Tetsuya Tatsuta

Carnosic acid suppresses the production of amyloid-β 1-42 by inducing the metalloprotease gene TACE/ADAM17 in SH-SY5Y human neuroblastoma cells.

A hallmark of Alzheimers disease (AD) is the aggressive appearance of plaques of amyloid beta (Aβ) peptides, which result from the sequential cleavage of amyloid precursor protein (APP) by the β- and γ-secretases. Aβ production is evaded by alternate cleavage of APP by the α- and γ-secretases. Carnosic acid (CA) has been proven to activate the transcription factor Nrf2, a main regulator of the antioxidant response. We investigated the effects of CA on the production of Aβ 1-42 peptide (Aβ42) and on the expressions of the related genes in SH-SY5Y human neuroblastoma cells. The treatment of cells with CA suppressed Aβ42 secretion (61% suppression at 30μM). CA treatment enhanced the mRNA expressions of an α-secretase TACE (tumor necrosis factor-α-converting enzyme, also called a disintegrin and metalloproteinase-17, ADAM17) significantly and another α-secretase ADAM10 marginally; however, the β-secretase BACE1 (β-site APP-cleaving enzyme-1) was not increased by CA. Knockdown of TACE by siRNA reduced soluble-APPα secretion enhanced by CA and partially recovered the CA-suppressed Aβ42 secretion. These results suggest that CA reduces Aβ42 production by activating TACE without promoting BACE1 in human neuroblastoma cells. The use of CA may have a potential in the prevention of Aβ-mediated diseases, particularly AD.

MDA5 and ISG56 mediate CXCL10 expression induced by Toll-like receptor 4 activation in U373MG human astrocytoma cells

Toll-like receptor (TLR) 4 is a pattern recognition receptor, and recognizes not only bacterial lipopolysaccharide (LPS) but also endogenous danger-associated molecular patterns released from dying or injured cells. It has been reported that TLR4 signaling in astrocytes plays an important role in various neurological diseases. However, details of TLR4 signaling in astrocytes are not fully elucidated. In the present study, we demonstrated that TLR4 signaling, induced by LPS, increases the expression of melanoma differentiation-associated gene 5 (MDA5) and interferon (IFN)-stimulated gene 56 (ISG56) in U373MG human astrocytoma cells. We also found that nuclear factor-κB, p38 mitogen-activated protein kinase and IFN-β are involved in the expression of MDA5 and ISG56 induced by LPS. RNA interference experiments revealed that MDA5 and ISG56 positively regulate the LPS-induced expression of a chemokine CXCL10, but not CCL2. In addition, it was suggested that MDA5 and ISG56 constitute a positive feedback loop. These results suggest that MDA5 and ISG56 may contribute not only to physiological inflammatory reactions but also to the pathogenesis of various neurological diseases elicited by TLR4 in astrocytes, at least in part, by regulating the expression of CXCL10.

ISG54 and ISG56 are induced by TLR3 signaling in U373MG human astrocytoma cells: possible involvement in CXCL10 expression.

Toll-like receptor (TLR) 3 is a pattern recognition receptor that recognizes double-stranded RNA (dsRNA). TLR3 signaling in astrocytes leads to the expression of interferon-β (IFN-β), and IFN-β regulates immune and inflammatory reactions by inducing IFN-stimulated genes (ISGs). We demonstrated in the present study that polyinosinic-polycytidylic acid (poly IC), an authentic dsRNA, up-regulated the expression of ISG54 and ISG56 in U373MG human astrocytoma cells. This reaction was confirmed to be mediated via the TLR3/IFN-β pathway. We also found that ISG56 positively regulates the expression of ISG54, retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). In addition, positive feedback loops were found between ISG54 and ISG56, and also between ISG54 and RIG-I. RNA interference experiments revealed that all of ISG54, ISG56, RIG-I and MDA5 were involved in the poly IC-induced expression of a chemokine CXCL10. These results suggest that ISG54 and ISG56 are involved in the induction of CXCL10 in TLR3/IFN-β signaling at least partly by co-operating with RIG-I and MDA5. ISG54 and ISG56 may contribute to immune and inflammatory reactions elicited by the TLR3/IFN-β signaling pathway in astrocytes, and may play an important role both in antiviral immunity and in neuroinflammatory diseases.

Tumor necrosis factor-α synergistically enhances polyinosinic-polycytidylic acid-induced toll-like receptor 3 signaling in cultured normal human mesangial cells: possible involvement in the pathogenesis of lupus nephritis

AbstractAimIt has been reported that tumor necrosis factor (TNF)-α plays dual controversial roles, beneficial or detrimental, in the pathogenesis of murine lupus nephritis (LN). However, its precise role in the development of human LN remains to be determined. MethodsWe examine the effect of pretreatment with TNF-α on the toll-like receptor 3 (TLR3) signaling induced by polyinosinic-polycytidylic acid (poly IC), a synthetic analog of viral dsRNA that makes “pseudoviral” infection in cultured normal human mesangial cells, and analyzed the expression of CC chemokine ligand 5 (CCL5) via TLR3/interferon (IFN)-β/retinoic acid-inducible gene-I (RIG-I) pathway by reverse transcriptase-polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay.ResultsWe found synergistic effect of TNF-α, even at low level, on the expression of CCL5 induced by poly IC in a concentration-dependent manner, in comparison with that by poly IC alone. Knockdown of either IFN-β or RIG-I decreased CCL5 expression induced by TNF-α followed by poly IC.ConclusionPretreatment with TNF-α leads marked activation of the TLR3/IFN-β/RIG-I/CCL5 axis induced by “pseudoviral” infection. Since chronic local activation of proinflammatory cytokines including TNF-α in resident renal cells may exist in patients with active lupus, synergistic effect of TNF-α and “pseudoviral” infection is possibly involved in the development of LN.

Perioperative complications of endoscopic submucosal dissection for early gastric cancer in elderly Japanese patients 75 years of age or older.

OBJECTIVE The number of endoscopic submucosal dissection (ESD) procedures for early gastric cancers among patients 75 years of age or older has been increasing. We herein examined both the outcomes and complications of ESD in elderly patients. METHODS We investigated the effects of underlying diseases, lesion characteristics, treatment outcomes and complications during and in the postoperative periods of ESD among elderly patients 75 years of age or older versus non-elderly patients less than 75 years of age. Patients A total of 318 early gastric cancers in consecutive 307 patients, all of whom underwent ESD for gastric cancer, were included in this study. RESULTS The number of patients with hypertension and ischemic heart disease was significantly higher in the elderly group than in the non-elderly group. The proportion of lesions with an absolute indication, extended indication or no indication was not significantly different between the groups. During the ESD procedure, the use of atropine sulfate for bradycardia was significantly more frequent in the elderly group. No significant differences were observed between the groups in terms of the treatment for hypertension, oxygen administration or incidence of perforation. In addition, there were no significant differences with respect to oxygen administration, postoperative bleeding or the occurrence of fever and/or pneumonia after the ESD procedure. CONCLUSION Although bradycardia was more frequently observed in the elderly patients during ESD in this study, ESD was performed safely and managed appropriately, with infrequent postoperative complications. ESD appears to be effective, even in elderly patients.

Expression of melanoma differentiation associated gene 5 is increased in human gastric mucosa infected with Helicobacter pylori

Helicobacter pylori infection is associated with gastroduodenal diseases. Melanoma differentiation associated gene 5 (MDA5) plays a role in antiviral host defense. We investigated the effect of H pylori infection on MDA5 expression in human gastric mucosa. Biopsy samples from the antrum and corpus were obtained from 33 patients. MDA5 mRNA and protein were examined by real-time PCR and immunohistochemical staining. Histological gastritis was graded according to updated Sydney System. MDA5 mRNA was significantly increased in the antrum infected with H pylori. MDA5 protein positively stained in infiltrating mononuclear cells. MDA5 mRNA expression was significantly correlated with the grade of glandular atrophy (rs = 0.767) and intestinal metaplasia (rs = 0.748) in the corpus with H pylori infection. These results indicate that MDA5 may be involved in innate immune reactions against H pylori and associate with glandular atrophy and intestinal metaplasia in patients with H pylori infection.

TLR4 signaling induces retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5 in mesangial cells.

BACKGROUND It is known that recognition of bacterial lipopolysaccharide (LPS) and various endogenous ligands by Toll-like receptor 4 (TLR4) induces inflammatory reactions. However, the role of TLR4 activation in mesangial inflammation remains to be elucidated. Retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are putative RNA helicases and are involved in immune and inflammatory reactions. The purpose of the present study was to investigate the implication of RIG-I and MDA5 in TLR4 signaling in mesangial cells. METHODS Normal human mesangial cells in culture were treated with LPS. Expression of RIG-I, MDA5, interferon-β (IFN-β), CXCL10 and CXCL8 was examined using real-time RT-PCR, Western blotting and ELISA. The cells were also subjected to RNA interference against TLR4, IFN-β, RIG-I or MDA5. RESULTS LPS induced the expression of IFN-β, RIG-I, MDA5, CXCL8 and CXCL10 in human mesangial cells. RNA interference against either TLR4 or IFN-β inhibited LPS-induced RIG-I and MDA5 expression. Knockdown of TLR4, IFN-β, RIG-I or MDA5 resulted in decreased induction of CXCL10, while only TLR4 knockdown inhibited CXCL8 induction. CONCLUSIONS TLR4 signaling induces the expression of RIG-I and MDA5 in mesangial cells. RIG-I and MDA5 may be involved in inflammatory reactions by regulating CXCL10 expression in the downstream of TLR4 signaling in human mesangial cells.

Interaction between Interferon-Stimulated Gene 56 and Melanoma Differentiation-Associated Gene 5 in Toll-Like Receptor 3 Signaling in Normal Human Mesangial Cells

Background/Aims: Toll-like receptor 3 (TLR3) is a pathogen recognition receptor against viral double-stranded RNA. TLR3 signaling is important in antiviral responses, but inappropriate TLR3 signaling may be related with inflammatory renal diseases. Interferon (IFN)-stimulated gene 56 (ISG56) is an IFN-inducible gene that encodes a multifunctional protein with 6 tetratricopeptide motifs and is thought to be involved in antiviral reactions, but the role of ISG56 in TLR3 signaling in mesangial cells is not known well. Methods: Normal human mesangial cells were cultured and treated with a synthetic TLR3 ligand polyinosinic-polycytidylic acid, and the expression of ISG56 was analyzed using real-time RT-PCR and Western blot analyses. Using an RNA-interfering technique, involvement of TLR3, IFN-β, melanoma differentiation-associated gene 5 (MDA5) or retinoic acid-inducible gene-I (RIG-I) in ISG56 expression, and of ISG56 in the expression of MDA5, RIG-I, CXCL10 and CCL5 was examined. Results: Treatment of cells with polyinosinic-polycytidylic acid induced ISG56. ISG56 induction was inhibited by knockdown of TLR3 or IFN-β, and knockdown of ISG56 resulted in the decreased expression of MDA5, RIG-I, CXCL10 and CCL5. RNA interference against MDA5 decreased ISG56 expression. Conclusion: ISG56 was induced by TLR3 signaling via newly synthesized IFN-β. ISG56 is involved in the expression of MDA5, RIG-I, CXCL10 and CCL5, and ISG56 and MDA5 may constitute a positive-feedback loop. ISG56 may play a role in immune and inflammatory reactions induced by TLR3 signaling in human mesangial cells.

CCL5 is induced by TLR 3 signaling in HuCCT1 human biliary epithelial cells: possible involvement in the pathogenesis of biliary atr esia

Biliary atresia (BA) is a disease of the newborn that is characterized by progressive, inflammatory and sclerosing cholangiopathy. Innate immune responses to viral components are thought to be involved in the pathogenesis of BA. It is also reported that some chemokines, such as CCL5, are possibly involved in the pathogenesis of experimental animal model of BA. We treated human biliary epithelial HuCCT1 cells with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA (dsRNA) which mimics viral RNA, and analyzed the CCL5 expression by quantitative reverse transcription-PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). To examine the regulation mechanisms of CCL5, we subjected the cells to RNA interference (siRNA) against Toll-like receptor 3 (TLR3), interferon (IFN)-β, NF-κB p65 and IFN regulatory factor (IRF) 3. Immunohistochemical staining for CCL5 was also performed in tissues from patients with BA. Poly IC induced CCL5 expression in HuCCT1 cells. CCL5 expression induced by poly IC was inhibited by the knockdown of TLR3, p65 or IRF3, but it was not affected by knockdown of IFN-β. Immunohistochemical staining showed that CCL5 was strongly expressed in biliary epithelial cells of patients with BA. The current study suggests that TLR3 signaling induces CCL5 expression via NF-κB and IRF3 in bile duct cells, and this pathway may be involved in the pathogenesis of BA.

Estimation of perioperative invasiveness of colorectal endoscopic submucosal dissection evaluated by energy metabolism

The aim of this study was to assess the perioperative invasiveness of endoscopic submucosal dissection for colorectal cancer quantitatively by using energy metabolism. In fifty-three patients who underwent endoscopic submucosal dissection for colorectal cancer, resting energy expenditure using an indirect calorimeter, body weight and basal energy expenditure using the Harris–Benedict equation before and after endoscopic submucosal dissection. Resting energy expenditure/body weight and resting energy expenditure/basal energy expenditure were 19.7 ± 2.5 kcal/kg/day and 0.96 ± 0.12 on the day of endoscopic submucosal dissection, whereas one day after the endoscopic submucosal dissection they increased to 21.0 ± 2.9 kcal/kg/day and 1.00 ± 0.13 (p<0.001 and p<0.05, respectively). The stress factor on the postoperative day 1 was computed as 1.06. The increase was lower comparing with that experienced for surgery, suggesting that the perioperative invasiveness of colorectal endoscopic submucosal dissection is lower in comparison to that during surgery. Furthermore, in spite of technical difficulty, stress factor of colorectal endoscopic submucosal dissection was approximately equal to that of gastric endoscopic submucosal dissection. (The study of the resting energy metabolism and stress factor using an indirect calorimeter in the perioperative period of endoscopic operation: UMIN000027135)