Takaya Yamada

Nonpathogenic Escherichia coli strain Nissle1917 prevents murine acute and chronic colitis

Background: Nonpathogenic Escherichia coli strain Nissle1917 has been used as a probiotics in human inflammatory bowel disease; however, there are few reports examining its therapeutic effect on animal colitis models, and its therapeutic mechanisms remain unknown. The aim of this study was to elucidate the therapeutic effect and mechanism of Nissle1917 using murine acute and chronic colitis models. Methods: Two models were used. (1) Acute model: colitis was induced by administration of 1.3% dextran sodium sulfate for 7 days. Nissle1917 or phosphate‐buffered saline were orally administered for 10 days. Mice were killed at day 10, and the colonic lesions were assessed macro‐ and microscopically. (2) Chronic model: IL‐10−/− mice were treated with Nissle1917 or phosphate‐buffered saline for 8 weeks. After 8 weeks of treatment, mice were killed to assess the colonic lesions macro‐ and microscopically. In the acute dextran sodium sulfate colitis model, viable, heat‐killed, or genomic DNA of Nissle1917 was orally administered for 10 days, and the therapeutic effect was assessed. Results: In the acute model, Nissle1917 ameliorated body weight loss, disease activity index, and macro‐ and microscopic damage. In the chronic model, it also suppressed the mucosal inflammatory findings and histologic damages. Moreover, heat‐killed Nissle1917 or its genomic DNA alone also ameliorated the acute DSS colitis and viable bacteria macro‐ and microscopically. Conclusions: Nonpathogenic E. coli strain Nissle1917 prevents both acute and chronic colitis, and its anti‐inflammatory effect is exhibited not only by viable bacteria but also by heat‐killed bacteria or its DNA.

A synthetic mimetic of CD4 is able to suppress disease in a rodent model of immune colitis

CD4+ mucosal T cells mediate the intestinal inflammation in Crohns disease and may serve as an important target for immune intervention. Here we assessed the therapeutic effect of a synthetic mimetic of CD4 designed to mimic both the sequence and conformation of the complementarity‐determining region 3 of murine CD4 V1 domain (rD‐mPGPtide) in a mouse colitis model using immunization with 2,4,6‐trinitrobenzene sulfonic acid (TNB). i.  v. administration of the rD‐mPGPtide but not control scrambled peptide could suppress severe inflammation in the chronic colitis mouse model. After treatment with the rD‐mPGPtide, a striking improvement of diarrhea and acute wasting disease was observed with decreased mortality. Serum anti‐TNB antibody titers, CD45RBlowCD4+ T cells in the lamina propria and IFN‐γ mRNA expression in the mucosa were significantly decreased with the rD‐mPGPtide treatment. Anti‐CD4 antibody also suppressed disease by depletion of CD45RBhighCD4+ T cells in the colonic mucosa. The observation that the synthetically engineered analogue of murine CD4 inhibits inflammation in a rodent disease model by different mechanisms than anti‐CD4 antibody suggests that a human version of this peptide has potential therapeutic utility in CD4+ mucosal T cell‐mediated intestinal inflammation in Crohns disease.

Nonpathogenic Escherichia coli Strain Nissle 1917 Inhibits Signal Transduction in Intestinal Epithelial Cells

ABSTRACT Although the probiotic Escherichia coli strain Nissle 1917 has been used for the treatment of inflammatory bowel diseases, the precise mechanisms of action of this strain remain unclear. In the present study, we estimated the anti-inflammatory effect of E. coli Nissle 1917 on inflammatory responses in vitro to determine the suppressive mechanism of Nissle 1917 on the inflammatory process. To determine the effect of E. coli Nissle 1917, the human colonic epithelial cell line HCT15 was incubated with or without E. coli Nissle 1917 or another nonpathogenic E. coli strain, K-12, and then tumor necrosis factor alpha (TNF-α)-induced interleukin-8 (IL-8) production from HCT15 cells was assessed. Enzyme-linked immunosorbent assays and real-time quantitative PCR showed that Nissle 1917 treatment suppressed TNF-α-induced IL-8 transcription and production. In addition, results from luciferase assays indicated that Nissle 1917 inhibited IL-8 promoter activity. On the other hand, these anti-inflammatory effects were not seen with E. coli K-12. In addition, heat-killed Nissle 1917 or its genomic DNA did not have this anti-inflammatory effect. Surprisingly, Nissle 1917 did not affect IL-8 transactivation pathways, such as NF-κB activation, nuclear translocation, and DNA binding, or even activation of other transcriptional factors. Furthermore, it also became evident that Nissle 1917 induced the anti-inflammatory effect without contact to epithelial cells. In conclusion, these data indicate that the nonpathogenic E. coli strain Nissle 1917 expresses a direct anti-inflammatory activity on human epithelial cells via a secreted factor which suppresses TNF-α-induced IL-8 transactivation through mechanisms different from NF-κB inhibition.

Immunogenic chemotherapy with cyclophosphamide and doxorubicin against established murine carcinoma

Mitigation of regulatory T cell-mediated immunosuppression and elicitation of immunogenic tumor cell death are crucial events for optimal anti-tumor immune activity in vivo. This study was designed to investigate the potential synergistic activity of the combined use of cyclophosphamide (CP) and doxorubicin (DR), both of which are known to resolve these two issues. BALB/c mice were inoculated subcutaneously with CT-26 carcinoma cells in the bilateral flank and treated with an intraperitoneal injection of a low dose of CP followed by an intratumoral injection of DR into one side of the tumor. We found that, in addition to a significant suppression of growth on the DR-treated side of the tumor, combination therapy suppressed the growth of DR-untreated remote tumors in both tumor-specific and T cell-dependent manners. Mitomycin C showed no such synergistic anti-tumor activity with CP treatment. Combination therapy increased the frequency of interferon (IFN)-γ-producing T lymphocytes specific to a CT-26-associated class I-binding tumor peptide in the tumor-draining lymph nodes. Real-time PCR analysis revealed that combination therapy led to an increase in IFN-γ and tumor necrosis factor-α mRNA expression; however, levels of Foxp3 and transforming growth factor-β within the remote tumor tissues were decreased. In addition, knock down of calreticulin expression in CT-26 cells using small interfering RNA attenuated anti-tumor vaccine effects induced by DR-treated CT-26 cells. These results provide an immunological rationale for the combined use of chemotherapeutic drugs, i.e., CP and DR, and further recommend their use with current cancer vaccines.

Butyric acid attenuates intestinal inflammation in murine DSS-induced colitis model via milk fat globule-EGF factor 8

Butyric acid, a short-chain fatty acid and one of the main metabolites of intestinal microbial fermentation of dietary fiber, has been shown to have an important role in maintaining the integrity of the intestinal mucosa, while it also has been shown to exert potent anti-inflammatory effects both in vitro and in vivo. However, the precise mechanisms underlying those effects have not been fully identified. We exposed colonic epithelial cells to butyric acid, then extracted total RNA samples, and subsequently hybridized them to microarray chips. Among the upregulated genes, milk fat globule-epidermal growth factor 8 (MFG-E8) was elevated by approximately fivefold. We previously reported that the potential therapeutic benefits of MFG-E8 in intestinal tissue injury were dependent not only on enhanced clearance of apoptotic cells but also required diverse cellular events for maintaining epithelial integrity. The influence of butyric acid on cell function is often attributed to its inhibition of histone deacetylases (HDACs). We found that acetylation on histone 3 lysine 9 (acetyl-H3K9) around the MFG-E8 promoter was significantly increased with butyric acid exposure. Experimental colitis was induced by administration of dextran sodium sulfate (DSS) in C57BL/6N (MFG-E8+/+) and MFG-E8−/− mice. Although the colonic bacterial compositions in wild-type (WT) and MFG-E8−/− mice were not significantly different, intrarectal administration of butyric acid during an acute phase of colitis attenuated intestinal inflammatory parameters and inhibited body weight loss in the WT mice. Our novel findings suggest that butyric acid has significant anti-inflammatory effects partly via MFG-E8 on DSS-induced murine experimental colitis.

A Novel Fusicoccin Derivative Preferentially Targets Hypoxic Tumor Cells and Inhibits Tumor Growth in Xenografts

Malignant cells in solid tumors survive under prolonged hypoxia and can be a source of resistance to current cancer therapies. Tumor hypoxia is also associated with a more malignant phenotype and poor survival in cancer patients. Recent progress in our understanding of the biology of tumor cells under hypoxia has led to increased attention on targeting hypoxia for cancer therapy. We report here that a novel fusicoccin derivative (ISIR-042), but not its parent or related compounds such as fusicoccin A and cotylenin A, is more cytotoxic to hypoxic cells than to normoxic cells. The hypoxia-induced accumulation of hypoxia-inducible factor (HIF)-1α and the phosphorylation of Akt were effectively inhibited by treatment with ISIR-042, suggesting that the preferential cytotoxicity toward hypoxic cells is associated with a reduction of HIF-1α and Akt activation. ISIR-042 inhibited the growth of human pancreatic cancer MIAPaCa-2 cells while sparing normal endothelial cells, and significantly inhibited the growth of MIAPaCa-2 cells as xenografts without apparent adverse effects. Pancreatic cancer cells expressing CD24 and CD44 exhibited characteristics of stem cells. Treatment with gemcitabine increased this stem cell-enriched population, and this effect was significantly inhibited by ISIR-042, suggesting that ISIR- 042 preferentially inhibits stem/progenitors in pancreatic cancer cell lines compared with chemotherapeutic agents. These results suggest that ISIR-042 may be a potential therapeutic agent for hypoxic tumors such as pancreatic cancer.

Isolation and Characterization of a cDNA Clone for Plant Nuclear Antigen 21D7 Associated with Cell Division

A cDNA clone was isolated from a carrot (Daucus carota L.) cDNA expression library using monoclonal antibody 21D7, which recognizes a nuclear antigen associated with cell division in plant cells. To show that the isolated cDNA encodes the 21D7 antigen, a polyclonal antiserum was raised against a recombinant fusion protein specified by the cDNA. Both the polyclonal antiserum and the monoclonal antibody 21D7 recognized the same plant protein on immunoblots, in immunoprecipitation experiments, and in peptide mapping. Analysis of the cDNA revealed that the deduced amino acid sequence has 45% identity to the predicted sequence of the mouse transplantation antigen P91A from mutant tumor cells that is responsible for the immune rejection of the corresponding cell clone in syngeneic mouse. The expression of the plant cDNA at the mRNA level was highly correlated with cell proliferation. In suspension cultures of Catharanthus roseus (L.) G Don. cells, the highest level of expression was observed during the midlogarithmic phase of growth. When auxin was added to stimulate cell division of auxin-starved cells arrested in the G1 phase, transcription was immediately enhanced, and the level of expression remained high throughout the G1 and S phases and dropped dramatically at the end of DNA replication.

Sublingual Immunotherapy Induces Regulatory Function of IL-10-Expressing CD4+CD25+Foxp3+ T Cells of Cervical Lymph Nodes in Murine Allergic Rhinitis Model

Sublingual immunotherapy (SLIT) has been considered to be a painless and efficacious therapeutic treatment of allergic rhinitis which is known as type I allergy of nasal mucosa. Nevertheless, its mechanisms need to be further investigated. In this study, we constructed an effective murine model of sublingual immunotherapy in allergic rhinitis, in which mice were sublingually administered with ovalbumin (OVA) followed by intraperitoneal sensitization and nasal challenge of OVA. Sublingually treated mice showed significantly decreased specific IgE responses as well as suppressed Th2 immune responses. Sublingual administration of OVA did not alter the frequency of CD4+CD25+ regulatory T cells (Tregs), but led to upregulation of Foxp3- and IL-10-specific mRNAs in the Tregs of cervical lymph nodes (CLN), which strongly suppressed Th2 cytokine production from CD4+CD25− effector T cells in vitro. Furthermore, sublingual administration of plasmids encoding the lymphoid chemokines CCL19 and CCL21-Ser DNA together with OVA suppressed allergic responses. These results suggest that IL-10-expressing CD4+CD25+Foxp3+ Tregs in CLN are involved in the suppression of allergic responses and that CCL19/CCL21 may contribute to it in mice that received SLIT.

The predictability of clinical antitumor effects using two distinctive in vitro chemosensitivity tests: An analysis of true positive cases

The results of two types of in vitro chemosensitivity tests, namely, the human tumor clonogenic assay (HTCA) and the succinic dehydrogenase inhibition assay (SDIA), for solid tumors, including stomach, colorectal and lung cancers, were analyzed and their correlation with clinical effects evaluated. The anticancer agents employed were mitomycin C (MMC), 5-fluorouracil (5-FU), adriamycin (ADM) and cisplatin (DDP). The evaluability rates of the assays were 54.5% for HTCA and 89.0% for SDIA. Among the 29 cases with evaluable lesions subjected to HTCA, there were 4 true positives, 9 false positives, and 16 true negatives, whereas among the 32 cases subjected to SDIA, the corresponding numbers were 2, 6, and 24, respectively. There were no false negatives for either assay, the accuracy of prediction for HTCA being 69.0% and for SDIA, 81.3%. The true positives of both assays included one complete response (CR) and five partial responses (PR), although the eventual outcome was cancer death in all cases. Interestingly, in five out of the six true positive cases, the agent involved was either ADM or DDP, both the which are usually regarded as “second line” anticancer agents for gastrointestinal carcinomas.

Crystallization of Nb3Sn by arc casting

Abstract Nb-Sn mixtures were cast in an arc furnace and the formation of Nb3Sn was investigated with respect to the starting composition. It was found that the Nb3Sn phase was produced in four forms: (i) as a filler between interdendrites of α-Nb, (ii) as dendrites, (iii) as polycrystals (grains) and (iv) as a mass (probably an aggregation of small dendrites). The lattice parameters of Nb3Sn were determined using the Debye-Scherrer method.