Melatonin modulates TLR4-mediated inflammatory genes through MyD88- and TRIF-dependent signaling pathways in lipopolysaccharide-stimulated RAW264.7 cells.

Increasing evidence demonstrates that melatonin has an anti-inflammatory effect. Nevertheless, the molecular mechanisms remain obscure. In this study, we investigated the effect of melatonin on toll-like receptor 4 (TLR4)-mediated molecule myeloid differentiation factor 88 (MyD88)-dependent and TRIF-dependent signaling pathways in lipopolysaccharide (LPS)-stimulated macrophages. RAW264.7 cells were incubated with LPS (2.0 µg/mL) in the absence or presence of melatonin (10, 100, 1000 µm). As expected, melatonin inhibited TLR4-mediated tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, IL-6, IL-8, and IL-10 in LPS-stimulated macrophages. In addition, melatonin significantly attenuated LPS-induced upregulation of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in macrophages. Further analysis showed that melatonin inhibited the expression of MyD88 in LPS-stimulated macrophages. Although it had no effect on TLR4-mediated phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular regulated protein kinase (ERK), melatonin significantly attenuated the activation of nuclear factor kappa B (NF-κB) in LPS-stimulated macrophages. In addition, melatonin inhibited TLR4-mediated Akt phosphorylation in LPS-stimulated macrophages. Moreover, melatonin significantly attenuated the elevation of interferon (IFN)-regulated factor-3 (IRF3), which was involved in TLR4-mediated TRIF-dependent signaling pathway, in LPS-stimulated macrophages. Correspondingly, melatonin significantly alleviated LPS-induced IFN-ß in macrophages. In conclusion, melatonin modulates TLR4-mediated inflammatory genes through MyD88-dependent and TRIF-dependent signaling pathways.

Spatio-temporal pattern of schistosomiasis in Anhui Province, East China: Potential effect of the Yangtze River - Huaihe River Water Transfer Project.

Anhui Province has been one of typical epidemic areas of schistosomiasis in East China as a wide range of large lake and marshland regions provide an ideal environment for growth and reproduction of the intermediate snail host. With the completion of the Yangtze River-Huaihe River Water Transfer Project (YHWTP), launched by the end of 2016, the epidemic areas are expected to expand and controlling schistosomiasis remains a challenge. Based on annual surveillance data at the county level in Anhui for the period 2006-2015, spatial and temporal cluster analyses were conducted to assess the pattern of risk through spatial (Local Moran's I and flexible scan statistic) and space-time scan statistic (Kulldorff). It was found that schistosomiasis sero-prevalence was dramatically reduced and maintained at a low level. Cluster results showed that spatial extent of schistosomiasis contracted, but snail distribution remained geographically stable across the study area. Clusters, both for schistosomiasis and snail presence, were common along the Yangtze River. Considering the effect of the ongoing YHWTP on the potential spread of schistosomiasis, Zongyang County and Anqing, which will be transected by the new water-transfer route, should be given a priority for strengthened surveillance and control. Attention should also be paid to Guichi since it is close to one of the planned inlets of the YHWTP.

Cadmium selectively induces MIP-2 and COX-2 through PTEN-mediated Akt activation in RAW264.7 cells.

Increasing evidence demonstrates that cadmium (Cd) induces inflammation, but its mechanisms remain obscure. The present study showed that treatment with CdCl2 selectively upregulates macrophage inflammatory protein (MIP)-2 and cyclooxygenase (COX)-2 in RAW264.7 cells. Concomitantly, Cd²âº markedly elevated the level of phosphorylated Akt in dose- and time-dependent manners. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), blocked Cd²âº-evoked Akt phosphorylation. Correspondingly, LY294002 significantly repressed Cd²âº-induced upregulation of MIP-2 and COX-2 in RAW264.7 cells. Further experiments showed that treatment with Cd²âº significantly reduced the level of PTEN protein in RAW264.7 cells. MG132, a specific proteasome inhibitor, blocked Cd²âº-induced reduction in PTEN protein as well as Akt phosphorylation, implicating the involvement of proteasome-mediated PTEN degradation. Of interest, Cd²âº-induced degradation of PTEN protein appears to be associated with PTEN ubiquitination. N-acetylcysteine, a glutathione (GSH) precursor, blocked Cd²âº-evoked PTEN degradation as well as Akt phosphorylation. By contrast, L-buthionine-S,R-sulfoximine, an inhibitor of cellular GSH synthesis, exacerbated Cd²âº-induced PTEN degradation and Akt phosphorylation. Alpha-phenyl-N-tert-butylnitrone and vitamin C, two antioxidants, did not prevent from Cd²âº-induced PTEN degradation and Akt phosphorylation. In conclusion, Cd²âº selectively induces MIP-2 and COX-2 through PTEN-mediated PI3K/Akt activation. Cellular GSH depletion mediates Cd²âº-induced PTEN degradation and subsequent PI3K/Akt activation in macrophages.

Maternal LPS exposure during pregnancy impairs testicular development, steroidogenesis and spermatogenesis in male offspring.

Lipopolysaccharide (LPS) is associated with adverse developmental outcomes including embryonic resorption, fetal death, congenital teratogenesis and fetal growth retardation. Here, we explored the effects of maternal LPS exposure during pregnancy on testicular development, steroidogenesis and spermatogenesis in male offspring. The pregnant mice were intraperitoneally injected with LPS (50 µg/kg) daily from gestational day (GD) 13 to GD 17. At fetal period, a significant decrease in body weight and abnormal Leydig cell aggregations were observed in males whose mothers were exposed to LPS during pregnancy. At postnatal day (PND) 26, anogenital distance (AGD), a sensitive index of altered androgen action, was markedly reduced in male pups whose mothers were exposed to LPS daily from GD13 to GD 17. At PND35, the weight of testes, prostates and seminal vesicles, and serum testosterone (T) level were significantly decreased in LPS-treated male pups. At adulthood, the number of sperm was significantly decreased in male offspring whose mothers were exposed to LPS on GD 13-17. Maternal LPS exposure during gestation obviously diminished the percent of seminiferous tubules in stages I-VI, increased the percent of seminiferous tubules in stages IX-XII, and caused massive sloughing of germ cells in seminiferous tubules in mouse testes. Moreover, maternal LPS exposure significantly reduced serum T level in male mice whose mothers were exposed to LPS challenge during pregnancy. Taken together, these results suggest that maternal LPS exposure during pregnancy disrupts T production. The decreased T synthesis might be associated with LPS-induced impairments for spermatogenesis in male offspring.

Solid-phase epitope recovery: a high throughput method for antigen identification and epitope optimization.

Self tolerance to MHC class I-restricted nonmutated self Ags is a significant hurdle to effective cancer immunotherapy. Compelling evidence is emerging that altered peptide ligands can be far more immunogenic than their corresponding native epitopes; however, there is no way to reliably predict which modifications will lead to enhanced native epitope-specific immune responses. We reasoned that this limitation could be overcome by devising an empirical screen in which the nearly complete combinatorial spectrum of peptides of optimal length can be rapidly assayed for reactivity with a MHC class I-restricted cytotoxic T cell clone. This method, solid-phase epitope recovery, quantitatively ranks all reactive peptides in the library and allows selection of altered peptide ligands having desirable immunogenic properties of interest. In contrast to rationally designed MHC anchor-modified peptides, peptides identified by the present method are highly substituted in predicted TCR contact residues and can reliably activate and expand effector cell populations in vitro which lyse target cells presenting the wild-type epitope. We demonstrate that solid-phase epitope recovery peptides corresponding to a poorly immunogenic epitope of the melanoma Ag, gp100, can reliably induce wild-type peptide-specific CTL using normal donor T cells in vitro. Furthermore, these peptides can complement one another to induce these responses in an overwhelming majority of normal individuals in vitro. These data provide a rationale for the design of superior vaccines comprising a mixture of structurally diverse yet functionally convergent peptides.