When a virus encounters a susceptible cell, the virus enters and initiates cytocidal, persistent, latent or abortive infection. We recently reported that influenza virus (IV) induces persistent infection accompanied by cell survival and virus production in cultured amnion cells. In addition, cytocidal infection accompanied by apoptotic cell death and virus production occurred in cultured chorion cells. Both cell cultures were prepared from human fetal membranes. 1)IV induces cytocidal infection in a variety of cultures, such as HeLa and Madin-Darby canine kidney (MDCK) cell lines and human peripheral blood monocytes in vitro, all of which die through the mechanism of apoptosis.2-5) Furthermore, IV infection induces gene expression of inflammatory cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-a and interferon (IFN)-a/b, and C-C and C-X-C chemokines such as monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1a, RANTES (regulated on activation, normal T cell expressed and secreted) and IFN-inducible protein (IP)-10, 6,7) and proapoptotic factors such as Fas and Fas ligand,8) which occur in infected host cells undergoing apoptosis. Apoptosis and inducible gene expression of inflammatory mediators therefore seem to be correlated. However, whether IV induces gene expression of inflammatory mediators during persistent infection remains unknown.We therefore extensively examined expression of mRNAs for inflammatory cytokines such as IL-1b, IL-6, TNF-a, IFN-a, IFN-b, IFN-g, macrophage colony-stimulating factor (M-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF), and Fas during cytocidal and persistent infections by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Of note is the fact that IV infection induced the gene expression of a set of inflammatory cytokines during cytocidal infection accompanied by apoptotic cell death, but this induction was not present during persistent infection in the absence of apoptosis. In this paper we report a noticeable difference in gene expression of a set of inflammatory cytokines between cytocidal and persistent infections. MATERIALS AND METHODSMaterials PCR primers for IFN-a, IFN-b, IFN-g, M-CSF and GM-CSF were originally designed according to published cDNA sequences. 9-13) PCR primers for Fas were based on the published DNA sequences for the primers. 14)PCR primers for IL-1b, IL-6, TNF-a and glycerol-3-phosphate dehydrogenase (G3PDH) were purchased from CLON-TECH Labs. Inc. (CA, U.S.A.). DNA sequences for the PCR primers are shown in Table 1. Neutralizing antibodies against Fas (Medical & Biological Labs. Co., Ltd., Nagoya, Japan), IFN-b (Yamasa Shoyu Co., Ltd., Tokyo, Japan), TNF-a and IFN-g (Genzyme Diagnostic, MA, U.S.A.) were purchased from the listed suppliers.Cell Culture and Virus Infection Primary cultured chorion and amnion cells were prepared from human fetal membranes obtained by cesarean section during the month of normal parturition as described.15) HeLa cell line was obtained from...

Dipeptidyl peptidase IV (DPP-IV) expression in visceral adipose tissue is reportedly increased in obese patients, suggesting an association of DPP-IV with inflammation. In this study, first, lipopolysaccharide (LPS)- or palmitate-induced elevations of inflammatory cytokine mRNA expressions in RAW264.7 macrophages were shown to be significantly suppressed by coincubation with a DPP-IV inhibitor, anagliptin (10 μM), despite low DPP-IV expression in the RAW264.7 cells. Regarding the molecular mechanism, LPS-induced degradation of IκBα and phosphorylations of p65, JNK, and p38, as well as NF-κB and AP-1 promoter activities, were revealed to be suppressed by incubation with anagliptin, indicating suppressive effects of anagliptin on both NF-κB and AP-1 signaling pathways. Anagliptin also acted on 3T3-L1 adipocytes, weakly suppressing the inflammatory cytokine expressions induced by LPS and TNFα. When 3T3-L1 and RAW cells were cocultured and stimulated with LPS, the effects of anagliptin on the suppression of cytokine expressions in 3T3-L1 adipocytes were more marked and became evident at the 10 μM concentration. Anti-inflammatory effects of anagliptin were also observed in vivo on the elevated hepatic and adipose expressions and serum concentrations of inflammatory cytokines in association with the suppression of hepatic NF-κB transcriptional activity in LPS-infused mice. Taking these observations together, the anti-inflammatory properties of anagliptin may be beneficial in terms of preventing exacerbation of diabetes and cardiovascular events.

Objective: Several chemokines play important roles in recruiting the monocyte/macrophage lineage into adipose tissues. We previously found CCL19 was highly expressed in adipocytes cocultured with macrophages stimulated by endotoxin. This study aimed to evaluate the role of CCL19-CCR7 axis on obesity and insulin resistance. Methods: Serum CCL19 concentration was examined in obese model mice challenged by endotoxin. CCL19 receptor-null, Ccr7 2/2 , mice and wild-type mice fed a high-fat diet or normal diet were used to investigate the role of CCL19 signals on obesity-associated inflammation. Results: CCL19 protein was elevated in the sera of obese model mice challenged by endotoxin. Ccr7 2/2 mice were protected from diet-induced obesity and insulin resistance. The adipose tissue and liver expression of inflammatory genes of Ccr7 2/2 mice was much lower than in diet-induced obese mice. Ccr7 2/2 mice were protected from fatty liver and dyslipidemia and exhibited increased thermogenesis on high-fat feeding. CCL19 attracts activated dendritic cells (DC). The expression of the DC markers, CD11b and 11c, was not observed in the adipose tissues of Ccr7 2/2 mice fed a high-fat diet, which might be closely associated with the protection of these mice from obesity. Conclusions: The CCL19-CCR7 pathway associates with the development of high-fat-induced obesity and insulin resistance.

Background Recently, clinical studies have shown the protective effects of sodium glucose co-transporter2 (SGLT2) inhibitors against progression of diabetic nephropathy, but the underlying molecular mechanisms remain unclear. Methods Diabetic mice were prepared by injecting nicotinamide and streptozotocin, followed by high-sucrose diet feeding (NA/STZ/Suc mice). The SGLT2 inhibitor canagliflozin was administered as a 0.03% (w/w) mixture in the diet for 4 weeks. Then, various parameters and effects of canagliflozin on diabetic nephropathy were investigated. Results Canagliflozin administration to NA/STZ/Suc mice normalized hyperglycemia as well as elevated renal mRNA of collagen 1a1, 1a2, CTGF, TNFα and MCP-1. Microscopic observation revealed reduced fibrotic deposition in the kidneys of canagliflozin-treated NA/STZ/Suc mice. Interestingly, the protein level of Pin1, reportedly involved in the inflammation and fibrosis affecting several tissues, was markedly increased in the NA/STZ/Suc mouse kidney, but this was normalized with canagliflozin treatment. The cells showing increased Pin1 expression in the kidney were mainly mesangial cells, along with podocytes, based on immunohistochemical analysis. Furthermore, it was revealed that canagliflozin induced AMP-activated kinase (AMPK) activation concentration-dependently in CRL1927 mesangial as well as THP-1 macrophage cell lines. AMPK activation was speculated to suppress mesangial cell proliferation and exert anti-inflammatory effects in hematopoietic cells. Conclusion Therefore, we can reasonably suggest that normalized Pin1 expression and AMPK activation contribute to the molecular mechanisms underlying SGLT2 inhibitor-induced suppression of diabetic nephropathy, possibly at least in part by reducing inflammation and fibrotic change.

SUMMARYActivation of adenosine A 2a receptors in cerebral neurons induces sleep in various mammals. It was previously found that Japanese sake yeast enriched in adenosine analogues activates A 2a receptors in vitro and induces sleep in mice. Here it is reported that sake yeast activated A 2a receptors in a cultured human cell line and improved human sleep quality in a clinical trial. Sake yeast activated A 2a receptors in HEK cells in a dose-dependent manner with an EC 50 of 40 lg mL À1 , and the activation was attenuated almost completely by the A 2a receptor antagonist ZM241385 with an IC 50 of 73 nM. In a double-blind placebo-controlled crossover clinical study, 68 healthy participants ingested tablets containing either 500 mg of sake yeast powder or a placebo (cellulose) 1 h before sleep for 4 days. Electroencephalograms were recorded during sleep at home with a portable device for 4 week days. Electroencephalogram analyses revealed that sake yeast supplementation significantly (P = 0.03) increased delta power during the first cycle of slow-wave sleep by 110%, without changing other sleep parameters. Sake yeast supplementation also significantly increased growth hormone secretion in the urine on awakening by 137% from 3.17 AE 0.41 (placebo) to 4.33 AE 0.62 (sake yeast) pg mg À1 creatinine (P = 0.03). Subjective sleepiness (P = 0.02) and fatigue (P = 0.06) in the morning were improved by sake yeast. Given these benefits and the absence of adverse effects during the study period, it was concluded that sake yeast supplementation is an effective and safe way to support daily high-quality, deep sleep.