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Testicular teratomas are the major histologic type of testicular germ cell tumors and their incidence continues to grow. Moreover, teratomas can develop from undifferentiated cells in induced pluripotent stem (iPS) cell transplantation therapy, seriously hampering the progress of regenerative medicine. Germinal center-associated nuclear protein (GANP) is thought to be important to the biogenetic control of primordial germ cells and is among the genes susceptible to testicular germ cell tumors. Thus, we analyzed the expression of GANP in human testicular postpubertal-type teratomas and established a novel mouse model to reveal the association between GANP and teratomagenesis. We analyzed 31 cases of human testicular postpubertal-type teratomas and, in all cases, GANP was overexpressed. The aberrant expression was also detected in germ cell neoplasia in situ accompanied by the teratoma. GANP expression was particularly high in the epithelia of the epidermis, cutaneous appendages, and trachea-like ciliated epithelium. To further clarify the association between GANP and teratomagenesis, we established a novel teratomagenesis mouse model (CAG-ganp

Macrophages are classified into two phenotypes, M1 and M2, based on their roles. M2 macrophages suppress inflammation and increase in proportion to the malignancy of brain tumors. Recently, macrophage extracellular traps (METs), which change into a network, have been reported as a unique form of macrophage cell death. In this study, immunohistochemical analysis of macrophages in METs in human glioblastoma was performed. To distinguish between M1 and M2 macrophages, multiple immunostainings with Iba1 combined with CD163 or CD204 were performed. M2 macrophages were present in small amounts in normal and borderline areas but showed an increasing trend as they shifted to tumor areas, and most of them were the activated- or phagocytic-type. We also successfully detected METs coexisting with fibrin and lactoferrin near the border between the tumor and necrotic area. M2 macrophages not only suppressed inflammation but also were involved in the formation of METs. This study found that M2 macrophages play various roles in unstable situations.

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.

We herein report a case of Behçet's disease with renal infarction due to mucormycosis. A 76-year-old man with entero-Behçet's disease had been treated with glucocorticoid and tumor necrosis factor (TNF) inhibitors. His entero-Behçet's disease was refractory to these treatments, and ileocecal resection was performed. After the operation, renal infarction that was unresponsive to anticoagulation therapy developed. He ultimately died of renal failure due to renal infarction. At the autopsy, histopathology of abundant hyphae in the renal vessel wall revealed mucormycosis. Renal mucormycosis is an important cause of renal failure with renal infarction in immunocompromised patients.

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

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 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

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

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 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 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 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

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.

Huntingtin-associated protein 1 (HAP1) is abundantly expressed in the neurons of the central nervous system and forms unique intracytoplasmic inclusions of unknown function called ‘stigmoid bodies’ (STBs). Transmission electron microscopy has revealed that the STBs are aggregates of granules containing cavities with a diameter of 0.5–3 µm. Small STBs fuse to form larger STBs, the size of which is said to vary depending on the developmental growth stage and brain region. Light microscopy can only reveal that these STBs have similar circular shapes due to its limited resolution. Therefore, light microscopy is only fit for the study of the STB distribution and quantitative changes. We, herein, suggest the adoption of correlative light and electron microscopy, which combines confocal laser scanning microscopy and scanning electron microscopy as the method allowing us to identify the huntingtin-associated protein 1–positive STBs in formalin-fixed paraffin-embedded (FFPE) sections. This approach allows us to study the three-dimensional morphology of immunolabeled objects in histopathological specimens. The STBs in FFPE sections of murine hypothalami reflected the transmission electron microscopic images of Epon-embedded STBs, although we were not able to observe any organelle covering the STBs of the FFPE sections. Furthermore, we were able to reconstruct the three-dimensional structure of the STB, and we identified it to be of spherical form, covered with mitochondria and rough endoplasmic reticulum, and bearing a cluster of cavities in the centre. In the future, we might gain new insights by comparing the three-dimensional structure of the STB between different neurons and under a variety of conditions.

Primary central nervous system lymphomas (PCNSLs) rarely exhibit intratumoral hemorrhage. The differential diagnosis of hemorrhagic neoplasms of the central nervous system (CNS) currently includes metastatic carcinomas, melanomas, choriocarcinomas, oligodendrogliomas, and glioblastomas. Here we present the clinical, radiological, pathological, and molecular genetic features of six cases of PCNSL associated with intratumoral hemorrhage. The median age of patients was 75 years, with male predominance. While conventional PCNSLs were associated with low cerebral blood volume (CBV), perfusion magnetic resonance imaging (MRI) revealed elevated CBV in three cases, consistent with vascular proliferation. All six cases were diagnosed pathologically as having diffuse large B-cell lymphoma (DLBCL) with a non-germinal center B-cell-like (non-GCB) phenotype; marked histiocytic infiltrates and abundant non-neoplastic T-cells were observed in most cases. Expression of vascular endothelial growth factor and CD105 in the lymphoma cells and the small vessels, respectively, suggested angiogenesis within the neoplasms. Neoplastic cells were immunohistochemically negative for programmed cell death ligand 1 (PD-L1), while immune cells in the microenvironment were positive for PD-L1. Mutations in the MYD88 gene (MYD88) (L265P) and the CD79B gene (CD79B) were detected in five and one case, respectively. As therapeutic modalities used for PCNSLs differ from those that target conventional hemorrhagic neoplasms, full tissue diagnoses of all hemorrhagic CNS tumors are clearly warranted.

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.

The group A rotavirus (RVA) genome comprising 11 double-stranded RNAs encodes six structural proteins (VP1-VP4, VP6, and VP7) and six non-structural proteins (NSP1-NSP6). Among these 12 rotaviral proteins, NSP6 has been less studied as to its function. We previously prepared a recombinant NSP6-deficient RVA derived from simian strain SA11-L2 by reverse genetics, and found that the NSP6-deficient virus grew well in cell culture, although its growth was less abundant than that of the parental SA11-L2 strain. In this study, we examined the potency of a recombinant RVA incapable of NSP6 expression to cause diarrhoea in suckling mice. The suckling mice infected with the NSP6-deficient virus apparently experienced diarrhoea, although the symptom was milder and the duration of diarrhoea was shorter than in the mice infected with the authentic SA11-L2 strain. Thus, together with the results obtained for cultured cells in the previous study, it can be concluded that NSP6 is not necessarily required for replication and pathogenicity in vitro and in vivo.

Indoleamine 2,3‐dioxygenase 1 (IDO1) is a key enzyme associated with immunomodulation through its regulation of the tryptophan‐kynurenine (Kyn) pathway in advanced cancers, including metastatic renal cell carcinoma (mRCC). However, the failure of IDO1 inhibitors when used in combination with immune checkpoint inhibitors (ICIs), as observed in clinical trials, raises a number of questions. This study aimed to investigate the association of tryptophan 2,3‐dioxygenase (TDO) and IDO1 with cancer development and resistance to immunotherapy in patients with RCC. In our analysis of RCC tissue samples, tissue Kyn levels were elevated in advanced‐stage RCC and correlated well with TDO expression levels in RCC tumor cells. In patients with mRCC, TDO rather than IDO1 was expressed in RCC tumor cells, showing a strong association with Kyn expression. Furthermore, immunohistochemical staining of TDO was strongly associated with the staining intensity of forkhead box P3, as well as ICI therapy response and survival in patients with mRCC. Our study is the first to show that TDO expression in tumor tissues is associated with progression and survival, confirming its potential as a predictive biomarker of primary resistance to immunotherapy in patients with mRCC. Our findings suggest that strategies aimed at inhibiting TDO, rather than IDO1, in combination with ICI therapy may aid in the control of mRCC progression., This study aimed to investigate the association of tryptophan 2,3‐dioxygenase (TDO) and indoleamine 2,3‐dioxygenase 1 (IDO1) with cancer development and resistance to immunotherapy in renal cell carcinoma (RCC) patients. Our results suggest that TDO expression in tumor cells is associated with kynurenine accumulation, progression, and survival, confirming its potential as a predictive biomarker of primary resistance to immunotherapy in RCC patients. We believe that our study makes a significant contribution to the literature because, to the best of our knowledge, it is the first to show that the accumulation of kynurenine induced by TDO in tumor tissues is associated with progression and survival. Further, we believe that this paper will be of interest to the readership of your journal because our results strongly recommend the testing of specific TDO inhibitors or dual inhibitors of IDO1 and TDO with ICI treatment in future preclinical and clinical trials focusing on mRCC.

Senile (atrophic) colpitis is microscopically characterized by the predominance of parabasal squamous cells and the paucity of superficial cells. The activated parabasal cells or dyskeratotic superficial cells may be confused with squamous intraepithelial lesion (SIL) in the routine cytology practice. A total of 29 cervical cytology specimens diagnosed as atypical squamous cells (ASC) or SIL were retrospectively sampled from 24 postmenopausal women (age range: 56–84 years, mean: 65.5, median: 65). Cytological diagnoses in the routine services included ASC-US 20, ASC-H 2, LSIL 4 and HSIL 3. All the specimens showed an atrophic background (senile colpitis). There were two microscopic patterns of senile colpitis with atypia: 1) parabasal cells were clustered (n = 23) and 2) dyskeratotic superficial cells were seen in a highly inflamed background (n = 6). Immunostaining for p16-INK4a (p16 in short) was performed, after the cells were transferred to trimethoxy[3-(phenylamino)propyl]silane-coated glass slides. Only three of the 29 cytology specimens (two of the 24 cases) judged as HSIL cytologically revealed p16 positivity in clustered atypical parabasal cells. Biopsy was performed in 11 cases, and chronic cervicitis without p16 expression was seen in nine. Two lesions showed p16-positive dysplasia (one mild dysplasia and another moderate dysplasia). In one lesion in an 84 year-old female, both cytology and histology specimens showed p16 positivity (HSIL). Discrepancy of p16 expression between the cytology and histology specimens was encountered in two lesions, representing sampling errors. It is of note that the overcytodiagnosis is avoidable with the aid of p16 immunostaining. Keywords: Atypical squamous cells (ASC), cell transfer technique, p16-INK4a, senile colpitis, squamous intraepithelial lesion (SIL).

An autopsy case (85-year-old Japanese male) of myeloperoxidase- (MPO-) positive acute myeloid leukemia with maturation (M1) accompanying megakaryocytic differentiation is presented. The patient manifested acute coronary syndrome. Even after emergent percutaneous coronary intervention, his performance status remained poor, so no chemotherapy against leukemia was given. The final white blood cell count reached 291,700/μL, and the platelet count was elevated to 510,000/μL. No cytogenetic studies were performed. He died at the 25th day of hospitalization. Autopsy revealed marked leukemic infiltration to the endocardium and subendocardial myocardium. Subendocardial myonecrosis was surrounded or replaced by the leukemic blasts, and neither granulation tissue reaction nor fibrosis was observed. In the cardiovascular lumen, lard-like blood clots were formed and microscopically consisted of leukemic blasts and platelets (leukemic thrombi). Infiltration of leukemic blasts was seen in the body cavities and systemic organs including the lung. The MPO-positive blasts lacked azurophilic granules and expressed the stem cell markers, CD34 and CD117 (c-kit). No features of myelofibrosis were seen in the 100% cellular marrow. In the endocardium, liver, lymph nodes, and bone marrow, megakaryocytic cells (CD42b/CD61+, MPO-) were distributed, while the small-sized blastic cells in the blood and tissues predominantly expressed MPO. The blasts lacked expression of CD42b/CD61. Megakaryocytic differentiation might be stimulated by certain tissue factors. AML accompanying megakaryocytic differentiation in certain tissues and organs should be distinguished from acute megakaryoblastic leukemia. The mechanisms provoking acute coronary syndrome in acute myeloid leukemia are discussed.

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.

Sarcoidosis is a rare disease of isolated or diffuse granulomatous inflammation. Although any organs can be affected by sarcoidosis, cardiac sarcoidosis is a fatal disorder, and it is crucial to accurately diagnose it to prevent sudden death due to dysrhythmia. Although endomyocardial biopsy is invasive and has limited sensitivity for identifying granulomas, it is the only modality that yields a definitive diagnosis of cardiac sarcoidosis. It is imperative to develop novel pathological approaches for the precise diagnosis of cardiac sarcoidosis. Here, we aimed to discuss commonly used diagnostic criteria for cardiac sarcoidosis and to summarize useful and novel histopathologic criteria of cardiac sarcoidosis. While classical histologic observations including noncaseating granulomas and multinucleated giant cells (typically Langhans type) are the most important findings, others such as microgranulomas, CD68+ CD163− pro-inflammatory (M1) macrophage accumulation, CD4/CD8 T-cell ratio, Cutibacterium acnes components, lymphangiogenesis, confluent fibrosis, and fatty infiltration may help to improve the sensitivity of endomyocardial biopsy for detecting cardiac sarcoidosis. These novel histologic findings are based on the pathology of cardiac sarcoidosis. We also discussed the principal histologic differential diagnoses of cardiac sarcoidosis, such as tuberculosis myocarditis, fungal myocarditis, giant cell myocarditis, and dilated cardiomyopathy.

Daily fat and sugar intake has increased in Japan, while total energy intake has decreased. However, the number of type 2 diabetes mellitus patients has increased, and this often causes renal injury characterized by autophagic vacuoles. Although many studies with comparisons of high fat or sugar versus a normal macronutrient balanced diet have been reported, there are few studies that equalized calorie intake and body weights. In the current study, AIN93M diets (CONT group) with matching energy content with lard derived high saturated fat (LARD group), soybean oil derived unsaturated fat (SOY OIL group) and sucrose (SUCROSE group) were provided to compare their effects on renal morphology in streptozotocin-injected CD-1 mice without causing obesity. The number of renal tubular vacuoles was higher in SUCROSE and slightly higher in LARD compared with CONT mice, and was higher in LARD and SUCROSE compared with SOY OIL mice. Most of those vacuoles were LAMP1-positive, a marker of lysosomal autophagy. These results suggest that despite identical energy contents, diets with high sucrose or saturated fat compared to unsaturated fat may aggravate lysosomal renal injury in a non-obese, streptozotocin-induced model of diabetes mellitus.

Fructose consumption is rising globally, but maternal high fructose intake might adversely affect offspring. Our previous report demonstrated that excess maternal fructose intake impairs hippocampa...

ObjectiveTo investigate the expression of basic fibroblast growth factor in the matrix of human acquired cholesteatoma compared to the deep meatal skin. This topic does not appear to have been fully investigated before.MethodsAn immunochemical study was conducted. Cholesteatoma tissues from adult patients were collected during surgery (n = 19). Control specimens were taken from the deep meatal skin (n = 8) and compared.ResultsA highly significant difference in basic fibroblast growth factor expression was identified between cholesteatoma and skin (mean ± standard error = 58.53 ± 3.6 per cent in cholesteatoma vs 40.6 ± 3.5 per cent in skin; p = 0.005). Both basal and parabasal keratinocytes were stained positive with basic fibroblast growth factor. Additionally, there was specific staining in the basal columnar middle-ear epithelium and mast cell membrane.ConclusionBasic fibroblast growth factor plays an active role in proliferative activity of cholesteatoma through its overexpression in basal and parabasal layers of cholesteatoma matrix. Moreover, its expression in the mast cell membrane supports its role in bone resorption activity.

Inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis, are chronic inflammatory disorders associated with oxidative stress. The intestines produce 5-hydroxytryptamine that may negatively affect disease state under inflammatory conditions when overproduced. 5-Hydroxytryptamine is a substrate for myeloperoxidase and is converted into reactive tryptamine-4,5-dione. Here, an experimental colitis model was established through oral administration of 5% dextran sulfate sodium to ICR mice for 7 days. Furthermore, the formation of tryptamine-4,5-dione in the colorectal mucosa/submucosa and colorectal tissue was analyzed by chemical and immunochemical methodologies. First, free tryptamine-4,5-dione in the homogenate was chemically trapped by o-phenylenediamine and analyzed as the stable phenazine derivative. Tryptamine-4,5-dione localization as adducted proteins in the colorectal tissue was immunohistochemically confirmed, and as demonstrated by both methods, this resulted in the significant increase of tryptamine-4,5-dione in dextran sulfate sodium-challenged mice compared with control mice. Immunohistochemical staining confirmed tryptamine-4,5-dione-positive staining at the myeloperoxidase accumulation site in dextran sulfate sodium-challenged mice colorectal tissue. The tryptamine-4,5-dione locus in the mice was partly matched with that of a specific marker for myeloperoxidase, halogenated tyrosine. Overall, the results possibly indicate that tryptamine-4,5-dione is generated by neutrophil myeloperoxidase in inflammatory tissue and may contribute to the development of inflammatory bowel disease.

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.

Although angiogenesis plays a crucial role in cancer growth and progression, no reliable method for assessing angiogenesis in tumor tissue sections currently is available. Using biomarkers with high specificity for proliferating endothelial cells could help quantify angiogenic activity. Thymidine kinase-1 (TK1) is an enzyme involved in the salvage pathway of DNA synthesis and its activity is correlated with cell proliferation. We investigated the use of double immunostaining for TK1 and CD31 for identifying activated tumor vessels. Differences in TK1/CD31 positive vessel rates (PVRs) between tumor and adjacent normal tissues were evaluated in 39 colorectal carcinoma (CRC) samples and compared with those of Ki67/CD31 double stained tissues. Mean TK1/CD31 PVR (23.6%) in CRCs was 13.9 fold greater than in adjacent normal tissues (1.7%)). By comparison, mean Ki67/CD31 PVR in CRCs was 20.0%, i.e. only 4.8 fold greater than in normal tissues (4.2%). Also, mean TK1/CD31 PVR in normal tissues was significantly less than mean Ki67/CD31 PVR. Our findings indicate that double immunostaining for TK1/CD31 can detect activated tumor vessels more accurately than staining for Ki67/CD31 and potentially could identify tumors that will respond to anti-angiogenic therapy.

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