ume extract attenuated the PCA reaction and mast cell degranulation. Furthermore, RBL-2H3 mast cells were used to identify anti-allergic ume compounds. The following ume compounds inhibited IgE-mediated mast cell degranulation: vanillin, syringic acid, protocatechuic aldehyde, lyoniresinol and p-coumaric acid. These results suggested that ume has the potential to inhibit mast cell degranulation and may be associated with reduced risk of allergic symptoms in women.

Gastric cancer (GC) is rich in many different histological types, but how the histological pattern is defined remains to be proved. The relation between GC histological types and the expression of nectin1, which is one of the cell adhesion molecules that composes adherens junction, has not been reported. According to a publicly available database of 406 GC patients, the median overall survival of Nectin1 high expression patients was 55.4 months and that of low expression patients was 25.6 months (P = 0.0246). Using surgically or endoscopically resected GC samples, nectin1 expression was analyzed by immunohistochemistry. Nectin1 expressed at adherens junction in all the normal epithelial cells. However, nectin1 expressed not at adherens junction but at apical membrane in epithelial cells in intestinal metaplasia. The expression pattern of nectin1 in intestinal type GC resembled to intestinal metaplasia. In order to analyze the difference in nectin1 expression between GC histological types, a total of 116 intestinal type GC and 33 diffuse type GC. The expression of necitin1 in diffuse type GC (3.0%) was remarkably decreased compared to that in intestinal type GC (65.5%) (P 0.0001). In conclusion, this is the first report showing an association between nectin1 expression and histological subtypes of GC.

We established a new “ecological” Grocott stain for demonstrating fungi, based upon a 4R principle of refusal, reduction, reuse, and recycle of waste management. Conventional Grocott stain employs environmentally harsh 5% chromic acid for oxidization. Initially, we succeeded in reducing the concentration of chromic acid from 5% to 1% by incubating the solution at 60°C and using five-fold diluted chromic acid solution at which point it was reusable. Eventually, we reached the refusal level where 1% periodic acid oxidization was efficient enough, when combined with preheating of sections in the electric jar, microwave oven, or pressure pan. For convenience sake, we recommend pressure pan heating in tap water for 10 min. Stainability of fungi in candidiasis and aspergillosis was comparable with conventional Grocott stain, while Mucor hyphae showed enhanced staining. The modified sequence was further applicable to detecting a variety of mycotic pathogens in paraffin sections. Our environmentally-friendly Grocott stain also has the advantage of avoiding risk of human exposure to hexavalent chromium solution in the histopathology laboratory. The simple stain sequence is can be easily applied worldwide.

Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme‐labeled antigen method is a novel histochemical technique that visualizes specific antibody‐producing cells in tissue sections by employing a biotin‐labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde‐fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme‐labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR‐detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti‐Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A‐reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.

We performed pre-embedding electron microscopic study for visualizing the antigen and genome of severe fever with thrombocytopenia syndrome (SFTS) virus in the cytoplasm of macrophages of the human splenic red pulp, both requesting preheating treatment of sections. To pursue this, coated glass slides with unique characteristics are needed. Namely, during staining they must prevent detaching off sections, but after staining the sections must be transferred to epoxy resin. Aminopropyltriexoxysilane-coated glass slides, widely used for immunostaining, were resistant to transfer to epoxy resin. In contrast, coated glass slides designated as Thinlayer Advanced Cytology Assay System (TACAS) were suitable for this purpose. The technique is also applicable to the coated glass slide-requiring cytology practice, in which immunocytochemical evaluation is needed after cell transfer to another glass slide.

Porphyromonas gingivalis is a keystone periodontal pathogen. Histologocally, the gingival tissue in periodontitis shows dense infiltration of plasma cells. However, antigens recognized by antibodies secreted from the immunocytes remain unknown. The enzyme-labeled antigen method was applied to detecting plasma cells producing P. gingivalis-specific antibodies in biopsied gingival tissue of periodontitis. N-terminally biotinylated P. gingivalis antigens, Ag53 and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro and Lys-hgp) were prepared by the cell-free protein synthesis system using wheatgerm extract. With these five labeled proteins as probes, 20 lesions of periodontitis were evaluated. With the AlphaScreen method, antibodies against any one of the five P. gingivalis antigens were detected in 11 (55%) serum samples and 17 (85%) tissue extracts. Using the enzyme-labeled antigen method on paraformaldehyde-fixed frozen sections of gingival tissue, plasma cells were labeled with any one of the five antigens in 17 (94%) of 18 specimens, in which evaluable plasma cells were detected. The positivity rates in periodontitis were significantly higher than those found previously in radicular cysts (20% in sera and 33% in tissue extracts with the AlphaScreen method, and 25% with the enzyme-labeled antigen method). Our findings directly indicate that antibodies reactive to P. gingivalis are locally produced in the gingival lesions, and that inflammatory reactions against P. gingivalis are involved in periodontitis.

Granulosa cells form ovarian follicles and play important roles in the growth and maturation of oocytes. The protection of granulosa cells from cellular injury caused by oxidative stress is an effective therapy for female infertility. We here investigated an effective bioactive compound derived from Prunus mume seed extract that protects granulosa cells from hydrogen peroxide (H2O2)-induced apoptosis. We detected the bioactive compound, 3,4-dihydroxybenzaldehyde (3,4-DHBA), via bioactivity-guided isolation and found that it inhibited the H2O2-induced apoptosis of granulosa cells. We also showed that 3,4-DHBA promoted estradiol secretion in granulosa cells and enhanced the mRNA expression levels of steroidogenic factor 1, a promoter of key steroidogenic enzymes. These results suggest that P. mume seed extract may have clinical potential for the prevention and treatment of female infertility.

Angiotensin II (Ang II) is a vasoactive hormone that has been implicated in cardiovascular diseases. Here, the effect of peach, Prunus persica L. Batsch, pulp extract on Ang II-induced intracellular Ca(2+) mobilization, reactive oxygen species (ROS) production and signal transduction events in cultured vascular smooth muscle cells (VSMCs) was investigated. Pretreatment of peach ethyl acetate extract inhibited Ang II-induced intracellular Ca(2+) elevation in VSMCs. Furthermore, Ang II-induced ROS generation, essential for signal transduction events, was diminished by the peach ethyl acetate extract. The peach ethyl acetate extract also attenuated the Ang II-induced phosphorylation of epidermal growth factor receptor and myosin phosphatase target subunit 1, both of which are associated with atherosclerosis and hypertension. These results suggest that peach ethyl acetate extract may have clinical potential for preventing cardiovascular diseases by interfering with Ang II-induced intracellular Ca(2+) elevation, the generation of ROS, and then blocking signal transduction events.

Synovial tissue in rheumatoid arthritis (RA) shows dense infiltration of plasmacytes. The purpose of the present study is to identify and localize autoantibodies produced in these immunocytes in RA synovitis. We developed a novel screening system for detecting specific autoantigens. Protein antigens recognized by antibodies in the serum and synovial tissue extract from five RA patients were screened with the AlphaScreen method. For screening, a biotinylated human autoantigen library was constructed by the wheat germ cell-free protein synthesis system. The AlphaScreen analysis of 2183 proteins detected a limited number of antigens reactive with the serum and synovial tissue extract. Eighteen biotinylated proteins, containing top five showing high signals in each synovitis tissue extract, were utilized as probes for the enzyme-labeled antigen method, in order to visualize the site of specific antibody production in synovial lesions. Specific antibodies against two proteins, tripartite motif-containing 21 (TRIM21, also known as SSA/Ro52) and F-box only protein 2 (FBXO2), were visualized in the cytoplasm of plasmacytes in two RA synovitis lesions, respectively. Absorption experiments using unlabeled proteins confirmed the specificity of staining. No positive signals against these two proteins were identified in the additionally evaluated RA and osteoarthritis synovial lesions. The present study indicated 1) the usefulness of screening the human autoantigen library with the AlphaScreen assay for detecting autoantibodies in RA synovitis, and 2) the applicability of biotinylated proteins to the enzyme-labeled antigen method for visualizing the site of autoantibody production within the lesion.

Neutrophil extracellular traps (NETs) are extracellular fibrillary structures composed of degraded chromatin and granules of neutrophil origin. In fibrinopurulent inflammation such as pneumonia and abscess, deposition of fibrillar eosinophilic material is a common histopathological finding under hematoxylin-eosin staining. Expectedly, not only fibrin fibrils but also NETs consist of the fibrillar material. The aim of the present study is to analyze immunohistochemically how NETs are involved in the inflammatory process. Archival formalin-fixed, paraffin-embedded sections accompanying marked neutrophilic infiltration were the target of analysis. Neutrophil-associated substances (citrullinated histone H3, lactoferrin, myeloperoxidase and neutrophil elastase) were evaluated as NETs markers, while fibrinogen gamma chain was employed as a fibrin marker. Light microscopically, the fibrils were categorized into three types: thin, thick and clustered thick. Lactoferrin represented a good and stable NETs marker. Thin fibrils belonged to NETs. Thick fibrils are composed of either mixed NETs and fibrin or fibrin alone. Clustered thick fibrils were solely composed of fibrin. Neutrophils were entrapped within the fibrilllar meshwork of the thin and thick types. Apoptotic cells immunoreactive to cleaved caspase 3 and cleaved actin were dispersed in the NETs. In conclusion, NETs and fibrin meshwork were consistently recognizable by immunostaining for lactoferrin and fibrinogen gamma chain.

The mammalian transcriptional factors, Cdx1 and Cdx2 (Cdx is caudal-type homeobox) are paralogues and critical for the cellular differentiation of intestinal or colorectal epithelia. It has been reported previously that in Cdx1 transgenic or knockout mice, endogenous Cdx2 levels are inversely correlated with Cdx1 levels. Recently, we found that exogenous Cdx1 expression can suppress Cdx2 in a human colorectal tumour cell line, SW480, although the underlying molecular mechanisms were unclear. In the present study, we show that several microRNAs induced by exogenous Cdx1 expression directly bind to the CDX2 mRNA 3′UTR (untranslated region) to destabilize these transcripts, finally leading to their degradation. Using microarray analysis, we found that several miRNAs that were computationally predicted to target CDX2 mRNAs are up-regulated by exogenous Cdx1 expression in SW480 cells. Among these molecules, we identified miR-9 , miR-16 and miR-22 as having the potential to suppress Cdx2 through the binding of the 3′UTR to its transcript. Importantly, simultaneous mutations of both the miR-9 - and miR-16 -binding sites in the CDX2 3′UTR were shown to be sufficient to block Cdx2 suppression. The results of the present study suggest a unique feature of miRNAs in which they contribute to homoeostasis by limiting the levels of transcription factors belonging to the same gene family. Abbreviations: CDX/Cdx, caudal-type homeobox; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miRNA, microRNA; RT, reverse transcription; UTR, untranslated region; VSV-G, vesicular stomatitis virus glycoprotein

Rebamipide is usually used for mucosal protection, healing of gastric ulcers, treatment of gastritis, etc., but its effects on gastric malignancy have not been elucidated. Using Lewis and Buffalo rat strains treated with peroral administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we evaluated the effect of rebamipide on the induction of tumor-suppressive dendritic cells, which are known to be heterogeneous antigen-presenting cells of bone marrow origin and are critical for the initiation of primary T-cell responses. Using CD68 as a marker for dendritic cells, the stomach pyloric mucosae of Lewis and Buffalo rats were immunohistochemically analyzed in the presence or absence of rebamipide and MNNG. After a 14-day treatment of rebamipide alone, no significant change in number of CD68-expressing cells was detected in either rat strain. However, after concurrent exposure to MNNG for 14 days, treatment with rebamipide slightly increased CD68-positive cells in the Lewis strain, and significantly increased them in the Buffalo strain. Analysis of two chemotactic factors of dendritic cells, IL-1β and TNF-α, in the gastric cancer cells showed that expression of IL-1β, but not TNF-α, was induced by rebamipide in a dose-dependent manner. A luciferase promoter assay using gastric SH-10-TC cells demonstrated that an element mediating rebamipide action exists in the IL-1β gene promoter region. In conclusion, rebamipide has potential tumor-suppressive effects on gastric tumorigenesis via the recruitment of dendritic cells, based on the upregulation of the IL-1β gene in gastric epithelial cells.

The chromatin remodeling complex SWI/SNF is an important epigenetic regulator that includes one Brm or BRG1 molecule as catalytic subunit. Brm and BRG1 do not function identically, so this complex can regulate gene expression either positively or negatively, depending on the promoter to which it is recruited. Notably, Brm attenuation due to posttranscription suppression occurs often in human tumor cells, in which this event contributes to their oncogenic potential. Here, we report that the 3′-untranslated region of Brm mRNA has two sites that are efficiently targeted by the microRNAs miR-199a-5p and -3p, revealing a novel mechanism for modulation of Brm-type SWI/SNF activity. Computational mapping of the putative promoter region of miR-199a-2 (miPPR-199a-2) has defined it as the major contributing genetic locus for miR-199a-5p and-3p production in these tumor cell lines. We validated this predicted region by direct promoter analysis to confirm that Egr1 is a strong positive regulator of the miR-199a-2 gene. Importantly, we also showed that Egr1, miR-199a-5p, and miR-199a-3p are expressed at high levels in Brm-deficient tumor cell lines but only marginally in Brm-expressing tumor cells. Finally, we also obtained evidence that Brm negatively regulates Egr1. Together, our results reveal that miR-199a and Brm form a double-negative feedback loop through Egr1, leading to the generation of these two distinct cell types during carcinogenesis. This mechanism may offer a partial explanation for why miR-199a-5p and -3p have been reported to be either upregulated or downregulated in a variety of tumors. Cancer Res; 71(5); 1680–9. ©2010 AACR.