Apolipoprotein E4 enhances brain inflammation by modulation of the NF-κB signaling cascade

Apolipoprotein E4 (apoE4), the major genetic risk factor of Alzheimer's disease (AD), is associated with enhanced brain inflammation. Genome-wide gene expression profiling was employed to study the effects of apoE genotype on hippocampal gene expression in LPS-treated mice, transgenic for either apoE4 or the AD benign allele, apoE3. This revealed that the expression of inflammation-related genes following intracerebroventricular injection of LPS was significantly higher and more prolonged in apoE4 than in apoE3 transgenic mice. Clustering analysis revealed gene clusters which responded differently in apoE4 and apoE3 mice and were significantly enriched in NF-kappaB response elements. Direct measurement of NF-kappaB-regulated genes revealed that their extent of activation was greater in the apoE4 mice. Immunohistochemistry experiments revealed that microglial and NF-kappaB activation were more pronounced in apoE4 than in apoE3 mice. These findings suggest that the increased brain inflammation in apoE4 mice is related to disregulation of NF-kappaB signaling pathway.     

Suppressive effect of inducible nitric oxide synthase (iNOS) expression by the methanol extract of Actinodaphne lancifolia

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) has played a crucial role in various pathophysiological processes including inflammation and carcinogenesis. Therefore, the inhibitors of NO synthesis or iNOS gene expression have been considered as potential anti-inflammatory and cancer chemopreventive agents. In our continuous search for iNOS inhibitors from natural products we have evaluated indigenous Korean plant extracts using an assay for inhibition of nitric oxide formation on lipopolysaccharide (LPS)-activated mouse macrophage RAW 264.7 cells. As a result, the methanolic stem extract of Actinodaphne lancifolia showed an inhibitory activity of NO production in a dose-dependent manner (IC50 = 2.5 microg/ml). Additional study demonstrated that the extract of Actinodaphne lancifolia significantly suppressed the iNOS protein and gene expression in a dose-dependent manner. These results suggest that Actinodaphne lancifolia could be a potential candidate for developing an iNOS inhibitor from natural products. Further elucidation of active principles for development of new cancer chemopreventive and/or anti-inflammatory agents could be warranted.     

Emphysema induced by elastase enhances acute inflammatory pulmonary response to intraperitoneal LPS in rats

Abnormalities in lungs caused by emphysema might alter their response to sepsis and the occurrence of acute lung injury (ALI). This study compared the extension of ALI in response to intraperitoneal lipopolysaccharide (LPS) injection in Wistar rats with and without emphysema induced by elastase. Adult male Wistar rats were randomized into four groups: control, emphysema without sepsis, normal lung with sepsis and emphysema with sepsis. Sepsis was induced, and 24 h later the rats were euthanised. The following analysis was performed: blood gas measurements, bronchoalveolar lavage (BAL), lung permeability and histology. Animals that received LPS showed significant increase in a lung injury scoring system, inflammatory cells in bronchoalveolar lavage (BAL) and IL‐6, TNF‐α and CXCL2 mRNA expression in lung tissue. Animals with emphysema and sepsis showed increased alveolocapillary membrane permeability, demonstrated by higher BAL/serum albumin ratio. In conclusion, the presence of emphysema induced by elastase increases the inflammatory response in the lungs to a systemic stimulus, represented in this model by the intraperitoneal injection of LPS.     

Distinct Expression of Cytokines and Mitogenic Inhibitory Factors in Semen of Fertile and Infertile Men

PROBLEM: To assess the effect of seminal plasma (SP) of fertile and infertile men on leukocyte mitogenic response, and the capability of sperm cells to produce IL-1. METHODS: This study included four groups: fertile men (donors, normal), infertile men with azoospermia (azoo), oligo-terato-asthenozoospermia (OTA), and OTA with genital infection (OTA-inf). Mouse spleen cell proliferation in response to lipopolysaccharide (LPS) or Concanavalin-A (Con-A) was examined in the presence of SP from the above four groups. Supernatants (sup) and lysates (lys) of sperm cells from fertile and oligoteratoasthenospermic (OTA) men were evaluated for IL-1 bioactivity by specific bioassay. RESULTS: Seminal plasma (SP) of the four groups were shown to inhibit the mitogenic response of mouse spleen cells to LPS and Con-A. SP of fertile men was significantly more inhibitory than SP from infertile men. Sperm cells from fertile and OTA infertile men constitutively produced IL-1. Sperm cells of both groups produced similar levels of IL-1 as examined in the supernatants and lysates. CONCLUSIONS: Seminal plasma of fertile men had more inhibitory mitogenic activity than that of OTA. Sperm cells constitutively produce IL-1. It is possible that the factors involved in this inhibition are not only anti-proliferative immune factors. Cytokines and inhibitory factors of mitogenesis in the seminal plasma may be involved in the physiology and pathophysiology of sperm functions and thus affect male fertility.     

槲皮素对LPS延迟中性粒细胞自发性凋亡效应的抑制作用

目的：研究槲皮素对接受细菌脂多糖(LPS)刺激的中性粒细胞(PMN)自发性凋亡的影响，探讨槲皮素的抗炎作用机制。方法：以人外周血PMN为研究对象，选择光镜和流式细胞术检测细胞凋亡情况，对裂解的片段化DNA进行定量(二苯胺测定法)和定性(琼脂糖凝胶电泳)分析。结果：槲皮素(1～100μmol／L)对PMN的自发性凋亡率并无影响，但却能部分恢复由LFS所延迟的PMN自发性凋亡进程。当槲皮素的浓度为40μmol／L时，其恢复作用达到最大。结论：槲皮素对LPS延迟PMN自发性凋亡的效应产生了抑制作用，减轻了因预激因子活化PMN而加重的炎症反应，部分揭示了槲皮素的抗炎作用机制。     

Control of Foxp3+ CD25+CD4+ regulatory cell activation and function by dendritic cells

Naturally occurring CD4+CD25+ regulatory T (TR) cells play crucial roles in normal immunohomeostasis. CD4+CD25+ TR cells exhibit a number of interesting in vitro properties including a 'default state' of profound anergy refractory to conventional T cell stimuli. We investigated the in vitro activation requirements of CD4+CD25+ TR cells using bone marrow-derived DC, which as professional antigen presenting cells (APC) can support the activation of normal naive T cells. Comparison of different APC types revealed that LPS-matured DC were by far the most effective at breaking CD4+CD25+ TR cell anergy and triggering proliferation, and importantly their IL-2 production. Examination of Foxp3, a key control gene for CD4+CD25+ TR cells, showed this to be stably expressed even during active proliferation. Although CD4+CD25+ TR cell proliferation was equivalent to that of CD25- cells their IL-2 production was considerably less. Use of IL-2-/- mice demonstrated that the DC stimulatory ability was not dependent on IL-2 production; nor did IL-15 appear crucial but was, at least in part, related to costimulation. DC also blocked normal CD4+CD25+ TR cell-mediated suppression partially via IL-6 secretion. DC therefore possess novel mechanisms to control the suppressive ability, expansion and/or differentiation of CD4+CD25+ TR cells in vivo.     

Validation of efficient high-throughput plasmid and siRNA transfection of human monocyte-derived dendritic cells without cell maturation

Transfection of primary immune cells is difficult to achieve at high efficiency and without cell activation and maturation. Dendritic cells (DCs) represent a key link between the innate and adaptive immune systems. Delineating the signaling pathways involved in the activation of human primary DCs and reverse engineering cellular inflammatory pathways have been challenging tasks. We optimized and validated an effective high-throughput transfection protocol, allowing us to transiently express DNA in naïve primary DCs, as well as investigate the effect of gene silencing by RNA interference. Using a high-throughput nucleofection system, monocyte-derived DCs were nucleoporated with a plasmid expressing green fluorescent protein (GFP), and transfection efficiency was determined by flow cytometry, based on GFP expression. To evaluate the effect of nucleoporation on DC maturation, the expression of cell surface markers CD86 and MHCII in GFP-positive cells was analyzed by flow cytometry. We established optimal assay conditions with a cell viability reaching 70%, a transfection efficiency of over 50%, and unchanged CD86 and MHCII expression. We examined the impact of small interfering RNA (siRNA)-mediated knockdown of RIG-I, a key viral recognition receptor, on the induction of the interferon (IFN) response in DCs infected with Newcastle disease virus. RIG-I protein was undetectable by Western blot in siRNA-treated cells. RIG-I knockdown caused a 75% reduction in the induction of IFNβ mRNA compared with the negative control siRNA. This protocol should be a valuable tool for probing the immune response pathways activated in human DCs.     

Molecular cloning, characterization and expression analysis of the tumor necrosis factor (TNF) superfamily gene, TNF receptor superfamily gene and lipopolysaccharide-induced TNF-α factor (LITAF) gene from Litopenaeus vannamei

In vertebrates, the tumor necrosis factor (TNF)-receptor (TNFR) system participates in diverse physiological and pathological events, such as inflammation and protective immune responses to microbial infections. There are few reports about the role of the invertebrate TNF-TNFR system in immune responses. Here, we isolated and characterized the TNF superfamily (LvTNFSF) gene, TNFR superfamily (LvTNFRSF) gene and lipopolysaccharide-induced TNF-α factor (LvLITAF) gene from Litopenaeus vannamei. LvTNFSF consists of 472 amino acids with a conserved C-terminal TNF domain and has 89.8% identity with the Marsupenaeus japonicus TNF superfamily gene. LvTNFRSF consists of 296 amino acids with a conserved TNFR domain and has 18.0% identity with Chlamys farreri TNFR, 14.6% identity with Drosophila melanogaster Wengen and 14.6% identity with Homo sapiens TNFR1. LvLITAF consists of 124 amino acids with the LITAF domain and shows 62.6% identity with D. melanogaster LITAF and 32.3% identity with H. sapiens LITAF. The promoter region of LvTNFSF was cloned and used to construct a luciferase reporter. In Drosophila S2 cells, the promoter of LvTNFSF can be activated by LvLITAF, L. vannamei NF-κB family proteins (LvRelish and LvDorsal) and LvSTAT. Unlike its mammalian counterparts, LvTNFRSF could not activate the NF-κB pathway in Drosophila S2 cells. Using real-time quantitative PCR, we obtained expression profiles of LvTNFSF, LvTNFRSF and LvLITAF in the gill, intestine and hepatopancreas of L. vannamei after challenge with Gram-negative Vibrio alginolyticus, Gram-positive Staphylococcus aureus, the fungus Candida albicans and white spot syndrome virus (WSSV). Taken together, our results reveal that LvTNFSF, LvTNFRSF and LvLITAF may be involved in shrimp immune responses to pathogenic infections.