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Background Particulate matter (PM) is one of the principal causes of human respiratory disabilities resulting from air pollution. Animal models have been applied to discover preventive and therapeutic drugs for lung diseases caused by PM. However, the induced severity of lung injury in animal models using PM varies from study to study due to disparities in the preparation of PM, and the route and number of PM administrations. In this study, we established an in vivo model to evaluate PM-induced lung injury in mice. Results PM dispersion was prepared using SRM2975. Reactive oxygen species were increased in MLE 12 cells exposed to this PM dispersion. In vivo studies were conducted in the PM single challenge model, PM multiple challenge model, and PM challenge with ovalbumin-induced asthma using the PM dispersion. No histopathological changes were observed in lung tissues after a single injection of PM, whereas mild to moderate lung inflammation was obtained in the lungs of mice exposed to PM three times. However, fibrotic changes were barely seen, even though transmission electron microscopy (TEM) studies revealed the presence of PM particles in the alveolar macrophages and alveolar capillaries. In the OVA-PM model, peribronchial inflammation and mucous hypersecretion were more severe in the OVA+PM group than the OVA group. Serum IgE levels tended to increase in OVA+PM group than in OVA group. Conclusions In this study, we established a PM-induced lung injury model to examine the lung damage induced by PM. Based on our results, repeated exposures of PM are necessary to induce lung inflammation by PM alone. PM challenge, in the presence of underlying diseases such as asthma, can also be an appropriate model for studying the health effect of PM.

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. Rag-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of Rag-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of Rag-2, we recently established a new Rag-2 knockout FVB mouse line (Rag-2-/-) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. Rag-2-/- mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in Rag-2-/- mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in Rag-2-/- mice. These findings indicate that our Rag-2-/- mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

Trefoil factor 1 (TFF1, also known as pS2) is strongly expressed in the gastrointestinal mucosa and plays a critical role in the differentiation of gastric glands. Since approximately 50% of all human gastric cancers are associated with decreased TFF1 expression, it is considered a tumor suppressor gene. Tff1 deficiency in mice results in histological changes in the antral and pyloric gastric mucosa, with severe hyperplasia and dysplasia of epithelial cells, resulting in the development of antropyloric adenoma. Here, we generated Tff1-knockout (KO) mice, without a neomycin resistant (NeoR) cassette, using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRSIPR/Cas9) system. Though our Tff1-KO mice showed phenotypes very similar to the previous embryonic stem (ES)-cell-based KO mice, they differed from the previous reports in that a reduction in body weight was observed in males. These results demonstrate that these newly established Tff1-KO mice are useful tools for investigating genetic and environmental factors influencing gastric cancer, without the effects of artificial gene insertion. Furthermore, these findings suggest a novel hypothesis that Tff1 expression influences gender differences.

Engineered ionizable lipid nanoparticles (LNPs) were developed for the LSEC-specific delivery of RNA therapeutics., Ionizable lipid nanoparticles (LNPs) have been widely used for in vivo delivery of RNA therapeutics into the liver. However, a main challenge remains to develop LNP formulations for selective delivery of RNA into certain types of liver cells, such as hepatocytes and liver sinusoidal endothelial cells (LSECs). Here, we report the engineered LNPs for the targeted delivery of RNA into hepatocytes and LSECs. The effects of particle size and polyethylene glycol–lipid content in the LNPs were evaluated for the hepatocyte-specific delivery of mRNA by ApoE-mediated cellular uptake through low-density lipoprotein receptors. Targeted delivery of RNA to LSECs was further investigated using active ligands. Incorporation of mannose allowed the selective delivery of RNA to LSECs, while minimizing the unwanted cellular uptake by hepatocytes. These results demonstrate that engineered LNPs have great potential for the cell type–specific delivery of RNA into the liver and other tissues.

Melanoma is a disease with a high recurrence rate and poor prognosis; therefore, the need for targeted therapeutics is steadily increasing. Oligodendrocyte transcription factor2 (Olig2) is a basic helix-loop-helix transcription factor that is expressed in the central nervous system during embryonic development. Olig2 is overexpressed in various malignant cell lines such as lung carcinoma, glioma and melanoma. Olig2 is known as a key transcription factor that promotes tumor growth in malignant glioma. However, the role of Olig2 in melanoma is not well characterized. We analyzed the role of Olig2 in apoptosis, migration, and invasion of melanoma cells. We confirmed that Olig2 was overexpressed in melanoma cells and tissues. Reduction of Olig2 increased apoptosis in melanoma cells by increasing p53 level and caspase-3/-7 enzyme activity. In addition, downregulation of Olig2 suppressed migration and invasion of melanoma cells by inhibiting EMT. Reduction of Olig2 inhibited expression of MMP-1 and the enzyme activity of MMP-2/-9 induced by TGF-β. Moreover, Olig2 was involved in the downstream stages of MEK/ERK and PI3K/AKT, which are major signaling pathways in metastatic progression of melanoma. In conclusion, this study demonstrated the crucial roles of Olig2 in apoptosis, migration, and invasion of melanoma and may help to further our understanding of the relationship between Olig2 and melanoma progression.

The compound 6:2 chlorinated polyfluorinated ether sulfonate (F-53B), a replacement for perfluorooctanesulfonate (PFOS) in the electroplating industry, has been widely detected in numerous environmental matrices, human sera, and organisms. Due to regulations that limit PFOS use, F-53B use is expected to increase. Therefore, in this study, we performed a subchronic oral toxicity study of F-53B in Sprague Dawley (SD) rats. F-53B was administered orally once daily to male and female rats for 28 days at doses of 5, 20, and 100 mg/kg/day. There were no toxicologically significant changes in F-53B-treated rats, except in the thyroid gland. However, F-53B slightly reduced the serum concentrations of thyroid hormones, including triiodothyronine and thyroxine, compared with their concentrations in the vehicle group. F-53B also induced follicular hyperplasia and was associated with increased thyroid hormone biosynthesis-associated protein expression. These results demonstrate that F-53B is a strong regulator of thyroid hormones in SD rats as it disrupts thyroid function. Thus, caution should be exercised in the industrial application of F-53B as an alternative for PFOS.

Skin irritation tests using reconstructed human epidermis (RhE) employ viability as an endpoint, but color interference or borderline results are often problematic. We examined whether the cytology of cells from treated RhE could determine skin irritancy. Six chemicals (three irritants; DnP, 1-B, PH, three non-irritants; DP, APA, HS) were evaluated in a RhE, Keraskin™. DP, HS, and PH were clearly classified with viability, but DnP, 1-B, and APA were often falsely determined, due to borderline values falling near the cutoff, 50%. In histology, the tissues treated with DnP, 1-B, and PH showed erosion of the stratum corneum, vacuolization, and necrosis in the basal layer. DP- and HS-treated tissues showed relatively normal morphology but APA induced necrosis similar to irritants. Cytology revealed that DnP, 1-B or PH depleted cells and induced irregular and abnormal cell shapes. In contrast, relatively regular and normal shapes and clear distinction between the nucleus and cytoplasm was observed for DP, APA and HS. To further confirm it, additional 10 substances, including false positives from OECD TG 439, were tested. Overall (16 substances in total), cytology: total area predicted the skin irritancy of test chemicals with the highest accuracy (87.5%) followed by cytology: cell count (81.3%), histology (75%) and viability (68.8%), confirming the utility of cytology as an alternative endpoint in the skin irritation test using RhE.

Phthalates widely used in the manufacture of plastics have deeply penetrated into our everyday lives. Recently, a concern over the toxicity of phthalates on thyroid, has been raised but in most of cases, the doses employed were unrealistically high. To investigate the effects of phthalates on thyroid, we investigated the effects of the repeated oral exposure to low to high doses (0.3, 3, 30 and 150 mg/kg) di-2-ethylhexylphthalate (DEHP) from weaning to maturity for 90 days in juvenile rats on the thyroid. The histological examination revealed that DEHP significantly induced hyperplasia in the thyroid from the doses of 30 mg/kg, which was confirmed with Ki67 staining. In line with this finding, increased mRNA expression of thyrotropin releasing hormone (Trh) was observed in the thyroid of female at 0.3 mg/kg and 150 mg/kg as determined by RNAseq analysis. Moreover, significantly increased expression of parathyroid hormone (Pth) in the female at 0.3 mg/kg, and thyroglobulin (Tg) and thyroid hormone responsive (Thrsp) in the male at 0.3 mg/kg were noted in the blood, of which changes were substantially attenuated at 150 m/kg, alluding the meaningful effects of low dose DEHP on the thyroid hormone regulation. Urinary excretion of mono-2-ethylhexyl-phthalate (MEHP), a major metabolite of DEHP was determined to be 4.10 and 12.26 ppb in male, 6.65 and 324 ppb in female at 0.3 and 30 mg/kg DEHP, respectively, which fell within reported human urine levels. Collectively, these results suggest a potential adverse effects of low dose phthalates on the thyroid.

Pancreatitis is an inflammatory disorder of pancreas which leads to varying degrees of pancreatic endocrine and exocrine dysfunction and manifests in either acute or chronic forms. Spontaneous pancreatitis in experimental animals has rarely been reported. Here, we found acute to chronic courses of spontaneous pancreatitis in spontaneously hypertensive rats (SHRs), showing the formation of tubular complexes (TCs) and enhanced islet regeneration. We investigated the expression pattern of clusterin in the pancreas of SHRs based on immunohistochemistry (IHC). IHC analysis revealed the strong expression of clusterin in dedifferentiated duct-like cells and regenerative islets of TCs. These results imply that clusterin might be involved in the formation of TCs and parenchymal regeneration during rat pancreatitis.

p16Ink4a/CDKN2A is a tumor suppressor that critically regulates the cell cycle. Indeed, p16Ink4a deficiency promotes tumor formation in various tissues. We now report that p16Ink4a deficiency in female mice, but not male mice, induces leanness especially in old age, as indicated by lower body weight and smaller white adipose tissue, although other major organs are unaffected. Unexpectedly, the integrity, number, and sizes of adipocytes in white adipose tissue were unaffected, as was macrophage infiltration. Hence, hypermobility appeared to be accountable for the phenotype, since food consumption was not altered. Histological analysis of the cerebellum and deep cerebellar nuclei, a vital sensorimotor control center, revealed increased proliferation of neuronal cells and improved cerebellum integrity. Expression of estrogen receptor β (ERβ) and PCNA also increased in deep cerebellar nuclei, implying crosstalk between p16Ink4a and ERβ. Furthermore, p16Ink4a deficiency expands LC3B+ cells and GFAP+ astrocytes in response to estrogen. Collectively, the data suggest that loss of p16INK4a induces sexually dimorphic leanness in female mice, which appears to be due to protection against cerebellar senescence by promoting neuronal proliferation and homeostasis via ERβ.

Phthalates are being suggested to be associated with altered thyroid function and proliferative changes, but detailed mechanisms remain unclear. Here, we examined the effects of di-(2-ethylhexyl) phthalate (DEHP) on DNA damage and proliferation in thyroid using thyroid carcinoma cell line, 8505C, in vitro and the rats orally treated with DEHP at 0, 0.3, 3, 30 and 150 mg/kg for 90 days from post-natal day 9 in vivo. Exposure to DHEP (1–50 μM) induced cellular proliferation, as evidenced by increased cell viability and DNA synthesis. Activation of γH2AX, a sensitive biomarker for DNA damage was observed following the exposure to DHEP (from 5 to 50 μM) with increased comet tail moment (5–100 μM) in comet assay, reflecting that DNA damage also occurred. When upstream signaling was examined, both thyrotropin receptor (TSHR)-ERK1/2 axis and TSHR-AKT axis were activated with upregulation of Pax8, a master transcriptional factor for thyroid differentiation and proliferation. Thyroid tissue from juvenile rats orally exposed to DEHP also confirmed DNA damage responses and the activation of TSHR signaling, which was evident from 0.3 to 3 mg/kg respectively. Notably, deletion of TSHR through siRNA attenuated these DEHP-induced events in vitro. Collectively these results suggest that DEHP induces DNA damage and cellular proliferation in thyroid, which appears to be from TSHR activation, providing an important insight into endocrine disrupting activities of phthalates on thyroid.

Numerous studies have attempted to develop a new in vitro eye irritation test (EIT). To obtain more reliable results from EIT, potential new biomarkers that reflect eye irritation by chemicals must be identified. We investigated candidate biomarkers for eye irritation, using a proteomics approach. Sodium lauryl sulfate (SLS) or benzalkonium chloride (BAC) was applied on a reconstructed human cornea-like epithelium model, MCTT HCE, and corneal protein expression was examined by two-dimensional gel electrophoresis. We found that ezrin (EZR) was significantly upregulated by SLS or BAC. In addition, upregulation of EZR in immortalized human corneal cells treated with SLS or BAC was confirmed by quantitative reverse transcription-PCR and western blot analysis. Furthermore, other well-known eye irritants such as cetylpyridinium bromide, Triton X-100, cyclohexanol, ethanol, 2-methyl-1-pentanol, and sodium hydroxide significantly increased EZR expression in immortalized human corneal cells. Induction of EZR promoter activity in irritant-treated human corneal cells was confirmed by a luciferase gene reporter assay. In conclusion, EZR expression may be a potential biomarker for detecting eye irritation, which may substantially improve the performance of in vitro EIT.

Doublecortin-like kinase 1 (Dclk1) is considered a reliable marker for tuft cells in the gastrointestinal tract. We investigated the dynamic changes of tuft cells associated with mouse models of oxyntic atrophy and metaplasia in the stomach. Increases in the numbers of Dclk1-positive tuft cells were observed in several models of parietal cell loss. However, the expanded population of Dclk1-expressing cells showed a morphologically distinct structure in apical microvilli and acetylated microtubules, which was not seen in the tuft cells present in the normal gastric mucosa. These microvillar sensory cells (MVSCs) showed no evidence of proliferation. The expansion of the MVSCs induced by oxyntic atrophy was reversible after the return of parietal cells. More important, expansion of MVSCs after induced parietal cell loss was not observed in Gast(-/-) mice. Although the Dclk1-expressing cells in the normal gastric mucosa were in part derived from Lrig1-expressing stem cells, the Lrig1-lineaged cells did not produce the expanded Dclk1-expressing cells associated with oxyntic atrophy. These studies indicate that loss of parietal cells leads to the reversible emergence of a novel Dclk1-expressing sensory cell population in the gastric mucosa.

Introduction A variety of in vitro tests to replace the Draize test have been developed; however, there is no available method for assessing the full spectrum of Globally Harmonized System (GHS) categories. Human cornea-like three-dimensional (3D) reconstructed tissue models are the most promising in vitro systems. The objective of this study was to evaluate the ocular toxicity of 11 test substances using the EpiOcular™ model after performing proficiency tests. We further evaluated the effectiveness of ezrin staining as a complementary marker in histological analysis to overcome the limitation of eye irritation tests using 3D reconstructed human corneal epithelium models. Methods The assessment of ocular toxicity was performed by the suggested OECD TG 492 procedure. After treatment with proficiency test chemicals and 10 test substances, EpiOcular™ tissue models were stained with hematoxylin and eosin and ezrin, and the histological changes were observed by immunofluorescence microscopy. Results The ocular toxicity assessment of 10 test chemicals using the EpiOcular™ eye irritation test were in accordance with the UN GHS classification of test chemicals. Histological analysis of ezrin staining showed that the cell membranes of models treated with 10 out of 11 non-irritant chemicals were maintained, whereas those of models treated with 14 eye irritant substances resulted in the apparent translocation of ezrins from the cell membrane to the cytoplasm or nucleus by destruction of cell membrane. Discussion Ezrin may be used as a complementary marker to more accurately assess ocular toxicity using 3D reconstructed human cornea-like epithelium models.

Accumulating evidence indicates that latency-associated Mycobacterium tuberculosis (Mtb)-specific antigens from the dormancy survival regulator regulon (DosR) may be promising novel vaccine target antigens for the development of an improved tuberculosis vaccine. After transcriptional profiling of DosR-related genes in the hyper-virulent Beijing Mtb strain K and the reference Mtb strain H37Rv, we selected Rv3131, a hypothetical nitroreductase, as a vaccine antigen and evaluated its vaccine efficacy against Mtb K. Mtb K exhibited stable and constitutive up-regulation of rv3131 relative to Mtb H37Rv under three different growth conditions (at least 2-fold induction) including exponential growth in normal culture conditions, hypoxia, and inside macrophages. Mice immunised with Rv3131 formulated in GLA-SE, a well-defined TLR4 adjuvant, displayed enhanced Rv3131-specific IFN-γ and serum IgG2c responses along with effector/memory T cell expansion and remarkable generation of Rv3131-specific multifunctional CD4+ T cells co-producing TNF-α, IFN-γ and IL-2 in both spleen and lung. Following challenge with Mtb K, the Rv3131/GLA-SE-immunised group exhibited a significant reduction in bacterial number and less extensive lung inflammation accompanied by the obvious persistence of Rv3131-specific multifunctional CD4+ T cells. These results suggest that Rv3131 could be an excellent candidate for potential use in a multi-antigenic Mtb subunit vaccine, especially against Mtb Beijing strains.

Coal-tar dyes in cosmetics may elicit adverse effects in the skin and eyes. Countries, like the US, have banned the use of coal-tar dyes in cosmetics for the eye area due to the potential for ocular irritation. We evaluated the eye irritation potential of 15 coal-tar dyes permitted as cosmetic ingredients in reconstructed human cornea-like epithelium (RhCEs [EpiOcular™ and MCTT HCE™]) tests and the short time exposure (STE) test. Eosin YS, phloxine B, tetrachlorotetrabromofluorescein, and tetrabromofluorescein were identified as irritants in RhCEs; dibromofluorescein and uranine yielded discrepant results. STE enabled further classification in accordance with the UN Globally Harmonized System of Classification and Labelling of Chemicals, as follows: eosin YS as Cat 2; phloxine B, Cat 1; and tetrachlorotetrabromofluorescein and tetrabromofluorescein, Cat 1/2. STE indicated dibromofluorescein (irritant in EpiOcular™) and uranine (irritant in MCTT HCE™) as No Cat, resulting in the classification of “No prediction can be made.” based on bottom-up approach with each model. These results demonstrated that in vitro eye irritation tests can be utilized to evaluate the potential ocular irritancy of cosmetic ingredients and provide significant evidence with which to determine whether precautions should be given for the use of coal-tar dyes in cosmetics or other substances applied to the eye area.

The main function of the skin is to protect the body from the external environment. The barrier function of the skin is mainly provided by the stratum corneum, which consists of corneocytes bound with the corneodesmosomes and lamellar lipids. Skin barrier proteins like loricrin and filaggrin also contribute to the skin barrier function. In various skin diseases, skin barrier dysfunction is a common symptom, and skin irritants like detergents or surfactants could also perturb skin barrier function. Many efforts have been made to develop strategies to improve skin barrier function. Here, we investigated whether the microfluidized lysates of Lactobacillus rhamnosus (LR), one of the most widely used probiotic species for various health benefits, may improve the skin barrier function in a reconstructed human epidermis, Keraskin&trade, Application of LR lysate on Keraskin&trade, increased the expression of tight junction proteins, claudin 1 and occludin as determined by immunofluorescence analysis, and skin barrier proteins, loricrin and filaggrin as determined by immunohistochemistry and immunofluorescence analysis and qPCR. Also, the cytotoxicity of a skin irritant, sodium lauryl sulfate (SLS), was alleviated by the pretreatment of LR lysate. The skin barrier protective effects of LR lysate could be further demonstrated by the attenuation of SLS-enhanced dye-penetration. LR lysate also attenuated the destruction of desmosomes after SLS treatment. Collectively, we demonstrated that LR lysate has protective effects on the skin barrier, which could expand the utility of probiotics to skin-moisturization ingredients.

Inflammatory bowel diseases such as ulcerative colitis represent serious health burdens, both because of the tissue-damaging disease itself, and because of an elevated risk of colon cancer. The increased expression of many members of the matrix metalloproteinase (MMP) family of enzymes that occurs in colitis, has long been associated with the destructive nature of the disease. Recent findings in cancer and other MMP-associated diseases, however, led us to question whether MMPs are indeed detrimental in the setting of colitis. Here, we focus on a single MMP family member, MMP10, and assess its role in a murine model of colonic tissue damage induced by dextran sulphate sodium (DSS) treatment. Using mice genetically deficient for MMP10, we find that absence of this enzyme leads to significantly worse disease scores and failure to resolve inflammation even after extended recovery periods. We show that MMP10 is produced predominantly by infiltrating myeloid cells in both murine and human colitis. Through bone marrow transplant experiments, we confirm that bone marrow-derived MMP10 contributes to colitis severity. Mice lacking MMP10 have a significantly higher propensity for development of dysplastic lesions in the colon after two rounds of DSS exposure. Thus, we conclude that MMP10 is required for resolution of DSS-induced colonic damage, and in its absence, chronic inflammation and ultimately dysplasia occurs.

The development of multifunctional reagents for simultaneous specific near-infrared (NIR) imaging and phototherapy of tumors is of great significance. This work describes the design of a cathepsin B-activated fluorescent probe (CyA-P-CyB) and its applications as an NIR imaging probe for tumor cells and as a phototherapy reagent for tumors. In vitro experiments demonstrated that CyA-P-CyB was activated via the cleavage of a peptide linker by cathepsin B in tumor cells to produce fluorescence in the NIR region based on a FRET mechanism. MTT assays showed that the phototoxicity of CyA-P-CyB toward cells depended on the activity of cathepsin B, and the probe exhibited specific phototoxicity toward tumor cells. CyA-P-CyB was also successfully applied to the in vivo imaging and phototherapy of tumors. Histological analysis indicated that CyA-P-CyB had no cytotoxic effects on seven mouse tissues (lung, liver, heart, kidney, pancreas, spleen and brain) after the CyA-P-CyB treatment and laser irradiation.

Objective Metaplastic lineages in the oxyntic mucosa of the stomach are critical preneoplastic precursors of gastric cancer. Recent studies have demonstrated that spasmolytic polypeptide-expressing metaplasia (SPEM) in the mouse oxyntic mucosa arises from transdifferentiation of mature gastric chief cells. Other investigations of intestinal progenitor cells have shown that cells demonstrating transcriptional activity for leucine-rich repeat containing G-protein-coupled receptor 5 (Lgr5) in the intestine, colon and gastric antrum function as adult stem cells. We have now investigated whether cells demonstrating Lgr5 transcriptional activity in the oxyntic mucosa of mice might be responsible for development of metaplasia. Design Lgr5-EGFP-IRES-Cre ERT2/+ ;Rosa26R mice were used to examine the distribution of Lgr5 transcriptionally active cells in the normal oxyntic mucosa as well as after treatment with DMP-777 or L-635 to induce acute SPEM. Lineage mapping was performed to determine if Lgr5-expressing cells gave rise to SPEM. Results Cells expressing transcriptional activity for Lgr5 in the oxyntic mucosa were present as scattered rare cells only along the lesser curvature of the stomach. These cells also stained for markers of chief cells (intrinsic factor and pepsinogen) but never showed any staining for proliferative markers (Ki-67). In Lgr5-EGFP-IRES-Cre ERT2/+ ;Rosa26R mice induced with tamoxifen, treatment with either DMP-777 or L-635 to induce acute oxyntic atrophy caused induction of SPEM, but no lineage mapping into SPEM from Lgr5-expressing cells was observed. Conclusion The results indicate that, while chief cells with Lgr5 transcriptional activity are present along the lesser curvature of the gastric oxyntic mucosa, they are not responsible for production of metaplasia.

Objectives:The objective of this study was to evaluate the syngeneic immunocompetent mouse model by using the micro-positron emission tomography with 2-[fluorine-18]-fluoro-2-deoxy-d-glucose (18F-FDG microPET) imaging of ovarian tumor growth.Methods:ID8 ovarian carcinoma cells derived from C57BL/6 mice were intraperitoneally injected into female C57BL/6 mice. Mice were injected with18F-FDG (7.4 MBq, intravenous injection), and microPET images were obtained 40 minutes later. Micro-computed tomographic images were also obtained immediately after microPET images for anatomical reference.18F-FDG microPET images were acquired at baseline and at 4, 8, 10, and 11 weeks after tumor cell injection. The maximum standardized uptake value (SUVmax) in each time point was obtained from the images and compared to follow the tumor growth.Results:Physiological uptake of18F-FDG was intensely found in the bladder and heart and frequently in the gastrointestinal tract. Diffused uptake of18F-FDG was observed in the peritoneal cavity of all tumor-bearing mice at 4 weeks, and high focal uptakes were developed in the peritoneal cavity at 8 to 11 weeks. High focal uptakes increased over time, correlating with a progressive increase in the SUVmaxof18F-FDG. At 11 weeks, the SUVmaxvalue was significantly increased (1.49 ± 0.10 at 11 weeks vs 0.29 ± 0.03 at baseline,P< 0.01). Tumors in the gut and peritoneum were confirmed by anatomical and histopathological examination.Conclusions:Our results demonstrate that the peritoneal tumor growth in the syngeneic ovarian cancer model can be detected by the18F-FDG microPET imaging.

CD47 (integrin-associated protein), a multi-spanning transmembrane protein expressed in all cells including red blood cells (RBCs) and leukocytes, interacts with signal regulatory protein α (SIRPα) on macrophages and thereby inhibits phagocytosis of RBCs. Recently, we generated a novel C57BL/6J CD47 knockout (CD47-/- hereafter) mouse line by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease, and here report their hematological phenotypes. On monitoring their birth and development, CD47-/- mice were born viable with a natural male-to-female sex ratio and normally developed from birth through puberty to adulthood without noticeable changes in growth, food/water intake compared to their age and sex-matched wild-type littermates up to 26 weeks. Hematological analysis revealed a mild but significant reduction of RBC counts and hemoglobin in 16 week-old male CD47-/- mice which were aggravated at the age of 26 weeks with increased reticulocyte counts and mean corpuscular volume (MCV), suggesting hemolytic anemia. Interestingly, anemia in female CD47-/- mice became evident at 26 weeks, but splenomegaly was identified in both genders of CD47-/- mice from the age of 16 weeks, consistent with development of hemolytic anemia. Additionally, helper and cytotoxic T cell populations were considerably reduced in the spleen, but not in thymus, of CD47-/- mice, suggesting a crucial role of CD47 in proliferation of T cells. Collectively, these findings indicate that our CD47-/- mice have progressive hemolytic anemia and splenic depletion of mature T cell populations and therefore may be useful as an in vivo model to study the function of CD47.

Sox2 is expressed in developing foregut endoderm, with highest levels in the future esophagus and anterior stomach. By contrast, Nkx2.1 (Titf1) is expressed ventrally, in the future trachea. In humans, heterozygosity for SOX2 is associated with anopthalmia-esophageal-genital syndrome (OMIM 600992), a condition including esophageal atresia (EA) and tracheoesophageal fistula (TEF), in which the trachea and esophagus fail to separate. Mouse embryos heterozygous for the null allele, Sox2 EGFP , appear normal. However, further reductions in Sox2, using Sox2 LP and Sox2 COND hypomorphic alleles, result in multiple abnormalities. Approximately 60% of Sox2 EGFP/COND embryos have EA with distal TEF in which Sox2 is undetectable by immunohistochemistry or western blot. The mutant esophagus morphologically resembles the trachea, with ectopic expression of Nkx2.1, a columnar, ciliated epithelium, and very few p63 + basal cells. By contrast, the abnormal foregut of Nkx2.1 -null embryos expresses elevated Sox2 and p63, suggesting reciprocal regulation of Sox2 and Nkx2.1 during early dorsal/ventral foregut patterning. Organ culture experiments further suggest that FGF signaling from the ventral mesenchyme regulates Sox2 expression in the endoderm. In the 40% Sox2 EGFP/COND embryos in which Sox2 levels are ∼18% of wild type there is no TEF. However, the esophagus is still abnormal, with luminal mucus-producing cells, fewer p63 + cells, and ectopic expression of genes normally expressed in glandular stomach and intestine. In all hypomorphic embryos the forestomach has an abnormal phenotype, with reduced keratinization, ectopic mucus cells and columnar epithelium. These findings suggest that Sox2 plays a second role in establishing the boundary between the keratinized, squamous esophagus/forestomach and glandular hindstomach.

Background & Aims: The loss of parietal cells from the gastric mucosa (oxyntic atrophy) is a critical step in the pathogenesis of chronic gastritis and gastric adenocarcinoma. Parietal cells are known to secrete epidermal growth factor receptor (EGFR) ligands, which are critical regulators of differentiation in the gastric mucosa. Although all of the actions of EGFR ligands are mediated through a common EGFR protein, individual ligands may produce different physiologic responses. Previous investigations have suggested that a deficit in EGFR signaling in waved-2 mice accelerates the emergence of metaplasia after induction of acute oxyntic atrophy. We sought to determine whether specific EGFR ligands regulate the metaplastic response to oxyntic atrophy. Methods: To induce spasmolytic polypeptide-expressing metaplasia (SPEM), amphiregulin (AR) and transforming growth factor-α–deficient mice and their wild-type littermates were treated with DMP-777 for 0–14 days and for 14 days followed by 14 days of recovery off drug. We evaluated the gastric mucosal response to oxyntic atrophy using cell lineage–specific markers. Results: Although loss of transforming growth factor-α did not influence the induction of SPEM, loss of AR caused an acceleration and amplification in the induction of SPEM after acute oxyntic atrophy. Trefoil factor family 2/spasmolytic polypeptide and intrinsic factor dual-immunostaining cells significantly increased in the SPEM of AR-deficient mice. At the bases of glands, intrinsic factor immunoreactive cells also were costained for 5-bromo-2'-deoxyuridine, suggesting their re-entry into the cell cycle. Conclusions: The absence of AR promoted the rapid emergence of SPEM in response to oxyntic atrophy.

Purpose: Helicobacter pylori infection can lead to gastric cancer, and cyclooxygenase-2 (COX-2) is overexpressed in the stomach during H. pylori infection. Therefore, we investigated whether nonsteroidal anti-inflammatory drugs might protect against this form of cancer. Specifically, we examined the chemopreventive effect of the COX-2 inhibitor nimesulide on H. pylori-associated gastric carcinogenesis in mice. Experimental Design: C57BL/6 mice were treated with the carcinogen N-methyl-N-nitrosourea (MNU) and/or H. pylori. To determine the effect of COX-2 inhibition, nimesulide was mixed with feed pellets and administered for the duration of the experiment. All of the mice were sacrificed 50 weeks after the start of the experiment. Histopathology, immunohistochemistry, and Western blotting for COX-2, Bax and Bcl-2 were performed in stomach tissues. In vitro experiments with the human gastric cancer cell line AGS were also performed to identify mechanisms underlying cancer chemoprevention by nimesulide. Results: Gastric tumors developed in 68.8% of mice that were given both MNU and H. pylori, whereas less than 10% developed gastric tumors when given either MNU or H. pylori alone. These findings indicate that H. pylori promotes carcinogen-induced gastric tumorigenesis. In mice treated with both MNU and H. pylori, nimesulide administration substantially reduced H. pylori-associated gastric tumorigenesis, whereas substantial inductions of apoptosis were observed. In vitro studies demonstrated that nimesulide and H. pylori when combined acted synergistically to induce more apoptosis than either alone. Conclusions: Our data show that nimesulide prevents H. pylori-associated gastric carcinogenesis, and suggest that COX-2 may be a target for chemoprevention of gastric cancer.

Overproduction of nitric oxide via inducible nitric oxide synthase (iNOS) is suggested to be a significant pathogenic factor in Helicobacter pylori induced gastritis. The purpose of this study was to examine the role of iNOS in H pylori associated gastric carcinogenesis.Two types of mice were used in this study: iNOS deficient mice (iNOS-/-) and wild-type littermates. Gastric cancer was generated in mice using a combination treatment comprising N-methyl-N-nitrosourea administration and H pylori infection. Fifty weeks after treatment, tumours in gastric tissues from both types of mice were examined using histopathology, immunohistochemistry, and immunoblotting for iNOS and 3-nitrotyrosine.The overall incidence of gastric cancer at week 50 was significantly lower in iNOS-/- compared with iNOS wild-type mice (p0.05). When analysed according to tumour pathology, the incidence of gastric adenocarcinoma was significantly lower in iNOS-/- compared with iNOS wild-type mice (p0.05). Immunostaining for iNOS was clearly observed in adenocarcinoma cells of iNOS wild-type mice, and was characterised by a strong cytoplasmic expression pattern. 3-Nitrotyrosine was expressed mostly in the area of the lamina propria of gastritis and adenoma lesions in iNOS wild-type mice. Immunoblotting analyses showed that iNOS and 3-nitrotyrosine were also expressed in both adenoma and adenocarcinoma tissues from iNOS wild-type mice. iNOS and 3-nitrotyrosine expression was greater in tumour tissues than in non-tumour tissues.These findings suggest that iNOS contributes to H pylori associated gastric carcinogenesis in mice.

The action of estrogen on target organs has been actively studied with the discovery of estrogen receptor (ER) beta. This study was carried out to examine the expression of ERalpha and ERbeta in the uterus and the vagina of immature Sprague-Dawley rats treated with 17-ethinyl estradiol (EE). Twenty days old rats were subcutaneously treated with EE at the doses of 0 (vehicle control), 0.03, 0.3, 1.0, 3.0, and 10.0 microg/kg/day for three consecutive days. The treatment of EE at the doses of 0.3, 1.0, 3.0 and 10.0 microg/kg/day significantly increased the weights of the uterus and vagina of rats (p

Objective Intestinal metaplasia and spasmolytic polypeptide-expressing metaplasia (SPEM) are considered neoplastic precursors of gastric adenocarcinoma and are both marked by gene expression alterations in comparison to normal stomach. Since miRNAs are important regulators of gene expression, we sought to investigate the role of miRNAs on the development of stomach metaplasias. Design We performed miRNA profiling using a quantitative reverse transcription-PCR approach on laser capture microdissected human intestinal metaplasia and SPEM. Data integration of the miRNA profile with a previous mRNA profile from the same samples was performed to detect potential miRNA-mRNA regulatory circuits. Transfection of gastric cancer cell lines with selected miRNA mimics and inhibitors was used to evaluate their effects on the expression of putative targets and additional metaplasia markers. Results We identified several genes as potential targets of miRNAs altered during metaplasia progression. We showed evidence that HNF4γ (upregulated in intestinal metaplasia) is targeted by miR-30 and that miR-194 targets a known co-regulator of HNF4 activity, NR2F2 (downregulated in intestinal metaplasia). Intestinal metaplasia markers such as VIL1, TFF2 and TFF3 were downregulated after overexpression of miR-30a in a HNF4γ-dependent manner. In addition, overexpression of HNF4γ was sufficient to induce the expression of VIL1 and this effect was potentiated by downregulation of NR2F2. Conclusions The interplay of the two transcription factors HNF4γ and NR2F2 and their coordinate regulation by miR-30 and miR-194, respectively, represent a miRNA to transcription factor network responsible for the expression of intestinal transcripts in stomach cell lineages during the development of intestinal metaplasia.

Objective The glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical regions within the stomach. We examined in detail the distribution of the full range of cell lineages within the human stomach. Design We determined the distribution of gastric gland cell lineages with specific immunocytochemical markers in entire stomach specimens from three non-obese organ donors. Results The anatomical body and antrum of the human stomach were defined by the presence of ghrelin and gastrin cells, respectively. Concentrations of somatostatin cells were observed in the proximal stomach. Parietal cells were seen in all glands of the body of the stomach as well as in over 50% of antral glands. MIST1 expressing chief cells were predominantly observed in the body although individual glands of the antrum also showed MIST1 expressing chief cells. While classically described antral glands were observed with gastrin cells and deep antral mucous cells without any parietal cells, we also observed a substantial population of mixed type glands containing both parietal cells and G cells throughout the antrum. Conclusions Enteroendocrine cells show distinct patterns of localisation in the human stomach. The existence of antral glands with mixed cell lineages indicates that human antral glands may be functionally chimeric with glands assembled from multiple distinct stem cell populations.

Gastric cancer is the second most common cause of cancer-related death in the world. A growing body of evidence indicates that inflammation is closely associated with the initiation, progression, and metastasis of many tumors, including those of gastric cancer. In addition, approximately 60% of the world's population is colonized by Helicobacter pylori, which accounts for more than 50% of gastric cancers. While the role of inflammation in intestinal and colonic cancers is relatively well defined, its role in stomach neoplasia is still unclear because of the limited access of pathogens to the acidic environment and the technical difficulties isolating and characterizing immune cells in the stomach, especially in animal models. In this review, we will provide recent updates addressing how inflammation is involved in gastric malignancies, and what immune characteristics regulate the pathogenesis of stomach cancer. Also, we will discuss potential therapeutics that target the immune system for the efficient treatment of gastric cancer.

The capacity to achieve sufficient concentrations of Ag-specific Ab of the appropriate isotypes is a critical component of immunity that requires efficient differentiation and interactions of Ag-specific B and Th cells along with dendritic cells. Numerous bacterial toxins catalyze mono(ADP-ribosyl)ation of mammalian proteins to influence cell physiology and adaptive immunity. However, little is known about biological functions of intracellular mammalian mono(ADP-ribosyl)transferases, such as any ability to regulate Ab responses. poly-(ADP-ribose) polymerase 14 (PARP14), an intracellular protein highly expressed in lymphoid cells, binds to STAT6 and encodes a catalytic domain with mammalian mono(ADP-ribosyl)transferase activity. In this article, we show that recall IgA as well as the STAT6-dependent IgE Ab responses are impaired in PARP14-deficient mice. Whereas PARP14 regulation of IgE involved a B cell–intrinsic process, the predominant impact on IgA was B cell extrinsic. Of note, PARP14 deficiency reduced the levels of Th17 cells and CD103+ DCs, which are implicated in IgA regulation. PARP14 enhanced the expression of RORα, Runx1, and Smad3 after T cell activation, and, importantly, its catalytic activity of PARP14 promoted Th17 differentiation. Collectively, the findings show that PARP14 influences the class distribution, affinity repertoire, and recall capacity of Ab responses in mice, as well as provide direct evidence of the requirement for protein mono-ADP-ribosylation in Th cell differentiation.

Gap junctions and their structural proteins, connexins (Cxs), have been implicated in carcinogenesis. To explore the involvement of Cx32 in gastric carcinogenesis, immunochemical analysis of Cx32 and proliferation marker Ki67 using tissue-microarrayed human gastric cancer and normal tissues was performed. In addition, after Cx32 overexpression in the human gastric cancer cell line AGS, cell proliferation, cell cycle analyses, and p21Cip1 and p27Kip1 expression levels were examined by bromodeoxyuridine assay, flow cytometry, real-time RT-PCR, and western blotting. Immunohistochemical study noted a strong inverse correlation between Cx32 and Ki67 expression pattern as well as their location. In vitro, overexpression of Cx32 in AGS cells inhibited cell proliferation significantly. G1 arrest, up-regulation of cell cycle-regulatory proteins p21Cip1 and p27Kip1 was also found at both mRNA and protein levels. Taken together, Cx32 plays some roles in gastric cancer development by inhibiting gastric cancer cell proliferation through cell cycle arrest and cell cycle regulatory proteins. [BMB Reports 2013; 46(1): 25-30]

Eradication of Helicobacter pylori correlates with regeneration of the gastric epithelium, ulcer healing and re-expression of the gastric morphogen Sonic Hedgehog (Shh). We sought to identify the role of Shh as a regulator of gastric epithelial regeneration during wound healing. A mouse model expressing a parietal cell-specific, tamoxifen-inducible deletion of Shh (HKCreERT2;Shhflox/flox or PC-iShhKO) was developed. Stomachs were collected and compared 7 to 150 days after the final vehicle or tamoxifen injection. Ulcers were induced in both controls and PC-iShhKO mice using acetic acid and ulcer size compared 1 and 7 days post induction. 1) Re-expression of Shh correlates with decreased hyperproliferation: Compared to controls, PC-iShhKO mice developed foveolar hyperplasia. Restoration of normal gastric epithelial architecture and differentiation correlated with the re-expression of Shh in PC-iShhKO mice 150 days after the final tamoxifen injection. At the tamoxifen dose used to induce Cre recombination there was no genotoxicity reported in either HKCreERT2 or Shhflox/flox control mouse stomachs. 2) Delayed wound healing in PC-iShhKO mouse stomachs: To identify the role of Shh in gastric regeneration, an acetic acid ulcer was induced in control and PC-iShhKO mice. Ulcers began to heal in control mice by 7 days after induction. Ulcer healing was documented by decreased ulcer size, angiogenesis, macrophage infiltration and formation of granulation tissue that correlated with the re-expression of Shh within the ulcerated tissue. PC-iShhKO mice did not show evidence of ulcer healing. Re-expression of Shh contributes to gastric regeneration. Our current study may have clinical implications given that eradication of Helicobacter pylori correlates with re-expression of Shh, regeneration of the gastric epithelium and ulcer healing.

Upper gastrointestinal neoplasia in the esophagus, stomach, and pancreas is associated with the formation of preneoplastic metaplasias. We have previously reported the up-regulation of human epididymis protein 4 (HE4) in all metaplasias in the stomach of humans and mice. We have now sought to evaluate the expression of HE4 in metaplasias/preneoplastic precursors and cancers of the human stomach, pancreas, and esophagus. Tissue microarrays for gastric cancers, pancreatic cancers, and esophageal adenocarcinoma were stained with antibodies against HE4. Immunostaining was quantified by digital imaging, and the results were evaluated to assess the expression in metaplasias, the expression in cancer pathological subtypes, and the effects of expression on survival in patients with cancer. In patients with gastric cancer from Korea, HE4 was detected in 74% of intestinal and 90% of diffuse cancers, whereas in a gastric cancer cohort from Johns Hopkins, HE4 was detected in 74% of intestinal-type and 92% of diffuse cancers. Nevertheless, in both cohorts, there was no impact of HE4 expression on overall survival. In the esophagus, we observed the expression of HE4 in scattered endocrine cells within Barrett esophagus samples, but Barrett columnar metaplasias and HE4 were detected in only 2% of esophageal adenocarcinomas. Finally, in the pancreas, HE4 expression was not observed in pancreatic intraepithelial neoplasia lesions, but 46.8% of pancreatic adenocarcinomas expressed HE4. Still, we did not observe any influence of HE4 expression on survival. The results suggest that HE4 is up-regulated during gastric and pancreatic carcinogenesis.

Early diagnosis and curative resection are the predominant factors associated with increased survival in patients with gastric cancer. However, most gastric cancer cases are still diagnosed at later stages. Since most pathologic specimens are archived as FFPE samples, the ability to use them to generate expression profiles can greatly improve cancer biomarker discovery. We sought to uncover new biomarkers for stomach preneoplastic metaplasias and neoplastic lesions by generating proteome profiles using FFPE samples. We combined peptide isoelectric focusing and liquid chromatography–tandem mass spectrometry analysis to generate proteomic profiles from FFPE samples of intestinal-type gastric cancer, metaplasia, and normal mucosa. The expression patterns of selected proteins were analyzed by immunostaining first in single tissue sections from normal stomach, metaplasia, and gastric cancer and later in larger tissue array cohorts. We detected 60 proteins up-regulated and 87 proteins down-regulated during the progression from normal mucosa to metaplasia to gastric cancer. Two of the up-regulated proteins, LTF and DMBT1, were validated as specific markers for spasmolytic polypeptide–expressing metaplasia and intestinal metaplasia, respectively. In cancers, significantly lower levels of DMBT1 or LTF correlated with more advanced disease and worse prognosis. Thus, proteomic profiling using FFPE samples has led to the identification of two novel markers for stomach metaplasias and gastric cancer prognosis.

SMAD proteins are downstream effectors of the TGF-β signaling pathway. Smad3-null mice develop colorectal cancer by 6 months of age. In this study, we have examined whether the loss of Smad3 promotes gastric neoplasia in mice. The stomachs of Smad3⁻/⁻ mice were compared with age-matched Smad3 heterozygous and wild-type mice. E-cadherin, Ki-67, phosphoSTAT3, and TFF2/SP expression was analyzed by immunohistochemisty. The short hairpin RNA (ShRNA)-mediated knockdown of Smad3 in AGS and MKN28 cells was also performed. In addition, we examined alterations in DCLK1-expressing cells. Smad3⁻/⁻ mouse stomachs at 6 months of age revealed the presence of exophytic growths along the lesser curvature in the proximal fundus. Six-month-old Smad3⁻/⁻ mouse stomachs showed metaplastic columnar glands initiating from the transition zone junction between the forestomach and the glandular epithelium along the lesser curvature. Ten-month-old Smad3⁻/⁻ mice all exhibited invasive gastric neoplastic changes with increased Ki-67, phosphoSTAT3 expression, and aberrant cytosolic E-cadherin staining in papillary glands within the invading submucosal gland. The shRNA-mediated knockdown of Smad3 in AGS and MKN28 cells promoted the expression of phosphoSTAT3. DCLK1-expressing cells, which also stained for the tuft cell marker acetylated-α-tubulin, were observed in 10-month-old Smad3⁻/⁻ mice within tumors and in fundic invasive lesions. In conclusion, Smad3-null mice develop gastric tumors in the fundus, which arise from the junction between the forestomach and the glandular epithelium and progress to prominent invasive tumors over time. Smad3-null mice represent a novel model of fundic gastric tumor initiated from forestomach/glandular transition zone along the lesser curvature.

Menetrier’s disease is a rare premalignant disorder of the stomach with no proven effective medical therapy. Increased epidermal growth factor receptor signaling has been implicated in the pathogenesis of Menetrier’s disease. We conducted a single-arm clinical trial with cetuximab, a monoclonal antibody that blocks epidermal growth factor receptor signaling, in nine individuals with clinically and histologically documented severe Menetrier’s disease that impaired quality of life to the extent that gastrectomy was being considered. Of the seven patients who completed the 1-month course of treatment, all showed statistically significant improvement both clinically (quality-of-life indices) and biochemically (increased parietal cell mass and gastric acidity). Furthermore, all seven patients who completed the 1-month trial elected to continue treatment, and four subsequently showed near-complete histological remission. Cetuximab should be considered as first-line therapy for Menetrier’s disease.

Transformation of epithelial cells is associated with loss of cell polarity, which includes alterations in cell morphology as well as changes in the complement of plasma membrane proteins. Rab proteins regulate polarized trafficking to the cell membrane and therefore represent potential regulators of this neoplastic transition. Here we have demonstrated a tumor suppressor function for Rab25 in intestinal neoplasia in both mice and humans. Human colorectal adenocarcinomas exhibited reductions in Rab25 expression independent of stage, with lower Rab25 expression levels correlating with substantially shorter patient survival. In wild-type mice, Rab25 was strongly expressed in cells luminal to the proliferating cells of intestinal crypts. While Rab25-deficient mice did not exhibit gross pathology, ApcMin/+ mice crossed onto a Rab25-deficient background showed a 4-fold increase in intestinal polyps and a 2-fold increase in colonic tumors compared with parental ApcMin/+ mice. Rab25-deficient mice had decreased beta1 integrin staining in the lateral membranes of villus cells, and this pattern was accentuated in Rab25-deficient mice crossed onto the ApcMin/+ background. Additionally, Smad3+/- mice crossed onto a Rab25-deficient background demonstrated a marked increase in colonic tumor formation. Taken together, these results suggest that Rab25 may function as a tumor suppressor in intestinal epithelial cells through regulation of protein trafficking to the cell surface.

Background & Aims The loss of parietal cells from the fundic mucosa leads to the emergence of metaplastic lineages associated with an increased susceptibility to neoplastic transformation. Both intestinal metaplasia (IM) and spasmolytic polypeptide (TFF2/SP) expressing metaplasia (SPEM) have been identified in human stomach, but only SPEM is present in most mouse models of gastric metaplasia. We previously determined that loss of amphiregulin (AR) promotes SPEM induced by acute oxyntic atrophy. We have now examined whether SPEM in the AR −/− mouse predisposes the stomach to gastric neoplasia. Methods Gross pathology of 18-month-old wild-type, AR −/− , and TGF-α −/− mice were examined. Ki-67, β-catenin, Pdx-1, TFF3, and TFF2/SP expression was analyzed by immunohistochemistry. Metaplastic gastric mucosa was analyzed by dual immunostaining for TFF2/SP with MUC2 or TFF3. Results By 18 months of age, more than 70% of AR −/− mice developed SPEM while 42% showed goblet cell IM labeled with MUC2, TFF3, and Pdx-1. A total of 28% had invasive gastric lesions in the fundus. No antral abnormalities were observed in AR −/− mice. Metaplastic cell lineages in AR −/− mice showed increases in cell proliferation and cytosolic β-catenin expression. Dual staining for TFF2/SP with MUC2 or TFF3 showed glands containing both SPEM and IM with intervening cells expressing both TFF2/SP and MUC2 or TFF2/SP and TFF3. Conclusions AR −/− mice develop SPEM, which gives rise to goblet cell IM and invasive fundic dysplastic lesions. The AR −/− mouse represents the first mouse model for spontaneous development of fundic SPEM with progression to IM.

3-Monochloropropane-1,2-diol (α-chlorohydrin, 3-MCPD) is a well-known contaminant, which has been detected in a wide range of foods and ingredients, and is also a suspected cause of cancer. In this study, the carcinogenicity of 3-MCPD in SD rats was investigated. Groups of 50 male and 50 female rats were exposed for two years to drinking water containing 0, 25, 100 or 400 ppm 3-MCPD. The body weights and water consumptions of the male and female rats given 400 ppm 3-MCPD were significantly lower than those of the controls. The incidences of renal tubule adenomas or carcinomas and Leydig cell tumors occurred with dose-related positive trends in male rats. The incidences of renal tubule carcinomas and Leydig cell tumors were significantly increased in male rats given 400 ppm 3-MCPD. The incidence of renal tubule adenomas showed a positive trend in female rats, which was significant in 400 ppm 3-MCPD group. In conclusion, there was clear evidence of the carcinogenic activity of 3-MCPD in male SD rats, based on the increased incidences of renal tubule carcinomas and Leydig cell tumors. There was some evidence of the carcinogenic activity of 3-MCPD in female SD rats, based on the increased incidence of renal tubule adenomas.

3-Monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant in a wide range of foods and ingredients and is a suspected cause of cancer. In this study, the 13-week toxicity of 3-MCPD was examined in B6C3F1 mice (10/sex/group) administered 3-MCPD doses of 0, 5, 25, 100, 200 and 400 ppm dissolved in their drinking water over a 13-week period. All the mice survived to the end of study. The mean body weight gains in the males and females given 400 ppm were significantly lower than those of the controls. The relative kidney weights of the males and females given 200 and 400 ppm were significantly higher than those of the controls without any corresponding histopathological changes. The sperm motility was lower in the 400 ppm group than the control, and there was a significant increase in the incidence of germinal epithelium degeneration in the 200 and 400 ppm groups. A delayed total estrus cycle length was observed in the 400 ppm group without any histopathological changes. Based on these results, the target organ was determined to be kidney, testis, and ovary. The no-observed-adverse-effect level (NOAEL) was found to be 100 ppm (18.05 mg/kg/day for males and 15.02 mg/kg/day for females).

Epidemiological evidence indicates that estrogens are one of the risk factors of breast cancer. However, there have been reports that pre-pubertal estrogen exposure is related to reduced breast cancer risk. These discrepancies made us investigate the time-point and duration of estrogen exposure. Our studies focus on the effect of estradiol-3-benzoate (EB) on the mammary gland that was exposed to carcinogens. Ninety-six female Sprague-Dawley rats were randomly divided into 6 groups. Animals at 7 weeks of age were injected with 7,12-dimethylbenz[a]anthracene (DMBA) in groups 1, 2 and 3 or N-methyl-N-nitrosourea (MNU) in groups 4, 5 and 6. One week later, the animals were subjected to sustained treatment with 0 micro g (groups 1 and 4), 30 micro g (groups 2 and 5) or 300 micro g (groups 3 and 6) of EB containing pellets for 4 weeks. All animals were sacrificed at 5 weeks or 21 weeks after carcinogen treatment, for the examination of mammary gland differentiation or mammary gland tumors, respectively. At 21 weeks after carcinogen treatment, the incidence of mammary tumors in group 2 was significantly decreased (P

The mouse ear swelling test (MEST) was developed as an alternative to guinea pig models for measuring the contact sensitization potential. However, the MEST relies on the quantitative measurement of ear swelling by micrometer as the means of determining the endpoint. The purpose of this study was to investigate the possibility of using cell proliferation in the ear and lymph node by bromodeoxyuridine (BrdU) immunohistochemistry as a reliable marker for MEST. Female Balb/c mice were treated by the topical application of various sensitizers, 2,4-dinitrochlorobenzene (DNCB), toluene diisocyanate (TDI) and α-hexylcinnamaldehyde (HCA) and an irritant, sodium lauryl sulfate (SLS) following the protocol of MEST. The proliferation of cells in the ear and auricular lymph node was analyzed by BrdU incorporations into cells. There were significant increases in the cell proliferations of the ear and auricular lymph node in mice treated with DNCB and TDI compared to the vehicle control. All allergens and the irritant were correctly identified by the MEST using BrdU immunohistochemistry of lymph node responses. The standard MEST assay showed positive results in the case of the strong sensitizers, DNCB and TDI. However, HCA and SLS were not correctly identified in the ear swelling assay. These results suggest that the measurement of cell proliferation in the auricular lymph node using BrdU immunohistochemistry could provide a reliable marker for MEST.