Murine model identifies tropomyosin as IgE cross-reactive protein between house dust mite and coho salmon that possibly contributes to the development of salmon allergy.

Background: Recently, we have developed a method to identify IgE cross-reactive allergens. However, the mechanism by which IgE cross-reactive allergens cause food allergy is not yet fully understood how. In this study, we aimed to understand the underlying pathogenesis by identifying food allergens that cross-react with house dust mite allergens in a murine model. Material and methods: Allergenic protein microarray analysis was conducted using serum from mice intraperitoneally injected with Dermatophagoides pteronyssinus (Der p) extract plus alum or alum alone as controls. Der p, Dermatophagoides farinae (Der f), coho salmon extract-sensitized and control mice were analyzed. Serum levels of IgE against Der p, Der f, coho salmon extract, protein fractions of coho salmon extract separated by ammonium sulfate precipitation and anion exchange chromatography, and recombinant coho salmon tropomyosin or actin were measured by an enzyme-linked immunosorbent assay. A murine model of cutaneous anaphylaxis or oral allergy syndrome (OAS) was established in Der p extract-sensitized mice stimulated with coho salmon extract, tropomyosin, or actin. Results: Protein microarray analysis showed that coho salmon-derived proteins were highly bound to serum IgE in Der p extract-sensitized mice. Serum IgE from Der p or Der f extract-sensitized mice was bound to coho salmon extract, whereas serum IgE from coho salmon extract-sensitized mice was bound to Der p or Der f extract. Analysis of the murine model showed that cutaneous anaphylaxis and oral allergic reaction were evident in Der p extract-sensitized mice stimulated by coho salmon extract. Serum IgE from Der p or Der f extract-sensitized mice was bound strongly to protein fractions separated by anion exchange chromatography of coho salmon proteins precipitated with 50% ammonium sulfate, which massively contained the approximately 38 kDa protein. We found that serum IgE from Der p extract-sensitized mice was bound to recombinant coho salmon tropomyosin. Der p extract-sensitized mice exhibited cutaneous anaphylaxis in response to coho salmon tropomyosin. Conclusion: Our results showed IgE cross-reactivity of tropomyosin between Dermatophagoides and coho salmon which illustrates salmon allergy following sensitization with the house dust mite Dermatophagoides. Our method for identifying IgE cross-reactive allergens will help understand the underlying mechanisms of food allergies.

YTHDC2 Promotes Malignant Phenotypes of Breast Cancer Cells.

YTH domain-containing 2 (YTHDC2) is known to be an important regulator for RNA metabolism. Here, we show that YTHDC2 is essential for breast cancer tumorigenesis and metastasis. We examined YTHDC2 expression levels by immunohistochemistry in human breast tumor tissues from 99 patients and found a significantly positive correlation between the YTHDC2 expression level and the tumor stage. We established YTHDC2-knocked-down cell lines using four breast cancer cell lines with different subtypes. Knockdown of YTHDC2 attenuated the sphere-forming and the metastatic ability of breast cancer cells. Although stemness and EMT markers, such as SOX2, c-MYC, and NANOG, were downregulated in several YTHDC2-knocked-down breast cancer cells, a common target gene of YTHDC2 in breast cancer cells was not identified. These findings suggest that while YTHDC2 is involved in malignant progression of breast cancers, the mechanism by which YTHDC2 regulates those phenotypes is different between subtypes of breast cancers.

SQAP, an acyl sulfoquinovosyl derivative, suppresses expression of histone deacetylase and induces cell death of cancer cells under hypoxic conditions.

Sulfoglycolipid, SQAP, is a radiosensitizing agent that makes tumor cells more sensitive to radiation therapy. A previous study revealed that SQAP induced the degradation of hypoxia-inducible factor-1α (HIF-1α) and inhibited angiogenesis in a hepatoma model mouse. Herein, we examined the biological activities of SQAP against hepatocarcinoma cells under low oxygen conditions. Cell growth inhibition of SQAP under hypoxic conditions was significantly higher than that under normoxic conditions. In addition, SQAP was found to impair the expression of histone deacetylase (HDAC) under low oxygen conditions. Our present data suggested that SQAP induced the degradation of HIF-1α and then decreased the expression of HDAC1. Unlike known HDAC inhibitors, SQAP increased the acetylation level of histone in cells without inhibition of enzymatic activity of HDACs. Our data demonstrated hypoxia-specific unique properties of SQAP.

Functional analysis of CD44 variants and xCT in canine tumours.

The cell surface glycoprotein CD44 has various types of splicing variants, which contribute to its multiple distinct cellular functions. Recently, it was reported that the CD44v8-10 isoform interacts with the system Xc(-) transporter-related protein (xCT), and inhibits the accumulation of reactive oxygen species by promoting the synthesis of the antioxidant glutathione in human tumour cells. In this study, we investigated the expression and function of CD44 variants and xCT in canine tumours. From semi-quantitative reverse transcription polymerase chain reaction analysis, the mRNA expression of the CD44v8-10 isoform was observed in canine tumour tissues as well as human cases. The overexpression of CD44v8-10 may promote the synthesis of glutathione and enhance the resistance to radiation of canine breast tumour cells. Furthermore, canine xCT mRNA expression was significantly upregulated in the canine breast tumour tissues as compared to the normal tissues surrounding the tumours. To investigate the function of canine xCT, we treated canine tumour cells with the xCT inhibitor sulfasalazine. Consequently, the sulfasalazine-treated cells were more sensitive to oxidative stress than the non-treated cells. Taken together, these results suggested that CD44v8-10 and xCT play important roles in the therapy resistance of canine tumours as well as human tumours.

The Metabolic Heterogeneity and Flexibility of Cancer Stem Cells.

Numerous findings have indicated that CSCs, which are present at a low frequency inside primary tumors, are the main cause of therapy resistance and cancer recurrence. Although various therapeutic methods targeting CSCs have been attempted for eliminating cancer cells completely, the complicated characteristics of CSCs have hampered such attempts. In analyzing the biological properties of CSCs, it was revealed that CSCs have a peculiar metabolism that is distinct from non-CSCs to maintain their stemness properties. The CSC metabolism involves not only the catabolic and anabolic pathways, but also intracellular signaling, gene expression, and redox balance. In addition, CSCs can reprogram their metabolism to flexibly respond to environmental changes. In this review, we focus on the flexible metabolic mechanisms of CSCs, and highlight the new therapeutics that target CSC metabolism.

Gut Environment and Dietary Habits in Healthy Japanese Adults and their Association with Bowel Movement.

INTRODUCTION: Constipation is a common symptom that impairs the quality of life (QOL). This study aimed to investigate the relationship between bowel movement and gut microbiota and dietary intake. METHODS: To investigate correlations among bowel movement, food intake, and gut environment, 60 healthy Japanese participants were recruited. Bowel movement was assessed using the Bristol stool form scale (BSFS) and constipation scoring system (CSS). Dietary habit was assessed with a self-administered questionnaire wherein the food intake frequency was classified into 8 categories for 72 food/food groups. Gut microbiota was analyzed using terminal restriction fragment length polymorphism analysis. RESULTS: The constipation rate was significantly higher in females than in males. The QOL was significantly impaired in the constipated group. The fecal count of Bacteroides was decreased and that of Clostridium cluster IV was increased in participants with constipation. The BSFS score was negatively associated with the fecal count of Clostridium cluster XI and positively associated with the fecal count of Clostridium cluster XVIII and consumption of green tea. The total CSS score was positively associated with the fecal Prevotella count and negatively associated with fecal acetate levels and consumption of vegetables. Discriminant analysis estimated that constipation could be predicted correctly in 83% (p < 0.001) of the participants based on fecal microbiota and fecal short-chain fatty acids. DISCUSSION/CONCLUSION: Bowel movement was strongly affected by gut environment and food intake in Japanese participants. Improvement in dietary habits could promote bowel movement through the improvement of the environment in the gut, resulting in ameliorated QOL issues in healthy adults.

PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

C-C chemokine receptor type 7 (CCR7) is essential for migration of dendritic cells (DCs) to draining lymph nodes. PU.1/Spi1 is a transcription factor playing a critical role in the gene regulation of DCs. PU.1 knockdown decreased the expression of CCR7 in bone marrow-derived DCs and subsequently attenuated migration in vitro and in vivo. Reporter assays, EMSA, and chromatin immunoprecipitation assays revealed that PU.1 binds to the most proximal Ets motif of the Ccr7 promoter, which is involved in transcriptional activation. The CCR7 expression level, which was higher in the programmed cell death 1 ligand 2 (PD-L2)+ population than in the PD-L2- population and was markedly suppressed by TGF-ß treatment, coincided with the binding level of PU.1 to the Ccr7 promoter. The PU.1 binding level in CCR7high mesenteric lymph nodes DCs was higher than in other DC subtypes. The involvement of PU.1 in the expression of the CCR7 gene was also observed in human DCs. We conclude that PU.1 plays a pivotal role in DC migration by transactivating the CCR7 gene via the Ets motif in the promoter in both humans and mice.-Yashiro, T., Takeuchi, H., Nakamura, S., Tanabe, A., Hara, M., Uchida, K., Okumura, K., Kasakura, K., Nishiyama, C. PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

Angiogenesis-related gene expression profile in clinical cases of canine cancer.

The balance between pro- and anti-angiogenic signalling is tightly regulated in normal tissues to maintain the functions of the vasculature. In contrast, the overproduction of angiogenic factors and enhanced angiogenesis are frequently observed in several types of tumours. Although there have been many reports on the correlation between tumour progression and angiogenesis in humans, little is known about tumour angiogenesis in canines. Hence, we attempted to clarify whether angiogenesis contributes to tumour progression in canines as well as humans. In this study, we investigated the expression of several angiogenesis-related genes, including CD34, VEGF-A, VEGFR-1, VEGFR-2, Ang-1, Ang-2, Tie1, and Tie2, in 66 canine tumour tissues and in the normal tissues surrounding the tumours by quantitative real-time PCR analysis. Our comparative analysis between canine tumour tissues and normal tissues revealed that several angiogenesis-related genes, such as vascular endothelial growth factor (VEGF) and VEGF-receptor genes, were significantly upregulated in canine tumour tissues when compared to the normal tissues. We also found that the angiopoietin (Ang)-1/Ang-2 gene expression ratio was lower in canine tumour tissues than in the normal tissues, suggesting less association between vascular endothelial cells and perivascular cells in the canine tumour tissues. Taken together, our results suggest that several angiogenesis-related genes may contribute to the malignant progression of canine tumours via tumour angiogenesis.

Nonbacterial thrombotic endocarditis involving all four cardiac valves.

This report describes a case of nonbacterial thrombotic endocarditis caused by Waldenström macroglobulinemia, with diffuse endocardial lesions and involvement of all 4 cardiac valves. A 77-year-old man presented with heart failure due to severe regurgitation of all 4 cardiac valves; surgical repair using bioprosthetic valves was indicated. A pathological study revealed fibrin-triggered thrombus formation that confirmed the diagnosis of nonbacterial thrombotic endocarditis. In cases of nonbacterial thrombotic endocarditis, the underlying cause should be investigated.

RNA helicase YTHDC2 promotes cancer metastasis via the enhancement of the efficiency by which HIF-1α mRNA is translated.

YTH domain containing 2 (YTHDC2) is a member of the DExD/H-box family of ATP-dependent RNA helicases. We previously found that YTHDC2 expression is up-regulated in several human cancer cells. In this study, we demonstrate novel roles for YTHDC2 in metastasis of colon tumor cells via translation-dependent pathway. Knockdown of YTHDC2 attenuated protein expression of metastasis-related genes, such as hypoxia-inducible factor-1alpha (HIF-1α), and inhibited metastasis of colon tumor cells in vitro and in vivo. To confirm that YTHDC2 promotes translation initiation by unwinding the 5'-untranslated region (5'UTR) of mRNA, we constructed a firefly luciferase reporter containing the 5'UTR of the HIF-1α mRNA and showed reduction in luciferase activity in YTHDC2-silenced cells. Furthermore, we examined expression levels of YTHDC2 by immunohistochemical staining in human colon cancer tissues from 72 patients and found a significantly positive correlation between YTHDC2 expression and the tumor stage, including metastasis. In conclusion, these results suggest that the RNA helicase YTHDC2 contributes to colon tumor metastasis by promoting translation of HIF-1α and that YTHDC2 is potentially a diagnostic marker and target gene for treating colon cancer patients.

Novel anticancer agent, SQAP, binds to focal adhesion kinase and modulates its activity.

SQAP is a novel and promising anticancer agent that was obtained by structural modifications from a natural compound. SQAP inhibits angiogenesis in vivo resulting in increased hypoxia and reduced tumor volume. In this study, the mechanism by which SQAP modifies the tumor microenvironment was revealed through the application of a T7 phage display screening. This approach identified five SQAP-binding proteins including sterol carrier protein 2, multifunctional enzyme type 2, proteasomal ubiquitin receptor, UV excision repair protein and focal adhesion kinase (FAK). All the interactions were confirmed by surface plasmon resonance analysis. Since FAK plays an important role in cell turnover and angiogenesis, the influence of SQAP on FAK was the principal goal of this study. SQAP decreased FAK phosphorylation and cell migration in human umbilical vein endothelial cells and A549 cancer cells. These findings suggest that inhibition of FAK phosphorylation works as the mechanism for the anti-angiogenesis activity of SQAP.

Risk factors for bleeding evaluated using the Forrest classification in Japanese patients after endoscopic submucosal dissection for early gastric neoplasm.

BACKGROUND/AIMS: Bleeding remains a serious complication after endoscopic submucosal dissection (ESD). Second-look endoscopy for hemostasis helps prevent post-ESD bleeding. We investigated the relationships between patient characteristics, tumor characteristics, and the Forrest classification for exposed vessels on artificial ulcers after ESD and evaluated whether hemostasis during second-look endoscopy was useful for preventing post-ESD bleeding. PATIENTS AND METHODS: We analyzed 250 patients (265 lesions) who underwent ESD for gastric neoplasms. Vessels classified by Forrest classifications during scheduled second-look endoscopy were analyzed for associations with patient characteristics, tumor characteristics, and recurrent bleeding. RESULTS: Two of 250 patients (0.8%) underwent emergency hemostatic endoscopy before scheduled second-look endoscopy. The remaining 248 patients (99.2%) underwent scheduled second-look endoscopy on the day after ESD. Patients with Forrest classification Ia, Ib, or IIa vessels had a significantly higher risk for recurrent bleeding after scheduled second-look endoscopy compared with patients with IIb or III vessels according to univariate analysis (P<0.05) and multivariate logistic regression analysis (odds ratio: 3.45; 95% confidence interval: 1.04-11.41; P=0.042). Univariate analysis indicated that hemodialysis correlated significantly with the presence of Ia, Ib, or IIa vessels compared with that of IIb or III vessels found during second-look endoscopy (P<0.05). Multivariate analysis indicated a significant relationship between hemodialysis and recurrent bleeding after second-look endoscopy (odds ratio: 10.05; 95% confidence interval: 1.97-51.26; P=0.006). CONCLUSION: Hemodialysis is a risk factor for post-ESD bleeding. Proper classification of exposed vessels within post-ESD ulcers according to the Forrest classification using second-look endoscopy might help predict or prevent recurrent bleeding.

Granulomatosis with Polyangiitis (Wegener's Granulomatosis) Complicated with Ruptured Posteromedial Papillary Muscle in the Absence of Coronary Angiographic Findings.

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis, associated with antineutrophil cytoplasmic autoantibody-associated systemic vasculitis, and it can affect many organ systems via the inflammation of small-to-medium-sized vessels. Cardiac involvements in GPA are relatively rare. We report a 75-year-old woman who was diagnosed with GPA and rapid progressive glomerulonephritis that resulted in a partial posteromedial papillary muscle rupture, but with no coronary angiographic findings. The surgical and pathological findings with regard to the ruptured papillary muscle revealed necrotic muscle and acute ischemic change. The mechanism of papillary muscle rupture in GPA is coronary vasculitis leading to myocardial infarction. The ischemic change is not always detected on coronary angiography, so assessment using an echocardiogram is important.

A case of diffuse esophageal spasm successfully treated by steroid therapy.

A 53-year-old man was admitted to our hospital with anterior chest pain and difficulty swallowing. Computed tomography revealed significant esophageal wall thickening. Esophageal intraluminal manometry revealed uncoordinated contraction and strong peristaltic pressure associated with the chest pain. The patient was subsequently diagnosed with diffuse esophageal spasm (DES). His serum immunoglobulin E level was high, and peripheral blood eosinophilia was observed. No eosinophilic infiltration was detected in the esophageal mucosa on endoscopic biopsy. It was presumed that this case of DES was induced by allergic disease. Treatment with 30 mg of oral prednisolone led to a prompt resolution of symptoms;the thickness of the esophageal wall decreased, and the simultaneous contractions disappeared. However, given the presence of a strong peristaltic wave, nutcracker esophagus (NE) was also suspected. This was a rare case of atypical DES induced by allergic disease and associated with NE.

EpsinR, a target for pyrenocine B, role in endogenous MHC-II-restricted antigen presentation.

While the presentation mechanism of antigenic peptides derived from exogenous proteins by MHC class II molecules is well understood, relatively little is known about the presentation mechanism of endogenous MHC class II-restricted antigens. We therefore screened a chemical library of 200 compounds derived from natural products to identify inhibitors of the presentation of endogenous MHC class II-restricted antigens. We found that pyrenocine B, a compound derived from the fungus Pyrenochaeta terrestris, inhibits presentation of endogenous MHC class II-restricted minor histocompatibility antigen IL-4 inducible gene 1 (IL4I1) by primary dendritic cells (DCs). Phage display screening and surface plasmon resonance (SPR) analysis were used to investigate the mechanism of suppressive action by pyrenocine B. EpsinR, a target molecule for pyrenocine B, mediates endosomal trafficking through binding of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Lentiviral-mediated short hairpin (sh) RNA downregulation of EpsinR expression in DCs resulted in a decrease in the responsiveness of CD4+ T cells. Our data thus suggest that EpsinR plays a role in antigen presentation, which provides insight into the mechanism of presentation pathway of endogenous MHC class II-restricted antigen.

Canine Orientia tsutsugamushi infection: report of a case and its epidemicity.

A lethargic household dog was referred to a private hospital in Japan. Diagnosis was carried out by the polymerase chain reaction (PCR) method developed for human Orientia tsutsugamushi infection using the dog's anticoagulated peripheral blood. Karp, Kato and Kuroki-type genomes were detected and the dog was diagnosed with O. tsutsugamushi infection. These findings demonstrate that dogs can act as a host for O. tsutsugamushi and the PCR method developed for human beings can be used for the diagnosis of canine O. tsutsugamushi infection. A concurrent epidemiological study examined 10 asymptomatic dogs that were fed in the same area as the sick dog. Kuroki-type genome in all dogs, Gilliam-type genome in 6 dogs and Kawasaki-type genome in 3 dogs were detected. These results provide further evidence that dogs can be naturally infected with O. tsutsugamushi outdoors and that dogs play a role as a host in the lifecycle of O. tsutsugamushi.

Critical Roles for PU.1, GATA1, and GATA2 in the expression of human FcεRI on mast cells: PU.1 and GATA1 transactivate FCER1A, and GATA2 transactivates FCER1A and MS4A2.

The high-affinity IgE receptor, FcεRI, which is composed of α-, ß-, and γ-chains, plays an important role in IgE-mediated allergic responses. In the current study, involvement of the transcription factors, PU.1, GATA1, and GATA2, in the expression of FcεRI on human mast cells was investigated. Transfection of small interfering RNAs (siRNAs) against PU.1, GATA1, and GATA2 into the human mast cell line, LAD2, caused significant downregulation of cell surface expression of FcεRI. Quantification of the mRNA levels revealed that PU.1, GATA1, and GATA2 siRNAs suppressed the α transcript, whereas the amount of ß mRNA was reduced in only GATA2 siRNA transfectants. In contrast, γ mRNA levels were not affected by any of the knockdowns. Chromatin immunoprecipitation assay showed that significant amounts of PU.1, GATA1, and GATA2 bind to the promoter region of FCER1A (encoding FcεRIα) and that GATA2 binds to the promoter of MS4A2 (encoding FcεRIß). Luciferase assay and EMSA showed that GATA2 transactivates the MS4A2 promoter via direct binding. These knockdowns of transcription factors also suppressed the IgE-mediated degranulation activity of LAD2. Similarly, all three knockdowns suppressed FcεRI expression in primary mast cells, especially PU.1 siRNA and GATA2 siRNA, which target FcεRIα and FcεRIß, respectively. From these results, we conclude that PU.1 and GATA1 are involved in FcεRIα transcription through recruitment to its promoter, whereas GATA2 positively regulates FcεRIß transcription. Suppression of these transcription factors leads to downregulation of FcεRI expression and IgE-mediated degranulation activity. Our findings will contribute to the development of new therapeutic approaches for FcεRI-mediated allergic diseases.

Transcriptional machinery of TNF-α-inducible YTH domain containing 2 (YTHDC2) gene.

We previously demonstrated that a cellular factor, cyclosporin A (CsA) associated helicase-like protein (CAHL) that is identical to YTH domain containing 2 (YTHDC2), forms trimer complex with cyclophilin B and NS5B of hepatitis C virus (HCV) and facilitates HCV genome replication. Gene expression of YTHDC2 was shown in tumor cell lines and tumor necrosis factor (TNF)-α-treated hepatocytes, but not in untreated. However, the function of YTHDC2 in the tumor cells and the mechanism by which the YTHDC2 gene is transcribed in these cells is largely unknown. We first evaluated that the role of YTHDC2 in the proliferation of hepatocellular carcinoma (HCC) cell line Huh7 using RNA interference and found that YTHDC2-downregulated Huh7 were significantly decreased cell growth as compared to control. We next demonstrated that the cAMP response element (CRE) site in the promoter region of the YTHDC2 gene is critical for YTHDC2 transcription. To further investigate the transcription factors bound to the CRE site, we performed chromatin immunoprecipitation assays. Our findings demonstrate that c-Jun and ATF-2 bind to the CRE site in Huh7, and that TNF-α induces the biological activity of these transcription factors in hepatocytes as well as Huh7. Moreover, treatment with the HDAC inhibitor, trichostatin A (TSA), reduces YTHDC2 expression in Huh7 and in TNF-α-stimulated hepatocytes. Collectively, these data show that YTHDC2 plays an important role in tumor cells growth and activation/recruitment of c-Jun and ATF-2 to the YTHDC2 promoter is necessary for the transcription of YTHDC2, and that HDAC activity is required for the efficient expression of YTHDC2 in both of hepatocyte and HCC cells.