Unstable carotid plaques cause cerebral emboli. Leptin promotes atherosclerosis and vessel wall remodeling. We hypothesized that carotid atherosclerotic lesion instability is associated with local leptin synthesis.P=0.001 and P=0.05, respectively). Symptomatic plaques showed preferential leptin, TNF-α, and plasminogen activator inhibitor type 1 transcript . Leptin mRNA and antigen in macrophages and smooth muscle cells were confirmed by in situ hybridization and immunohistochemistry. Plasma leptin levels were not significantly different between groups (P=1.0), whereas TNF-α was significantly increased in symptomatic patients (P=0.006). Human aortic smooth muscle cell culture stimulated by TNF-α, lipopolysaccharide, or lipoteichoic acid revealed 6-, 6.7-, and 6-fold increased secreted leptin antigen, respectively, at 72 hours (P<0.05).Carotid endarterectomy plaques from symptomatic (n=40) and asymptomatic patients with progressive stenosis (n=38) were analyzed for local expression of leptin, tumor necrosis factor (TNF)-α, and plasminogen activator inhibitor type 1. All lesions exhibited advanced atherosclerosis inclusive of thick- and thin-cap fibroatheromas or lesion rupture. Symptomatic lesions exhibited more plaque ruptures and macrophage infiltration (Neurologically symptomatic patients overexpress leptin mRNA and synthesize leptin protein in carotid plaque macrophages and smooth muscle cells. Local leptin induction, presumably by TNF-α, could exert paracrine or autocrine effects, thereby contributing to the pathogenesis of lesion instability.www.Clinicaltrials.gov. Unique identifier: NCT00449306.URL:  Many proatherogenic and proteolytic effects have been attributed at least in part to leptin activity. Thus, in addition to its pleiotropic hormonal functions regulating metabolic, immune, and endocrine functions,5 leptin has been implicated in the process of atherosclerosis via multiple avenues. For example, leptin has been shown to stimulate endothelial cells to increase oxidative stress through the generation of reactive oxygen species.6 Those enhance the expression of intracellular adhesive molecules7 and monocyte chemoattractant protein-1,8 both known to be involved in atherogenesis. Reactive oxygen species also exert elastolytic activity on the vessel wall capable of remodeling it. Leptin activates matrix metalloproteinase-2, upregulates matrix metalloproteinase-9, and stimulates endothelial cells to generate pathological angiogenesis via vascular endothelial growth factor-A synthesis,9 thus promoting plaque destabilization.10 Leptin also enhances atherosclerosis by inducing vascular smooth muscle cell (SMC) proliferation and migration through mitogen-activated protein kinase and phosphatidylinositol 3-kinase activation.11 SMCs also are stimulated to synthesize matrix metalloproteinase, tissue inhibitor of metalloproteinases,12 and angiotensin II receptor type 1.13 Human leptin increases cytokine production15 and stimulates proliferation of human circulating monocytes16 and T lymphocytes in peripheral blood.17 Leptin stimulates various immune cells involved in atherosclerosis to produce proinflammatory cytokines, which in turn form a feedback loop by promoting leptin synthesis in vivo,18 which suggests an autocrine/paracrine mode of action.Unstable carotid plaques frequently undergo rupture with intraluminal thrombosis, resulting in downstream cerebral emboli and stroke. Complex mechanisms involved in lesion progression and instability include induction of oxidative stress and the recruitment and activation of inflammatory/immune cells capable of synthesizing proatherogenic cytokines.21 and numerous clinical studies. Notably, clinical data have revealed that leptin expression is associated with obesity, insulin resistance, and metabolic syndrome.22 Moreover, hyperleptinemia is a surrogate biomarker for coronary vascular events,23 coronary restenosis after balloon angioplasty,24 and hemorrhagic stroke.25The impact of leptin on atherosclerosis and neointimal hyperplasia has been confirmed in a few experimental animal models26 with the activity of leptin at the tissue level being mediated through an interaction with the leptin receptor (ObR).27 A recent study of carotid atherosclerotic plaques has revealed that ObR is preferentially synthesized in lesions from neurologically symptomatic patients.28 Thus, it is reasonable that circulating leptin could be interacting with accumulated ObR in symptomatic carotid plaques. We hypothesized that leptin can be synthesized locally within carotid atherosclerotic lesions and could in part be responsible for the underlying plaque instability that leads to neurological symptoms. In addition, in light of numerous clinical data suggesting a causative linkage between inflammation and enhanced atherosclerosis,29 we further hypothesized that tumor necrosis factor (TNF)-α might be involved in leptin induction within the carotid plaque.Adipocytes currently are considered the sole source of plasma leptin,30 and their clinical presentation was transient ischemic attacks (n=27) or stroke (n=13) within the 3 months preceding tissue collection. The remaining 38 patients were clinically asymptomatic, exhibiting progressive ipsilateral internal carotid artery stenosis exceeding 80% on serial Doppler examinations. This study was approved by the local ethics committee, and written informed consent was obtained from all patients.Carotid plaques were collected from 78 patients undergoing endarterectomy for extracranial high-grade internal carotid artery stenosis . Forty of the patients were symptomatic, as defined by the North American Symptomatic Carotid Endarterectomy Trial (NASCET) classification,Clinical data , macrophage marker CD68 , TNF-α , and TNF-α receptor-1 . For identification of leptin and its specific receptor (ObR), histological sections were incubated overnight at 4°C with a rabbit polyclonal antibody against human leptin  or human leptin receptor . When necessary, antigen retrieval was performed by steam heat with EDTA (pH 8.0) or citrate (pH 6.0) buffer. Antibody binding was visualized by a polymer-based horseradish peroxidase substrate , with diaminobenzidine as the chromogen and Gill's hematoxylin as counterstain. Sections of normal gastric fundus mucosa and replacement of the primary antibodies by phosphate-buffered saline served as positive and negative controls for leptin-associated antibodies, respectively.For colocalization of leptin and ObR in SMCs and macrophages, antileptin antibodies were applied as described above, followed by a biotinylated goat anti-rabbit secondary antibody. Sections were stained with the use of a steptavidin-alkaline phosphatase detection system , and antibody staining was visualized with the Vucan Fast Red chromogen substrate . Subsequently, antibodies against either SMC (1:80) or CD68 (1:200) were applied overnight at 4°C after a sequential application of avidin/biotin  and protein blocks (Dako), and then sections were labeled and developed as described above.32 The presence of macrophage infiltrates and intraplaque vasa vasorum also was assessed by routine light microscopy, performed by 2 investigators in a blinded experiment.The lesions were classified on hematoxylin and eosin– and Movat's pentachrome–stained sections according to the American Heart Association (AHA) criteria, as defined by Stary and modified by Virmani.33 Purified complementary DNA was cloned into the pCR II-TOPO vector , linearized with BamHI and ApaI to generate sense and antisense strands, respectively, and verified for correct insertion and sequence . Riboprobes were generated by in vitro transcription and labeled by digoxigenin 11-UTP incorporation  by using either SP6 (antisense) or T7 (sense) RNA polymerase. The labeled probes were digested with deoxyribonuclease, extracted with phenol/chloroform, and ethanol precipitated. Leptin mRNA expression in situ was examined with a protocol modified from Breitschopf et al.34 Briefly, 5-μm-thick paraffin sections were digested with proteinase K , followed by overnight incubation with freshly labeled probes at 55°C. Positive signals were visualized by incubation with antidigoxigenin AP  and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphatase (Dako) until color development and were counterstained with Nuclear Fast Red. Human adipose tissue served as positive (labeled antisense) and negative (labeled sense probes) controls.Leptin cDNA was generated from human adipose tissue.Total RNA was isolated with the Trizol reagent (Invitrogen) and quantified with a spectrophotometer. First-strand complementary DNA (cDNA) was synthesized by using random hexamers with Superscript II . The cDNA prepared from the total RNA was used for quantitative real-time polymerase chain reaction (PCR) analysis for leptin, TNF-α, and plasminogen activator inhibitor type 1 (PAI-1).Abl (Abelson) with the fluorescent probe 5′-Fam-CTGGCCCAACGATGGCGA-BHQ-3′.35 The primers used were: forward, 5′-GGAGATAACACTCTAAGCATAACTAAAGG-3′, and reverse, 5′-GATGTAGTTGCTTGGGACCCA-3′. Gene expression results for leptin and TNF-α in the plaques were expressed in arbitrary units. Results for leptin mRNA in SMC lysates were presented as differences of normalized Ct values compared to control samples (ΔΔCt). Data were analyzed in SDS 2.3 (Applied Biosystems) and Excel  software.Reactions were performed on cDNA with an ABI PRISM 7900HT Sequence Detection System  and Universal PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions. The TaqMan probes and primers for leptin (assay ID number HS00174877), TNF-α (assay ID number HS00174128), and PAI-1 (assay ID number Hs00167155) were assay-on-demand gene expression products (Applied Biosystems). Total RNA from normal donor carotid arteries (n=3) was used to establish a baseline mRNA level for each gene, defined as 1. All samples from symptomatic and asymptomatic patients were normalized by using the endogenous control gene Plasma samples were collected from all patients within 2 hours before surgery and stored at −70°C. Circulating levels of leptin were determined with radioimmunoassay kits . The sensitivity of the leptin assay was 0.5 μg/L, and the interassay coefficient of variation was 3% to 6%. TNF-α plasma levels were assayed with solid-phase enzyme-linked immunosorbent assay .Normal human SMCs from aorta were purchased from PromoCell  and were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum. Fibroblast growth factor-2 (3 ng/mL) was added every other day until 80% confluence. The assays were performed on confluent cultures (6 to 9 days old) in 35-mm tissue culture dishes  that were preincubated in fresh medium  for 24 hours. Human venous SMCs were isolated by explantation from saphenous veins obtained for cardiac bypass surgery. Cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal calf serum and were used between passages 3 and 6. When cells reached 90% confluence, they were preincubated in Dulbecco's modified Eagle medium containing 1% fetal calf serum for 24 hours.SMC cultures then were challenged with TNF-α , lipopolysaccharide , or lipoteichoic acid  for 24, 48, and 72 hours, respectively, and nonstimulated aortic SMCs served as controls. Conditioned media were collected and assayed for leptin antigen level using a Human Leptin ELISA kit  according to manufacturer's instructions, where the minimum detectable dose of leptin is typically <6 pg/mL. Cell lysates from human aortic SMC cultures were used to isolate total RNA with an RNeasy kit  and were reverse-transcripted by Superscript II (Invitrogen).2074 AA GAG GTT TGG GGT CT, and C2134 CC ACT GTG TGA CAA AAA C; 18S, 5′-ATGGCCGTTCTTAGTTGGTG-3′ and 5′-GAACGCCACTTGTCCCTCTA-3′.Quantitative PCR was performed for leptin mRNA in a MyiQ Single-Color Real-Time PCR system with SYBR Green I . Leptin mRNA levels were normalized to 18S. The primer sequences were: leptin, AIn vitro leptin antigen studies were performed on 2 commercially available batches of human aortic SMC primary cultures and 3 primary saphenous vein SMC cultures from different individuals.P<0.05 was considered statistically significant.Differences in categorical variables  and 2 weP=0.001).The purpose of this study was to investigate leptin synthesis in carotid atherosclerotic plaques in association with clinical neurological symptoms. Clinical criteria with regard to demographics, cardiovascular risk factors, and medications  were simP=0.001), intraluminal thrombosis (P=0.02), and macrophage infiltration (P=0.05). Plaques from symptomatic patients exhibited a larger necrotic core that occupied >25% of total plaque area, increased angiogenesis, and intraplaque hemorrhage. However, the difference between the groups with regard to those features did not reach statistical significance. Also, the incidences of fibrosis and calcification were not significantly different between groups.Carotid lesions from both groups were assessed histologically. In both groups, lesions showed features of advanced atherosclerosis, with a similar prevalence of fibroatheromas, with or without a thick cap and calcification . SymptomThe presence of leptin and leptin receptor (ObR) antigen were studied by immunohistochemistry on parallel sections of the carotid plaques. Leptin and ObR were localized to the lesion's shoulder regions and areas within or bordering the necrotic core , also exTo address the possibility of local leptin synthesis, in situ hybridization analysis was performed and demonstrated the expression of leptin transcript mostly within inflammatory cells, identified as monocytes/macrophages, bordering areas of necrotic core and cholesterol clefts A. In addP=0.03; 2-sided Wilcoxon sum test, P=0.044; 2-sided Wilcoxon sum test, P=0.004; Pearson's correlation test). Notably, mRNA transcript for PAI-1, the physiological inhibitor of fibrinolysis, was increased in symptomatic plaques     and yielded no difference between symptomatic and asymptomatic groups  D. Furtheficient) E. No corum test) F. These P=0.048) and a 5.95±2.19-fold increase after 72 hours ; after treatment with LPS (100 ng/mL), there was a 5.02±2.02-fold increase in leptin antigen level after 48 hours  and a 6.70±2.16-fold increase after 72 hours ; and after treatment with LTA (10 μg/mL), there was a 4.2±2.98-fold increase in leptin antigen level after 48 hours  and a 6.04±2.61-fold increase after 72 hours . Results of one representative experiment are displayed in To investigate leptin induction in human vascular SMC cells, both aortic and saphenous vein SMCs were stimulated with TNF-α or bacterial cell wall components (LPS or LTA). Leptin antigen levels were increased in culture media in response to stimulation with any of the 3 agonists. Those were assessed after 24, 48, and 72 hours, exhibiting a similar pattern in both aortic and saphenous vein SMC cultures. There was no increase after 24 hours. Repeated-measures analysis revealed the following fold increases: After treatment with TNF-α (50 ng/mL), there was a 4.93±1.95-fold increase in leptin antigen level after 48 hours  and was correlated with body mass index. There was no correlation between elevated plasma leptin and documented diabetes mellitus, hypertension, or angiotensin-converting enzyme inhibitor therapy, and the prevalence rates of all these parameters were not significantly different between groups. These findings suggest that the paracrine effects of leptin at the plaque level might not respond to feedback mechanisms related to obesity, insulin resistance, or hypertension.37 We found PAI-1 transcript to be significantly increased in the symptomatic plaques in our study, consistent with a previous report.38 These results suggest a hypofibrinolytic nature of symptomatic lesions in our series, rendering them unstable and likely to embolize.Interestingly, plaques from both groups were similarly graded according to the AHA classification. However, when Virmani's criteria for plaque vulnerability were applied, a clear tendency toward increased instability was evident in symptomatic lesions. The latter exhibited significantly higher macrophage infiltration, a trend toward higher necrotic core size, and increased incidence of subsequent plaque rupture. To assess additional features characteristic of lesion instability, which also are attributable to symptomatic plaques, we determined PAI-1 mRNA in both groups. PAI-1 is known to correlate with the degree of atherosclerosis.39 We confirmed that carotid plaque macrophages and SMCs are capable of synthesizing leptin. Several modes of leptin induction were demonstrated in adipocytes, including exposure to insulin, dexamethasone,40 angiotensin II,41 glucose, and hypoxia.42 TNF-α and interleukin-1β also were shown to induce leptin in cultured adipocytes and preadipocytes.43 Angiotensin II recently was shown to induce leptin in human coronary SMCs44 and in human aortic aneurysm wall–derived SMCs in our laboratory.45 To examine putative candidate mediators responsible for the local induction of leptin, we chose to assess the presence of TNF-α within both the plaque tissue and the systemic circulation. Our hypothesis about the potential role of TNF-α was based on a range of converging clinical and experimental data implicating this proinflammatory cytokine in atherosclerosis. For instance, plasma TNF-α levels correlate with atherosclerotic disease incidence46 and with early carotid atherosclerosis in nonsymptomatic patients.47 In addition, chronic exposure to bacterial infections, associated with elevated TNF-α, has been linked to carotid atherosclerosis in a large prospective study.48 Analogously, a higher prevalence of recurrent infections was reported in patients suffering from symptomatic coronary atherosclerosis.49 Finally, this paradigm is further supported by a study that identified a broad variety of bacterial molecular signatures in coronary atherosclerosis specimens, many of which are known to stimulate TNF-α production.50 Our analysis reveals a concomitant preferential increase in TNF-α and leptin mRNA transcripts in carotid plaques from clinically symptomatic patients. Strong signal for TNF-α and its receptor (TNF-αR1) was also evident in carotid plaque macrophages and SMCs. At the systemic level, a concomitant increase in levels of circulating TNF-α antigen were recorded in symptomatic patients. It should be emphasized that plasma samples were collected from all patients 1 to 2 hours before surgery—days to weeks after stroke or transient ischemic attack occurred in symptomatic patients. It is thus unlikely that any elevation of systemic TNF-α level could have resulted from inflammation related to the acute neurological event, plaque rupture, or subsequent instant intraluminal thrombosis. At the same time, this sequence of events could suggest a preceding infection or inflammatory condition that might have promoted both systemic and intraplaque TNF-α synthesis. Notably, elevated levels of TNF-α also could have resulted from activation of resident macrophages by oxidized low-density lipoprotein,51 spontaneously released from local lysis of macrophages, or in turn, TNF-α induction by leptin.52Leptin is synthesized primarily by white adipocytes but also is produced in other cell types, such as fibroblasts, placental trophoblasts, skeletal muscle, and gastrointestinal tract epithelium.53 However, this could have resulted from leptin induction in response to stimulation of the adipocyte cell mass. Our cell culture experiments were focused on leptin induction by cytokines in human vascular SMCs, a major cellular component of the vessel wall, and the atherosclerotic plaque. The present in vitro data indicate direct linkage between TNF-α, Gram-negative bacterial LPS, or Gram-positive bacterial LTA and leptin induction in SMCs. Also, the potential presence of a paracrine/autocrine mechanism is suggested from experimental data on the release of TNF-α in response to LPS and TNF-α stimulation in SMC cultures.54 Our study does not present leptin induction in primary human macrophage cultures when stimulated with the same agonists or in macrophage cultures exposed to hypoxia . However, it is reasonable to assume that under in vivo conditions, cell-to-cell interaction could provide the essential milieu for leptin induction in macrophages, to account for the presence of leptin mRNA and antigen in carotid plaque macrophages .In summary, leptin and ObR are preferentially synthesized in clinically symptomatic carotid plaques by resident SMCs and macrophages. This induction might be activated by cytokines like TNF-α when available within the plaque and in the systemic circulation. Cytokine upregulation has been speculated to originate from bacterial infection or repeated inflammatory stimulation. Oversynthesized leptin within the plaque could exert paracrine or autocrine effects causing destabilization of the atherosclerotic lesion, resulting in embolic complications.This study has some limitations. Because plaque tissue was totally consumed for histological analysis and total RNA extraction, none was available for protein extraction. Undoubtedly, quantification of protein levels could have complemented leptin and TNF-α synthesis in situ data. A major disadvantage was the lack of medical records to account for preceding infections or severe inflammation in symptomatic or asymptomatic patients. Moreover, given that leptin might mediate several processes involved in plaque destabilization, the clinical time course from leptin's local synthesis to the occurrence of an embolic event is unclear. Although the present study provides additional data to suggest an alternative avenue to account for the linkages among infection/inflammation, atherosclerosis, and plaque instability, it does not set out to downplay the contribution of other mechanisms involved in plaque destabilization.


Three miRNAs  modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells.MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α). Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human  Obesity and insulin resistance are characterized by several disturbances in white adipose tissue (WAT) function including increased basal (i.e. non-hormone stimulated) lipolysis and a chronic low-grade inflammation. The latter results in an increased release of pro-inflammatory factors including interleukin-6 (IL-6), chemo-attractant protein chemokine (C-C motif) ligand 2  and tumour necrosis factor-alpha (TNF-α) which can be produced by both adipocytes and infiltrating leucocytes (e.g. macrophages) see .In adipocytes, TNF-α affects lipolysis via multiple mechanisms mediated via its cognate receptor TNF-α-receptor-1 (TNFR1) The regulation of TNF-α production and secretion is complex and involves an extensive cross-talk at the intra- and extracellular level, including a self-regulatory loop MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level in vitro) studies were carried out in subcutaneous WAT obtained from healthy men and women undergoing liposuction for cosmetic reasons. None of the subjects was on any regular medication and there was no selection for age, sex or body mass index (BMI). Isolation, culture and in vitro differentiation of human adipocyte progenitor cells from subcutaneous WAT were performed as described previously g for 10 min and the supernatant (containing mature adipocytes and collagenase solution), was removed. The stroma-vascular fraction (containing pre-adipocytes) was re-suspended in erythrocyte lysis buffer for 10 min, filtered through a nylon mesh and centrifuged as described above. The supernatant was discarded and the cell pellet was re-suspended in an inoculation DMEM/F12 medium supplemented with 10% fetal bovine serum, 100 µg/mL penicillin-streptomycin and was subsequently filtered through a 70 µm pore size filter. Cells were plated at the density of 30.000–50.000 cells/cm2 in inoculation medium to allow cells attachment. After 24 h, the medium was changed to differentiation medium . Rosiglitazone was included first 3–6 days and then removed from the differentiation medium. In vitro differentiated adipocytes obtained from different subjects were not pooled. Each experiment was repeated in cells isolated from at least three separate individuals. All subjects were informed about this study and written consent was obtained. This study was approved by the Research Ethics Committee at Karolinska Institutet Huddinge (Sweden).Experimental  and HiperFect Transfection Reagent  at day 10–12 of differentiation according to the manufacturers’ protocols. After appropriate incubation times (6–12–24–48 h), cells were harvested for RNA or protein analysis and conditioned media was collected. Optimal transfection conditions were determined in separate titration experiments and transfection efficiency was assessed with quantitative real-time PCR (qRT-PCR) using miRNA probes . All experiments showed at least, 100–200 fold-change up-regulation of the transfected miRNA. We did not observe any morphological change on adipocyte phenotype and transfected adipocytes remained functional 48 h post-transfection . To rule out unspecific effects, control cells were transfected with miRIDIAN miRNA Mimic Negative Controls (Thermo Fisher Scientific).For RNAi experiments, human differentiated adipocytes were transfected as described above with ON-TARGETplus SMARTpool siRNA against TNFR1 (Thermo Fisher Scientific) and appropriate negative control for 24 h prior to co-transfection with miRIDIAN miRNA Mimics (Neg. Cntl/miR-145) for additional 48 h.Glycerol release into cell culture medium was determined as an index of lipolysis using a bioluminescence method as described ΔCt-target gene/2ΔCt-reference gene with 18S and/or LRP10 as internal control. For miRNA expression experiments (transfection efficiency), miRNA expression was normalized to reference gene RNU48Cells were harvested in QIAzol Lysis Reagent (Qiagen) and total RNA was isolated using miRNeasy Mini Kit (Qiagen) according to manufacturer instructions. RNA concentration as well as purity was measured spectrophotometrically using a Nanodrop ND-2000 Spectrophotometer (Thermo Fisher Scientific) and high quality RNA was confirmed using the Agilent 2100 Bioanalyzer . Reverse transcription and qRT-PCR were performed as described in vitro differentiated adipocytes by ELISA according to manufacturer’s instructions .TNF-α secretion levels were determined in conditioned media from in vitro differentiated adipocytes were harvested and nuclear extracts were prepared as described previously 6 cells/mL in solution A , and then allowed to swell for 15 min at 0°C. Thereafter, the cell suspension was shaken vigorously and mixed immediately to 1∶1 (vol/vol) with solution B (solution A supplemented with 0.6 M sucrose). The nuclei were pelleted by centrifugation at 14.000 rpm for 90 sec. Nuclei were examined under a light microscope for purity and integrity using TrypanBlue/NaCl (150 mM) solution. Nuclei from 1×106 cells were subsequently re-suspended in 40–60 µL of nuclei lysis buffer  supplemented with 5 mM NaF, 1 mM Na3VO4, protease inhibitor cocktail and benzonase (Sigma). Concentration of nuclear proteins was measured using BC/RC kit . Translocation assay was performed according to manufacturer instructions. Nuclear extracts (2.5 µg) were used in binding reactions and provided Jurkat nuclear extract (1.25 µg) was used as positive control in translocation assay. Wild-type and mutated consensus oligonucleotides were used in the assay as competitors of NF-κB binding in order to assure specificity of the assay. Wild-type (but not mutated) oligonucleotides inhibited NF-κB p65 activation.To determine NF-κB activation by miR-145, we used TransAM NF-κB p65 kit from Active Motif . After appropriate transfection time with mimics of miR-145, Cells were lysed in RIPA buffer as described previously 10log-transformed to become normally distributed. Results were analysed by unpaired Student t-test. In miR-145 over-expression experiments, relative values of glycerol, TNF-α secretion and mRNA expression were corrected by transfection efficiency and analysed by linear regression.Data displayed in figures and tables are mean ± standard error of the mean (SEM). When appropriate, the data was in vitro differentiated adipocytes followed by measures of glycerol release (an indicator of lipolysis). While five out of the eleven miRNAs had no effect on adipocyte lipolysis, four  significantly increased and two (miR-26a and let-7d) significantly decreased glycerol release . This was followed by a time-dependent increase in TNF-α secretion from 12 h, 24 h (p<0.05) to 48 h (p<0.001). These changes preceded the effects observed on lipolysis since glycerol levels remained unchanged at early time points (6 h –12 h –24 h) and were significantly increased only after 48 h (p<0.001). MiR-145 over-expression levels were similar at all studied time-points  post-transfection . TNF-α mints see . This suNF-κB pathway is an important regulator for TNF-α-induced lipolysis in human adipocytes The results in vs. the soluble form (17 KDa) of TNF-α at 48 h  of the eleven tested miRNAs had a concordant effect on TNF-α secretion and glycerol release. This suggests involvement of TNF-α in their lipolytic effect although we cannot exclude TNF-α-independent mechanisms as well. Importantly, the concentration of TNF-α in conditioned media from miR-26a, let-7d and miR-145 overexpressing cells varied between 0.2 pg/mL (for miR-26a) and 1.1 pg/mL (for miR-145) which is within the physiological concentration interval of TNF-α in the interstitial space of human adipose tissue In the over-expression experiments, miRNAs were >100 fold up-regulated. At this high-level, there might be off-target effects. This is generally a methodological issue in miRNA over-expression experiments. However, we always compare cells treated with specific mimics miRNA with mimics Negative Control. Furthermore, in a recent study In order to study the axis miRNA-TNF-α lipolysis more in detail, we selected miR-145 because it was the only miRNA that stimulated both TNF-α and glycerol release. Several lines of evidence suggest that miR-145 stimulates lipolysis by increasing the endogenous production of TNF-α. First, there was a strong correlation between changes in TNF-α mRNA/TNF-α secretion and lipolysis following miRNA over-expression. Second, TNFR1 knock down abrogates miR-145-induced lipolysis suggesting a causal relationship between the effects of miR-145 and TNFR1-mediated signaling. Importantly, the finding that TNFR1 depletion in fat cells attenuates lipolysis suggests that endogenous TNF-α production is important for basal adipocyte lipolysis. Third, miR-145-induced effects on adipocyte lipolysis occurred after induction of TNF-α gene and protein secretion. Fourth, NF-κB, which stimulates TNF-α transcription in an auto−/paracrine loop, was activated prior to the peak in TNF-α secretion/mRNA expression following miR-145 transfection. This indicates that NF-κB induction may be a mechanism through which miR-145 regulates TNF-α expression. Moreover, since TNF-α induction by miR-145 was not dependent on TNFR1 expression, it is plausible that the TNF-α/NF-κB feedback loop might be mediated via TNFR2 while TNF-α-induced lipolysis is primarily induced via TNFR1. Nevertheless, the direct link between miR-145 and NF-κB is not clear at the moment. Since miRNAs are down-regulators of gene expression, there are probably several steps up-stream of NF-κB activation linking miR-145 to TNF-α production which could include one or more negative regulators of NF-κB. However, to identify this/these candidates might prove difficult and was out of the scope of this study.TNF-α can either be present in soluble or a cell membrane-bound state. Although both forms are biologically active, membrane-bound TNF-α is thought to be responsible for cell-to-cell interaction and activate TNF-α signaling pathways in adjacent cells thereby promoting the development of an intense inflammatory response within the cellular micro-environment Final steps in TNF-α-mediated lipolysis activation include HSL Our screen included eleven miRNAs out of which only three displayed concordant effects on lipolysis and TNF-α production. For the remaining candidates, a few attenuated TNF-α without altering lipolysis . This could possibly depend on the fact that it is sometimes difficult to assess reductions (compared with increases) in glycerol levels. It is therefore possible that increasing the number of experiments would identify  reductions in glycerol and TNF-α release also with these miRNAs. Moreover, a few miRNAs increased lipolysis without significant changes in TNF-α levels  or even with a reduction in its release (miR-193b). These data suggest that specific miRNAs may alter lipolysis via TNF-α-independent mechanisms. However, investigating these alternative pathways was out of the scope of this present study.On the basis of the present findings, we propose that TNF-α production and lipolysis can be regulated by miRNAs. MicroRNA-145 enhances the production/release of TNF-α and increases the amount of membrane bound TNF-α via inhibition of ADAM17. It also activates NF-κB, decreases PDE3B expression and enhances HSL phosphorylation. All these events promote lipolysis activation and taken together, suggest that the effects of miR-145 on lipolysis are, at least in part, mediated by the effects on TNF-α.If the present findings are relevant for other species, remains to be established. Future studies are also needed to elucidate the mechanisms mediating the effects of the other miRNAs identified in this study.Figure S1Quantification of over-expression of miR-145 in human differentiated pre-adipocytes. Human differentiated adipocytes were transfected with miR-145 Mimics and collected at several time-points post-transfection (6 h –12 h –24 h –48 h) as described in material and methods. Cells were harvested for RNA and relative miR-145 expression levels were determined. Results are indicative of three biological/independent experiments. Values are shown as mean ± SEM and expressed as relative fold change vs. Neg. Cntl. of each time-point.(TIF)Click here for additional data file.Figure S2Quantification of TNFR1 mRNA levels after specific gene silencing with siRNA. TNFR1 was silenced with siRNA in human differentiated adipocytes as described in material and methods. Cells were harvested for RNA and relative TNFR1 mRNA expression levels were determined. Results are indicative of three biological/independent experiments. Values are shown as mean ± SEM. Statistical differences were analyzed by Student t-test: ***p<0.001.(TIF)Click here for additional data file.Figure S3Quantification of PDE3B mRNA levels miR-145 over-expression time-course. MiR-145 was over-expressed in human differentiated adipocytes for 6 h –12 h –24 h –48 h. Cells were harvested for RNA and PDE3B mRNA expression levels were determined. Results are indicative of three biological/independent experiments. Values are shown as mean ± SEM. Statistical differences were analyzed by Student t-test: **p<0.01.(TIF)Click here for additional data file.


Here, we demonstrate that systemic administration of (R)-DOI can block the systemic effects of TNF-α in whole animal, with potent anti-inflammatory effects in the aortic arch and small intestine. This includes blockade of TNF-α-induced expression of pro-inflammatory cell adhesion , cytokine , and chemokine  genes, and expression of VCAM-1 protein in the intestine. Further, systemic (R)-DOI also prevents the TNF-α-induced increase of circulating IL-6. Importantly, utilizing receptor selective antagonists, we have demonstrated that the mechanism underlying the systemic anti-inflammatory effects of (R)-DOI is activation of serotonin 5-HT2A receptors. Our results highlight a powerful new role for the serotonin 5-HT2A receptor in inflammatory processes, and indicate that agonism of serotonin receptors may represent an effective and novel approach to develop powerful small molecule therapeutics for inflammatory diseases and conditions such as atherosclerosis and inflammatory bowel disease.Tumor necrosis factor alpha (TNF-α) plays a key role in inflammation, and its production and signaling contribute to many inflammatory related diseases. Recently, we discovered that selective activation of serotonin 5-HT Serotonin  is a neurotransmitter and hormone whose effects are mediated through interactions at seven different families of receptor proteins, comprised of 14 different subtypes, consisting of 13 G-protein coupled receptors and one ligand-gated ion channel R)-1--2-aminopropane [(R)-DOI], a highly selective agonist at serotonin 5-HT2 receptors, super-potently inhibits tumor necrosis factor alpha (TNF-α) induced inflammation in primary rat aortic smooth muscle (RASM) cells Icam-1, Vcam-1), cytokines (Il-6), nitric oxide synthase activity, and activation and nuclear translocation of NFκB in RASM cells with an IC50 of ∼15 picomolar R)-DOI are exclusively mediated through activation of serotonin 5-HT2A receptors Recently, we discovered that (R)-DOI has potent anti-inflammatory effects against the inflammation produced by systemic TNF-α in mice. The anti-inflammatory effects are most prominent in the aortic arch and small intestine. Importantly, we demonstrate that 5-HT2A receptor activation is mediating the anti-inflammatory response.Here, we have translated these findings to the whole animal, and show that systemic (R)-DOI was generously provided by Dr. David E. Nichols . Each was dissolved in sterile physiological saline prior to use.TNF-α was from Shenandoah Biotechnology . (th week of age (mean weight of mice ± SEM on the day of treatment: 25.8±1.2 g). All animals were maintained in the animal care facility at LSUHSC in ventilated cages housed in a pathogen-free animal facility with free access to food and water. Animal protocols were prepared in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at Louisiana State University Health Sciences Center.Young adult male C57BL/6J mice were purchased from The Jackson Laboratory  and used for experiments in their 10R)-DOI as detailed: Group SS = Saline/Saline; Group ST = Saline/TNF-α (10 µg/kg); Group D3S = (R)-DOI (0.3 mg/kg)/Saline; Group D1T = (R)-DOI (0.01 µg/kg)/TNF-α (10 µg/kg); Group D2T = (R)-DOI (0.1 µg/kg)/TNF-α (10 µg/kg); and Group D3T = (R)-DOI (0.3 µg/kg)/TNF-α (10 µg/kg). Each mouse received intraperitoneal injections spaced 30 minutes apart, with saline or (R)-DOI first and TNF-α second. Five hours after the final treatment, mice were euthanized by a single injection of pentobarbital followed by cardiac puncture, exsanguination, and collection of blood. Blood was centrifuged in heparin coated tubes  to isolate plasma, which was immediately stored at −80°C. Tissue samples were dissected and immediately frozen on dry ice and stored at −80°C until processing. The dose of TNF-α and timing of tissue harvesting was based on previous literature examination of TNF-α on proinflammatory markers in the mouse For the dose response experiments, mice were treated with sterile saline, TNF-α, and (R)-DOI (0.3 mg/kg)/TNF-α (10 µg/kg); Group MDT = M100907 (1.0 mg/kg)/(R)-DOI (0.3 µg/kg)/TNF-α (10 µg/kg); Group MT = M100907 (1.0 mg/kg)/Saline/TNF-α (10 µg/kg); and Group M = M100907 (1.0 mg/kg)/Saline/Saline. Each mouse received intraperitoneal injections spaced 30 minutes apart, with M100907 or vehicle first, (R)-DOI or saline second, and TNF-α or saline third. Vehicle = 10% DMSO in sterile saline. Five hours after the final treatment, mice were euthanized by a single injection of pentobarbital followed by cardiac puncture. Blood and tissues were collected as described above.For the antagonist experiment, separate groups of mice were treated as follows: Group S = Vehicle/Saline/Saline; Group T = Vehicle/Saline/TNF-α (10 µg/kg); Group DT = Vehicle/. Probe library probes from Roche Diagnostics  were as follows: U10, U83, U22, U80 U6, U38, and U3 for Icam-1, Vcam-1, Mcp-1, Cx3cl1, Il-6, Il-1b, and Htr2a respectively. Quantitative determination of gene expression levels using a two-step cycling protocol was performed on either a StepOne Plus  to gather the data shown in Gapdh expression as determined using the Mouse Gapdh Gene Assay (Roche) in multiplex experiments. Additional information regarding sample preparation and QPCR protocols are provided in Cytokines in mouse blood plasma were assayed using the Milliplex Mouse Cytokine/Chemokine Panel kit  following manufacturer’s directions on the Biorad Bioplex system . For all tissues, RNA was extracted using TRI Reagent-RT from Molecular Research Center, Inc.  following protocols supplied by the manufacturer. First-strand cDNA was generated using the ImProm-II cDNA synthesis kit  following the manufacturer’s protocols with 500 ng total RNA per reaction. Quantitative real-time polymerase chain reaction (QPCR) was performed as described in The organic phase of the TRI Reagent from the RNA isolations described above was collected and purified by dialysis using Slide-A-Lyzer G2 Dialysis Cassettes (Thermo Scientific) as described by Hummon et al. All statistical analysis was performed using GraphPad Prism .in vivo, we pretreated normal young adult male mice with (R)-DOI (i.p.), followed 30 minutes later by treatment with a low dose of TNF-α (i.p.) that was predicted to produce a moderate systemic inflammatory response R)-DOI: 0.01 mg/kg, 0.1 mg/kg, and 0.3 mg/kg. The highest dose we used, 0.3 mg/kg, is equal to the minimal dose needed to produce discrimination in the sensitive two-lever drug discrimination behavioral assay of 0.3 mg/kg Icam-1 and Vcam-1), cytokines , and chemokines (Mcp-1 and Cx3cl1).To explore the effects of systemic serotonin receptor activation on systemic TNF-α mediated inflammation R)-DOI in different tissues. We saw no effect of (R)-DOI on TNF-α-mediated inflammation in the kidney, liver, or adipose tissues (data not shown). Consistent with our observations in cell culture with vascular cells, we observed a significant anti-inflammatory effect in the aortic arch for some markers where 0.3 mg/kg (R)-DOI -DOI  blocked pression .R)-DOI (0.01 mg/kg), mirroring the super potencies observed in vitro -DOI in the intestine. Inverted U dose response curves for drugs at GPCRs are commonly observed. One possibility is that multiple pathways are being engaged at different drug levels that together contribute to the overall effect. Somewhat surprising, considering the results in the small intestine, was that no effects of (R)-DOI were observed in the colon -DOI . Of these, the TNF-α-induced increase of IL-6 levels were significantly and completely blocked by the 0.1 and 0.3 mg/kg dose of (R)-DOI .R)-DOI in the whole animal, we examined relative 5-HT2A receptor expression in each of the tissues by QPCR. There appears to be no relationship between 5-HT2A receptor mRNA expression and the ability of (R)-DOI to block the effects of TNF-α -DOI observed in brain.Because of the observed differential effects of -DOI were mediated through 5-HT2A receptor activation. To address the primary mechanism underlying the anti-inflammatory effects of (R)-DOI in whole animal we pretreated mice with the 5-HT2A receptor selective antagonist M100907 30 minutes prior to (R)-DOI. As is shown in R)-DOI against TNF-α, indicating that the primary anti-inflammatory mechanism of (R)-DOI in whole animals is indeed through 5-HT2A receptor activation.In our previous R)-DOI to block TNF-α mediated increases in protein expression and measured Vcam-1 levels in the small intestine by western blot. We used tissue preparations from the same group of animals that were also treated with M100907 described above. The expression increase in Vcam-1 protein mediated by TNF-α was found to be more modest in comparison to increases in mRNA levels (2.5-fold vs. 7-fold) in the same set of animals. Nevertheless, pretreatment with (R)-DOI blocked TNF-α mediated increases in Vcam-1 levels (R)-DOI and M100907 resulted in the potentiation of the TNF-α mediated increase in Vcam-1. These data raise the possibility that there are potentially multiple and interacting pathways modulating inflammation from the 5-HT2A receptor.To further confirm anti-inflammatory effects, we examined the ability of -DOI has potent anti-inflammatory effects in whole animal. These results are consistent with our previous in vitro study, where we reported that (R)-DOI super-potently blocks TNF-α mediated inflammation in primary aortic smooth muscle cells R)-DOI is through activation of the 5-HT2A receptor. We chose three different pretreatment doses of (R)-DOI to administer to mice to examine the dose response profile. Our highest dose of 0.3 mg/kg is the minimal necessary to produce any statistically significant behavioral effect in the highly sensitive two-lever drug discrimination assay in vitro studies. The typical dose of DOI used for behavioral studies in mice is in the 1–3 mg/kg range We show here that the highly selective serotonin 5-HTR)-DOI. Consistent with our examination of aortic smooth muscle cells in vitro, we observed a trend that became significant at the 0.3 mg/kg dose for partial to complete blockade of proinflammatory marker gene expression induced by TNF-α in the aortic arch. The aortic arch has long been recognized as key vascular tissue affected by inflammation that ultimately leads to atherosclerosis R)-DOI. It is not possible for us at this point to determine the site of action for blockade of IL-6 synthesis and release, but it is may be related to components of the vasculature, which are known to produce IL-6 like vascular smooth muscle cells. TNF-α mediated inflammation in vascular smooth muscle cells, as well as macrophages and endothelial cells, is believed to be a major mechanism underlying the pathophysiology of atherosclerosis 2A receptors and TNF-α receptors are known to be expressed in immune related cells, and there may be a contribution from action at these cells contributing towards the overall observed effect of (R)-DOI. We show in R)-DOI (0.3 mg/kg) significantly and completely blocks TNF-α-induced Mcp-1, IL-1b, and Cx3cl1 in the aortic arch. These correspond to proteins linked to the inflammation that leads to the development of atherosclerotic plaques Icam-1, Vcam-1, and Mcp-1 gene production R)-DOI in whole aortic arch, 5-HT2A receptor activation may be an effective approach to develop novel therapeutics for atherosclerosis. Importantly, therapeutic levels of this drug are far below those predicted to produce unwanted behavioral or other physiological effects.Analysis of the dissected tissues and collected blood revealed that some tissues did indeed respond to the anti-inflammatory effects of (R)-DOI. The lowest dose of (R)-DOI, 0.01 mg/kg, consistently produced a maximal effect that was equivalent to the highest dose of drug, which for some markers meant complete blockade of the effects of TNF-α. Interestingly, no anti-inflammatory response to (R)-DOI was observed in the descending colon. Serotonin was identified in the gut before its role as a neurotransmitter was established, and has a long history of study in this tissue. Indeed, the vast majority of serotonin in the body is released from enterochromaffin cells within the intestinal mucosa of the gut, where it performs a number of functions 2A receptor function is involved in Irritable Bowel Syndrome, as it has been reported that certain gene polymorphisms are associated with an increased risk R)-DOI in the small intestine suggest that 5-HT2A receptor agonism may be an effective novel small molecule therapeutic strategy to treat inflammatory bowel disorders.In the small intestine we observed super-potent anti-inflammatory effects of -DOI, here in vivo. Other agonists (e.g. LSD), or even 5-HT itself may have anti-inflammatory activity in vivo through activation of the 5-HT2A receptor. As mentioned previously, the role of 5-HT in inflammation is not entirely clear. Serotonin can be either pro or anti-inflammatory depending on the tissue where it is acting. For example, in the bowel mucosa serotonin has pro-inflammatory effects whereas neuronal enteric serotonin has anti-inflammatory effects Although we used a panel of five agonists at the 5-HT2A receptors with the selective 5-HT2A receptor antagonist M100907 has anti-inflammatory effects against TNF-α mediated Vcam-1 protein increases, but not Vcam-1 mRNA increases suggest that there may be a separate antagonist mediated anti-inflammatory pathway that acts post-transcriptionally. There are reports that sarpogrelate, a 5-HT2 receptor antagonist, has anti-inflammatory properties R)-DOI+M100907+TNF-α group. That the combination of both (R)-DOI and M100907 can produce an enhancement of TNF-α mediated gene or protein expression further supports the notion of separate effector pathways, such that when the two treatments are combined it somehow produces an enhancement of the effects of TNF-α mediated inflammation. The nature of these potential interactions, however, remain to be elucidated in work beyond this study.Our results showing that blockade of 5-HT2A receptor mRNA expression detected in a particular tissue did not correlate with the anti-inflammatory effect of (R)-DOI. For example, although receptor expression in the aortic arch was nearly 10 fold higher than in the small intestine, the anti-inflammatory effects were found to be greater in the intestine. Further, we did not observe an anti-inflammatory effect in many tissues examined where receptor mRNA is expressed. These observations may indicate that anti-inflammatory pathway effectors are either more abundant or couple more efficiently to the receptor only in certain tissues like the small intestine to produce the desired anti-TNF-α effects through functionally selective mechanisms R)-DOI in the liver, DOI has recently been shown to induce profound regeneration of the liver through activation of VEGF pathways R)-DOI i.p., which is near the gut, and this may have had an influence on distribution to more distal tissues within 30 minutes. Further analysis with different routes of administration has indicated anti-inflammatory effects in additional tissues. For example, inhaled (R)-DOI has anti-inflammatory effects in the lung .We also observed that the level of 5-HTR)-DOI activation of serotonin receptors and find potent anti TNF-α effects in whole animal. Blockade of systemic TNF-α-induced proinflammatory marker gene expression by (R)-DOI is most pronounced in the small intestine, and significant in the aortic arch. Furthermore, the TNF-α-induced increase in circulating IL-6, and expressed Vcam-1 protein in the small intestine are also entirely blocked by (R)-DOI. Importantly, using a 5-HT2A receptor selective antagonist, we show that the primary mechanism for this anti-inflammatory activity is through activation of the serotonin 5-HT2A receptor. Because we only used a 5-HT2A receptor selective antagonist, it is not possible to rule out a role for the 5-HT2B or 5-HT2C receptors, but 5-HT2A activity is both necessary and sufficient for the anti-inflammatory effects of (R)-DOI in whole animal. Further, there is a high density of 5-HT2A receptors in the brain, not only in cortex R)-DOI may have some action centrally that contributes to peripheral anti-inflammatory activity. Together, our results indicate that agonism of 5-HT2A receptors is a novel approach to developing small-molecule based therapeutics for inflammatory diseases that involve TNF-α, especially for those of the vasculature and gut such as atherosclerosis and inflammatory bowel disease. Importantly, levels of drug needed to produce these beneficial effects are predicted to be up to orders of magnitude less than what are necessary to induce unwanted behavioral intoxication, and other physiological effects known to be influenced by 5-HT2A receptor activity like vasoconstriction in the vasculature and gut function.In summary, we have further characterized the anti-inflammatory effects of (Table S1Summary table of QPCR primer information.(XLSX)Click here for additional data file.QPCR Information S1Information relevant to MIQE standards for QPCR.(DOCX)Click here for additional data file.


Tumor necrosis factor-α (TNF-α) is one of the most typical pro-inflammatory cytokines with both beneficial and destructive properties for the central nervous system. Increasing evidences have demonstrated the important role of TNF-α in the development of ischemic stroke, but studies examining the possible association with stroke or direct functional effects of polymorphisms in TNF-α have been contradictory.In vitro evaluation of potential interaction between variants of the TNF-α gene  demonstrated that these three polymorphisms could interact together to determine the overall activity of the TNF-α gene.In this study, a 2-kb length of the proximal promoter of the TNF-α was screened and four polymorphisms were investigated in the case–control study. Our data confirmed the association between -308G/A variant with stroke in 1,388 stroke patients and 1,027 controls and replicated in an independent population of 961 stroke patients and 821 controls (odds ratio (OR) = 1.34, 95% confidence interval (CI) =1.02 to 1.77 and OR = 1.56, 95% CI = 1.09 to 2.23, respectively). To reconcile the association between polymorphisms and stroke and to give a comprehensive picture of the genetic architecture of this important gene, we performed a meta-analysis of 15 published studies in an Asian population. Our results demonstrated an association between rs1800629 and ischemic stroke . Another meta-analysis results of 14 studies demonstrated that ischemic stroke patients have higher serum TNF-α level than the control subjects (standardized mean difference (SMD) = 2.33, 95% CI = 1.85 to 2.81). These findings strongly implicate the involvement of TNF-α in the pathogenesis of stroke. Inflammation has important roles in the development and rupture of atherosclerotic lesions leading to cardiovascular disease events . The stuTNF-α is one of the most typical pro-inflammatory cytokines with both beneficial and destructive properties for the central nervous system ,7. TNF-αThere is increasing evidence that inflammatory variables are associated with atherogenesis and predict risk of cardiovascular disease . SeveralIn this study, we investigated the relationship between common variants in TNF-α gene and TNF-α level, cardiovascular risk factors, and stroke in a large collaborative analysis of Chinese Han populations, to explore the randomized allocation of alleles to better understand the nature of the association between TNF-α and stroke.n = 1,388) were recruited between November 2004 and January 2009 from five hospitals in Wuhan, China, according to the International Classification of Diseases . Ethnically and geographically matched controls  were randomly recruited from the population by a house-to-house recruitment protocol. All control subjects were free of neurological conditions and followed the same exclusion criteria as cases. For the purpose of replication, we introduced the second independent case–control cohort that has been fully described elsewhere [In the initial study, we investigated the association between common variants in TNF-α gene and stroke using a case–control cohort that has been described previously . In brielsewhere  and compA total of 376 healthy controls  were selected for serum TNF-α concentration determination. They were recruited randomly from the general population in Dongxi Lake community and all the subjects were free of cardiovascular disease. Additional exclusion criteria were referred to those reported previously . All theA 2-kb length of the human TNF-α gene promoter region between −2,000 and +30 base pairs (GenBank Accession: NG_007462) were identified by direct sequencing of genomic DNA derived from 96 randomly selected individuals from the control samples. Amplification and primers used for genetic variation screening are available in Additional file An ABI PRISM 7900HT Sequence Detection System  was used for genotyping by TaqMan-based assays. TaqMan probes and PCR primers are available from the Additional file Serum samples were collected after overnight fasting and stored at −80°C. TNF-α levels in serum were determined in 376 unrelated healthy individuals by OPTEIA ELISA kit , according to the manufacturer’s instructions. All samples were analyzed in duplicate and recombinant standards were included on every plate. The arithmetic mean of the duplicate samples was considered for analysis. The intra-assay coefficient of variation was 1.9 ± 0.8% and inter-assay variation was 2.7 ± 1.2%.To study the functionality of the polymorphisms in the promoter region of TNF-α, 10 different constructs were made. A 1,607 base pairs fragment was amplified by PCR from DNA from one individual with no variant in this promoter region using Oligonucleotides was as follows: forward primer, 5’ GGCCAGATCTCGACCAGAGCCCCACACG 3’; reverse primer, 5’ GGGGAAGCTTGGCTGAGGAACAAGCACCG 3’. Oligonucleotides were designed with BglII and GGCC tail at the 5’ end of the forward oligonucleotide and HindIII and GGGG tail at the 5’ end of the reverse oligonucleotide. PCR products were TA-cloned into pMD® 18-T vector system following the manufacturer’s conditions . This construct was then served as the template to generate other constructs that contains different mutations in the promoter region. Mutations were generated through overlap PCR with specially designed oligonucleotides. All the promoter fragments were subsequently cloned into pGL3 basic vector  through double digested ligation reaction. These constructs were finally confirmed by sequencing with Applied Biosystems 3130xl capillary sequencer . All the oligonucleotides which were used to create the site mutation were listed in the Additional file The human hepatoblastoma cells (HepG2) were cultured in 100 mm dishes in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS).4 cells/well) were transiently transfected with 20 ng of the appropriate plasmids or control vector, 1 ng of pRL-TK co-transfector, and 0.5 μL of Lipofectamine 2000 , and the cells were lysed 48 h after transfection. Luciferase activity was assayed using the Dual-Luciferase® Reporter (DLRTM) Assay System . Firefly luciferase expression levels were adjusted with reference to Renilla luciferase activity. Three independent experiments were performed for each reporter.HepG2 cells in 96-well format  of the variant. Differences of quantitative variables between groups were analyzed using the Student’s t-test. Logistic regression models were performed to estimate ORs and 95% CI for the association between genotypes and stroke. The Bonferroni correction method was applied for correction for multiple testing.The distributions of quantitative variables were tested for normality by use of a 1-sample Kolmogorov-Smirnov test. A χBecause observed TNF-α level was not normally distributed, to test for association between the polymorphisms within TNF-α gene and TNF-α level, statistical significance was calculated using the Mann–Whitney U test for non-parametric variables. Due to the small number of individuals homozygous for the less frequent allele, homozygotes have been pooled with the corresponding heterozygotes for the statistical analysis.n = 125) represents the lower quartile, Q2 (n = 125) represents the moderate quartile, and Q3 (n = 126) represents the higher quartile. For the TNF-α promoter variant, serum TNF-α level, and traditional cardiovascular risk factor including age, sex, BMI, blood pressure, total cholesterol, and HDL cholesterol, unadjusted mean values of traditional cardiovascular risk factor with their respective standard deviations were obtained for each genotype. Mean differences in the traditional cardiovascular risk factor for each genotype and quartile were calculated.We divided the distributions of the TNF-α level among control subjects into three quartiles: Q1 (P <0.05 was taken as significant (two-tailed).One-way ANOVA was used to compare the relative luciferase activities of the different polymorphisms of TNF-α promoter. All statistics was performed with the SPSS 13.0 package . A value of http://www.cnki.net/index.htm with the keywords ‘tumor necrosis factor alpha or TNF-α’, ‘stroke’, ‘TNF-α level and stroke’, ‘TNF-α polymorphism and stroke’, ‘cytokine polymorphism and stroke’, ‘inflammatory factor’, and the specific names and abbreviations of each gene. The analyzed data cover all English and Chinese publications from April 2000 to March 2012.The publications included in the analysis were selected from PubMed, Google Scholar, and from 2 (percentage of effect size attributable to heterogeneity) [Meta-analysis was carried out using the Stata software 10.0  and the Q statistic was calculated to test for heterogeneity followed by calculation of Igeneity) . Resultsgeneity) . The rangeneity)  were useIn this study, the region from +30 to −2000 base pairs of the human TNF-α gene promoter has been analyzed by direct DNA sequencing. A total of eight polymorphisms located at positions −308, -238, -857, -863, -986, -1031, -1376, and −1671 were identified in the Chinese Han population. All polymorphisms were in Hardy-Weinberg equilibrium in our sample. The distribution of the genotypes and the relative allele frequencies is shown in Additional file P >0.05). Two single nucleotide polymorphisms (SNPs) (rs1800629 and rs1799964) were significantly associated with increased risk of stroke in the first study independent of traditional cardiovascular risk factors in dominant models . The significant genetic associations were observed under dominant genetic model in the second study for the SNP rs1800629 , but this was not replicated for rs1799964  . No publication bias was found by using asymmetry analysis in the funnel plot . Sensitivity analyses showed that the pooled effect estimates were positive in spite of omitting any one of selected studies, which indicated that there was no significant trend that the overall result was influenced by any individual study  were selected for genotyping in the case–control study. The frequencies of these four tested variants did not deviate significantly from the HWE in cases and control subjects . No association was observed between the other two SNPs (rs351525 and rs1799724) and serum TNF-α level in our populations. Combined analyses revealed a lower serum TNF-α level in subjects with -308G/A, -857C/T, and -1031T/C combination carriers than those with wild type (P <0.05) . The Egger’s test and Begg’s funnel plot were applied for comparison to assess the publication bias of the literatures, and a possibility of publication bias for this test was observed (P = 0.001) .To test whether the polymorphisms in the TNF-α promoter region were functionally important for the regulation of TNF-α transcription, we performed functional analyses comparing the activities of four SNPs (MAF > 0.02) -308G/A, -238A/G, -857C/T, -1031T/C, and a novel SNP -1376T/C alleles in HepG2 cells. The -863C/A was in strong LD with -1031T/C and was not applied for the further functional analysis. As shown in Figure P <0.05, P <0.05, P <0.05, respectively)  may act cooperatively, another four constructs were made for transient transfection, including -308G/A / -857C/T, -308G/A / -1031T/C, -857C/T / -1031T/C, -308G/A / -857C/T / -1031T/C. We found that the relative luciferase activity of the rare allele combination -308G/A / -1031T/C, -857C/T / -1031T/C, -308G/A / -857C/T / -1031T/C were significantly lower compared to wild-type ones  Figure , which sThe present systematic study examined the polymorphism of TNF-α gene promoter, circulating TNF-α levels, and traditional cardiovascular risk factor for ischemic stroke in the Chinese Han population. Our findings show that the polymorphisms of TNF-α promoter may function as an important risk factor for ischemic stroke and our meta-analysis data confirmed that patients with acute ischemic stroke compared to controls without stroke have significantly higher median serum levels of TNF-α. TNF-α level was also positively associated with some established traditional cardiovascular risk factors, age, BMI, total cholesterol, blood pressure, and inversely with HDL in our study. Additional functional analyses showed that variants in TNF-α gene promoter region may interact together to determine the overall activity of the TNF-α gene promoter. The flow chart illustrating our study process was shown in Additional file et al. in a European population [SNPs at the promoter region of the TNF-α gene have been commonly studied. Resequencing of 96 unrelated Chinese Han individuals revealed high linkage disequilibrium between the −863 C/A and the -1031T/C and no other SNPs have any significant LD with each other. But complete allelic associations were observed between the -238G/A and the -1031T/C, and between the −863 C/A and the -1031T/C in Europeans . In addiet al. [In our next case–control study and meta-analysis, we confirmed the significant association between TNF-α promoter variant -308G/A and stroke in the Asian population. Recently, several GWASs for stroke have been reported -34, but et al. . Recentlet al.  did not Inflammatory processes have increasingly been shown to be involved in the pathogenesis of cerebrovascular diseases, including ischemic stroke and cerebral hemorrhage. We demonstrate elevated serum TNF-α level was associated with higher risk of ischemic stroke in the Chinese Han population. By contrast, the TNF-α was not associated with risk of stroke in a British prospective study . Anotheret al. [To date, there is no consensus regarding the functional significance of TNF gene polymorphisms. A number of explanations for this discrepancy have been offered including cell types, stimulants, and reporter gene constructs. Therefore, these polymorphisms may serve as markers for additional polymorphisms in the TNF-α locus or neighboring genes that may influence disease severity or functional activity. In this study, we provided additional evidence that limited data reported so far do not provide strong evidence in favor of a physiological role of -308G/A and -238G/A in the regulation of TNF-α promoter activity. We also detected a -1031C allele had a significantly reduced luciferase activity compared to -1031 T allele which had a strong partial LD with -863C/A polymorphism and the similar results were reported by Skoog Our conclusions must be interpreted in the context of important potential limitations of our study. Not all variants at the TNF-α gene were assessed in this study. Complete sequencing will be necessary for systematic identification of potentially causative mutations in TNF-α whole gene function region. Another limitation of the present study is the relatively small sample size used for TNF-α level analysis, which may give rise to false associations by chance (type one error), or may fail to detect true differences. Serum TNF-α levels are affected by multiple environmental, genetic factors and their interactions. Although we have observed the effects of several metabolism characters and SNP in TNF-α on serum levels in this study, there are still many other correlative environmental and genetic factors need to be determined. Our meta-analysis results must be viewed with careful because of potential biases. Perhaps because positive results have a better chance of being accepted for publication than small studies with non-significant or negative findings. Therefore, conclusions based on these published work might be misleading . FinallyTo conclude, this may be the first systematic and comprehensive analysis of TNF-α and stroke especially in an Asian population. Our results strongly support the involvement of TNF-α in the pathogenesis of stroke, and may have potentially important scientific, clinical, and public health implications.The authors declare that they have no competing interests.GC carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. HW and RL participated in the sequence alignment and was involved in critically revising the manuscript for importand intellectual content. ZL, YW and RH participated in the design of the study and performed the statistical analysis. HD and DW conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.This work was supported by ‘973’ projects  and ‘863’ project (No. 2012AA02A510).Table S1. Sequences of PCR primers used for amplification and sequencing of TNF-α promoter. Table S2. TaqMan Primer and Probe Sequences. Table S3. Oligonucleotides used to create the site mutation. Table S4. Frequency of TNF-α promoter polymorphisms. Table S5. Characteristics of eligible studies included in the meta-analysis. Table S6. TNF-α levels of individual studies included in the meta-analysis. Figure S1. LD Structure and Haplotype Blocks of the TNF-α promoter. Figure S2. Funnel plot of selected studies. Figure S3. Sensitivity analyses of selected studies. Figure S4. Serum TNF-α concentration in corresponding with different combinations of TNF-α promoter polymorphisms. Figure S5. Flow chart illustrating our study process.Click here for file


The gut mucosal homing integrin receptor α4β7 present on activated CD4+ T-cells interacts with the HIV-1 gp120 second variable loop (V2). Case control analysis of the RV144 phase III vaccine trial showed that antibodies induced by the vaccine bound to a MuLV-gp70 scaffolded V1V2 loop of gp120 (V1V2-gp70) and correlated inversely with infection. These, and other data, generate the hypothesis that the vaccine-elicited antibodies may have been involved in limiting HIV-1 acquisition. We have developed a high-throughput assay to evaluate antibodies that block α4β7 binding. We have named this the RAP assay.Plates were coated with either MAdCAM-1, the natural ligand of α4β7, or streptavidin followed by the addition of biotinylated cyclic-V2 peptides (strain 92TH023 or MN). Plasma and purified IgG antibodies from RV144 volunteers, conformational mAbs specific for V2 (697), for V2 and V3 , or for V2 linear epitopes  were then added to the peptide-coated plates followed by RPMI8866 cells, which constitutively express α4β7. Cell binding/inhibition was assessed by AlamarBlue. Anti-α4β7-specific mAb (ACT-1) served as a control. In separate experiments, plasma, IgG, or mAbs were tested in a competition assay using V1V2-gp70.ACT-1 inhibited the binding of both MAdCAM-1 and cyclic-V2 peptides to α4β7 by 65-85%, while CH58 and CH59 inhibited the α4β7-cyclic-V2 peptide binding by 37-45% in a dose-dependent manner. PG9, PG16, and 697 did not inhibit the binding of V2-peptides to the cells. However, in the competition assay, 697 and PG9 mAbs inhibited V1V2-gp70 binding to α4β7. Some of the RV144 plasma and IgG inhibited binding to 92TH023 or MN-V2-peptides.We have developed a novel high-throughput reproducible assay for assessing α4β7-specific blocking antibodies. The above results raise the hypothesis that anti-V2 loop antibodies may play a role in regulation of gp120-α4β7 interaction.


Toll-like receptors (TLRs) play a fundamental role in the immune system by detecting pathogen associated molecular patterns (PAMPs) to sense host infection. Ethanol at doses relevant for humans inhibits the pathogen induced cytokine response mediated through TLRs. The current study was designed to investigate the mechanisms of this effect by determining whether ethanol inhibits TLR3 and TLR4 mediated TNF-α secretion through inhibition of transcription factor activation or post-transcriptional effects. In NF-κB reporter mice, activation of NF-κB in vivo by LPS was inhibited by ethanol . Inhibition of protein synthesis by cycloheximide revealed that poly I:C- or LPS-induced secreted TNF-α is synthesized de novo, not released from cellular stores. Using real time RT-PCR, we found inhibition of LPS and poly I:C induced TNF-α gene transcription by ethanol. Using an inhibitor of tumor necrosis factor alpha converting enzyme (TACE), we found that shedding caused by TACE is a prerequisite for TNF-α release after pathogen challenge. Flow cytometry was used to investigate if ethanol decreases TNF-α secretion by inhibition of TACE. In cells treated with LPS, ethanol decreased both TNF-α cell surface expression and secretion. For example, 4.69±0.60% of untreated cells were positive for cell surface TNF-α, LPS increased this to 25.18±0.85%, which was inhibited by ethanol (86.8 mM) to 14.29±0.39% and increased by a TACE inhibitor to 57.88±0.62%. In contrast, cells treated with poly I:C had decreased secretion of TNF-α but not cell surface expression. There was some evidence for inhibition of TACE by ethanol in the case of LPS, but decreased TNF-α gene expression seems to be the major mechanism of ethanol action in this system. We and others have reported that ethanol, at concentrations relevant to human exposure, inhibits signaling through toll-like receptors The study described here focuses on TLR3 and TLR4. The TLR4 molecule senses lipopolysaccarides (LPS) from the outer membrane of gram-negative bacteria. The natural ligand for TLR3 is double stranded RNA (dsRNA) of viral origin. It also senses the synthetic dsRNA analog polyinosinic:polycytidylic acid (poly I:C). All TLRs apart from TLR3 recruit the adapter protein MyD88 (myeloid differentiation primary response gene 88) to the TIR domain Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine and a major player in the regulation of the immune response. It is mainly produced by macrophages and other cells of the immune system upon pathogen challenge. Pro-TNF-α is a homotrimeric type II transmembrane protein Inhibition of TACE and inhibition of signaling by ethanol could both contribute to the inhibition of the release of soluble TNF-α. The study described here was designed to evaluate both mechanisms in the same experimental system. The major novel finding of this study was that inhibition of TACE by ethanol was not the major mechanism that decreased production of soluble TNF-α. In particular, we found that ethanol decreased both cell surface and released TNF-α, unlike a known TACE inhibitor, which increased cell surface TNF-α but decreased the released form. This suggest that inhibition of signaling and TNF-α gene expression (which were confirmed here) was a more important mechanism of action in this experimental system.The protein synthesis inhibitor cycloheximide interferes with the translocation step at the ribosome, thereby blocking translational elongation. To test if TNF-α is available from cellular stores, synthesis of new protein was inhibited by cycloheximide. If TNF-α protein were available in the cell, blocking protein synthesis would not significantly affect the amount of TNF-α measured in cell culture supernatants after challenge with a TLR ligand.The effects of LPS or poly I:C alone and in combination with cycloheximide were examined. Cycloheximinde was added to cultures of RAW264.7 cells either at the same time point as LPS or poly I:C, or 30 min after the addition of LPS or poly I:C to the cell culture . Two houAdded to the cell culture supernatant as long as 30 min after LPS or poly I:C, cycloheximide caused a major decrease in TNF-α, verified by ELISA. Cycloheximide almost completely abrogated TNF-α synthesis after challenge with TLR ligands. As a control, cycloheximide was also incubated with the TNF-α ELISA standard, and it was shown not to interfere with the ELISA system. From these findings it can be inferred that de novo synthesis of TNF-α happens after pathogen challenge, and that the TNF-α on the cell membrane does not represent a sufficient “storage” form of TNF-α, which is ready for secretion upon stimulation. Therefore, the mechanism by which ethanol decreases TNF-α secretion after pathogen challenge must include effects other than or in addition to preventing its release from storage. Ethanol could act at the transcriptional or post-transcriptional level.To investigate if ethanol suppresses NF-κB signaling in vivo, we looked at the effect of LPS alone or in combination with ethanol in transgenic mice. The NF-κB reporter mice carry the luciferase gene driven by an NF- κB responsive promoter. Challenge with TLR ligands activates the NF- κB dependent pathway causing expression of luciferase in the reporter mice which is then detected by injecting luciferin, resulting in light emission in tissues with high NF-κB activation. One group of mice was gavaged with ethanol 5 min before LPS injection, and one group was only injected with LPS. A control mouse did not receive either ethanol or LPS. After LPS injection , light emission was measured with the IVIS imaging system .Mice treated with LPS show significantly more NF-κB activation than mice treated with EtOH+LPS, measured by overall light emission in each mouse (P<0.05). These findings corroborate the hypothesis, that EtOH inhibition is effective at the level of NF-κB dependent signaling, and is not limited to post-transcriptional effects.A decrease in NF-κB activation by ethanol should be reflected by decreased cytokine gene transcription. Therefore, the effect of ethanol on the LPS- and poly I:C-induced TNF-α mRNA expression was evaluated by real time RT-PCR in RAW264.7 cells. Appropriate groups were incubated with ethanol for 30 min, and then with LPS or poly I:C for further two hours. Untreated naive cells served as control. RNA was isolated from cell lysates, and real time RT-PCR was preformed .Escherichia coli in mice is almost eliminated by ethanol at both 4 g/kg and 6 g/kg in mice A concentration of 43.4 mM EtOH and higher in the poly I:C group, and 86.8 mM in the LPS group, significantly inhibited the pathogen induced TNF-α mRNA expression in RAW264.7 cells (P<0.01). It should be noted that the dosage of ethanol used in the NF-κB reporter mice (6 g/kg) yields a peak blood ethanol concentration of ∼86 mM Tumor necrosis factor alpha converting enzyme (TACE/ADAM17), a metalloprotease, is located in the cell membrane and cleaves pro-TNF-α from the cell surface. TAPI-0 (TNF-α processing inhibitor-0) is an inhibitor of metalloproteases. In this experiment, the extent of TACE contribution to the level of TNF-α shed into the cell culture supernatant was evaluated . In the 6/ml. Appropriate groups were treated with 43.4 and 86.8 mM ethanol for 30 min, and then with 100 ng/ml LPS and/or 20 µg/ml TAPI-0 for further 2 h. Supernatants were collected and subjected to ELISA . Two independent experiments were conducted . Surface TNF-α expression is indicated as % gated . An ELISIt has been widely accepted that excessive ethanol consumption increases susceptibility to infections, especially pneumonia. However, the mechanisms underlying the effects of alcohol on host defense are not fully understood. Studies in mouse models show that TNF-α plays a key role in host defense A major reason for conducting this study were reports indicating that TNF-alpha production was inhibited by ethanol primarily at the post-transcriptional level Zhang and colleagues To better understand the dynamics of TNF-α production and release of the soluble form of TNF-α, we inhibited protein synthesis with cycloheximide after challenge with TLR ligand. It was shown that TNF-α is primarily synthesized de novo after activation with LPS or poly I:C. Even though the cycloheximide experiment does not exclude that TNF-α protein is stored in the cells , it is unlikely, because TNF-α synthesis was abrogated even when protein synthesis was inhibited 30 min after TLR activation.Ethanol has been shown by western blot and electrophoretic mobility shift assay (EMSA) to inhibit LPS induced NF-κB activation in human monocytes In real time RT-PCR studies we evaluated if NF-κB inhibition by ethanol leads to a decrease in TNF-α mRNA. We found that the upregulation of TNF-α mRNA by LPS and poly I:C was consistently decreased by ethanol. This is consistent with our recent results indicating suppression by ethanol of TLR signaling and the production of a wide range of inflammation-related cytokines and chemokines TACE releases TNF-α from the cell surface by shedding We showed by flow cytometry that treatment with LPS, but not treatment with poly I:C significantly increased cell surface TNF-α. Corresponding ELISA, however, showed increased free TNF-α after treatment with both, LPS and poly I:C, possibly due to an increase in TACE activity as well as upregulation of production of TNF-α. An upregulation of TACE mRNA with a peak after 2 h, and increased TACE surface expression measured by flow cytometry in human alveolar macrophages by LPS and IFN-γ after 20 h was reported by Armstrong et al. This LPS-induced TACE expression was found to be downregulated by IL-10 Our results indicate that inhibition of NF-κB signaling and decreased TNF-α mRNA expression by ethanol occurred and acted to decrease surface TNF-α. The ethanol effect on TNF-α surface expression was more prominent in LPS treated cells. The free TNF-α in corresponding cell culture supernatants was significantly decreased by 43.4 mM ethanol in both LPS and poly I:C treated cells, which may be partly due to some inhibition of TACE activity. The effect of ethanol on TNF-α surface expression in the absence (or near absence) of TACE activity was revealed when shedding was inhibited by TAPI-0. In LPS and poly I:C treated cells. Ethanol at only 43.4 mM significantly decreased TNF-α surface expression, supporting the PCR results showing decreased TNF-α synthesis. Although we cannot exclude TACE inhibition caused by ethanol, our results indicate that decreased TNF-α production has more effect than any decrease in TACE activity that ethanol may cause.Taken together, in this study we have shown, that new protein synthesis is required for maximum production of secreted TNF-α, and that TNF-α is not stored in the cell in a form ready for secretion. Our results indicate that inhibition of LPS- or poly I:C-induced TNF-α production by ethanol starts at the signaling pathway and is not limited to post-transcriptional effects (such as cleavage by TACE). Decreased transcription, not just TACE inhibition, is involved in decreased TNF-α production, as indicated by comparing the changes of surface and secreted TNF-α in ethanol and TAPI-0 treated cultures. The above findings give new insights into the mechanisms of ethanol inhibition of TLR signaling. However, it is possible, that ethanol interferes with other proteins in the TLR signaling cascade.2 tissue culture flasks  at 37° C and 5% CO2.The RAW264.7 macrophage like cell line was purchased from American Type Culture Collection (ATCC). The RAW264.7 cells were cultured in RPMI 1640 with L-glutamine  supplemented with 10% FBS  and penicillin-streptomycin . They were used before the 10th passage. Cells were grown in 75 cm6/ml, 1 ml/well, and grown for 24 h before the experiment. Appropriate wells were treated with 86.8 mM ethanol and incubated 30 min. Then, 100 ng/ml LPS or 50 µg/ml poly I:C were added to appropriate wells, and incubated further 2 h. Supernatants were collected or cells harvested for further procedures that were performed within 30 min after harvesting. For the protein synthesis inhibitor experiment, cells were cultured as described above, and appropriate wells (6 samples per group) were treated with 5 µg/ml cycloheximide.Poly I:C (# tlrl-pic-5) was purchased from Invivogen . Ultra Pure lipopolysaccharide (LPS) from E. coli serotype O111:B4 was obtained from List Biological Laboratories . The protein synthesis inhibitor cycloheximide was purchased from Sigma Aldrich . The TACE inhibitor TAPI-0 was purchased from Peptides International . For all cell culture assays, cells were seeded into NUNC 24-well plates  at 1×10A mouse cytokine ELISA kit was obtained from BD Biosciences . The assay was carried out according to the manufacturer's specifications. NUNC maxisorp 96 well ELISA plates were used .Transgenic mice of C57BL/6J X CBA/J genetic background carrying the luciferase gene driven by an NF- κB response element, as described by Carlsen et al. Samples of RAW264.7 cells were lysed, RNA was purified with the RNeasy Plus Mini Kit 50 . Cell lysates were homogenized with QiaShredders . Samples were prepared with the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit with ROX reference dye . Real time RT-PCR was performed with the Stratagene Mx3005P QPCR System . Primers were designed with the Roche primer design software .Mouse TNF-α:5′-TGCCTATGTCTCAGCCTCTTC-3′forward: 5′-GAGGCCATTTGGGAACTTCT-3′reverse: Mouse 18S:5′-AAATCAGTTATGGTTCCTTTGGTC-3′forward: 5′-GCTCTAGAATTACCACAGTTATCCAA-3′reverse: All samples were normalized to 18S. Data were analyzed using the ΔΔCt method.Cultures of RAW264.7 cells were treated with ethanol, LPS or poly I:C as described above. In the initial experiments with TACE inhibition, 1 mg TAPI-0 was reconstituted in 20 µl DMSO to a final concentration of 5 mg/ml, as recommended. Appropriate wells were treated with 25 µg/ml TAPI-0 or 5 µl/ml DMSO right before addition of LPS or poly I:C. Supernatants were harvested and further analyzed by mouse TNF-α ELISA. In later experiments, TAPI-0 was dissolved directly in complete medium to double the final concentration needed and added to each culture to dilute it to the final concentration, 20 µg/ml.2 tissue culture flasks and transferred into sterile, endotoxin free Eppendorf tubes at 2.5×106/ml. Appropriate tubes were treated with 86.8 mM EtOH for 30 min, and with 20 µg/ml TAPI-0, 100 ng/ml LPS, or 50 µg/ml poly I:C for another 2 h. The caps were left open and loosely covered with sterile lids to ensure air supply to the cells. Then, cells were pelleted at 1500 rpm at 4°C for 4 min and re-suspended in 200 µl FACS buffer. Cells were then transferred into a 96 well V-bottom plate, pelleted and resuspended in 200 µl FACS buffer. Appropriate wells were labeled with 1 µl fluorochrome conjugated primary antibody and incubated for 20 min on ice. Antibodies were obtained from Invitrogen: Monoclonal rat anti mouse TNF-α FITC conjugate (# RM9011), and rat IgG FITC isotype control (# R101). Samples were washed twice and resuspended in 200 µl FACS buffer after final wash. Experiments were carried out on a BD FACS Calibur flow cytometry system .FACS buffer at pH 7.2–7.4 was prepared with sterile phosphate buffered saline , 0.5% sodium azide, and 2% fetal bovine serum. In some cultures TAPI-0 was added, as noted above. The RAW264.7 cells were grown in 75 cmReal time RT-PCR results were analyzed with the delta delta CT method, using Microsoft Excel 2004 for Mac, version 11.0 . For the NF-κB reporter study, the unpaired t-test was used to compare the LPS-treated group and the group treated with LPS plus ethanol. Although one untreated control animal was included, this was not included in the statistical analysis, because it was included only to confirm that the instrument settings were appropriate and were not yielding substantial emissions in the absence of treatment. For all experiments with more than two groups, results were analyzed by one way analysis of variance with the Newman-Keuls multiple comparison test to compare the significance of the difference of each mean value to every other mean value. A P value<0.05 was considered significant. Prism 4.0 software  was used for analysis.


Neorickettsia are obligate intracellular bacterial endosymbionts of digenean parasites present in all lifestages of digeneans. Quantitative information on the transmission of neorickettsial endosymbionts throughout the complex life cycles of digeneans is lacking. This study quantified the transmission of Neorickettsia during the asexual reproductive phase of a digenean parasite, Plagiorchis elegans, developing within naturally parasitized lymnaeid pond snails.Lymnaea stagnalis snails were collected from 3 ponds in Nelson County, North Dakota and screened for the presence of digenean cercariae. Cercariae were identified to species by PCR and sequencing of the 28S rRNA gene. Neorickettsia infections were initially detected using nested PCR and sequencing of a partial 16S rRNA gene of pooled cercariae shed from each parasitized snail. Fifty to 100 single cercariae or sporocysts were isolated from each of six parasitized snails and tested for the presence of Neorickettsia using nested PCR to estimate the efficiency at which Neorickettsia were transmitted to cercariae during asexual development of the digenean.L. stagnalis were collected and 240 (39%) shed digenean cercariae. Of these, 18 (8%) were Neorickettsia-positive. Six Neorickettsia infections were selected to determine the transmission efficiency of Neorickettsia from mother to daughter sporocyst and from daughter sporocyst to cercaria. The prevalence of neorickettsiae in cercariae varied from 11 to 91%. The prevalence of neorickettsiae in sporocysts from one snail was 100%.A total of 616 Neorickettsia infection in cercariae of Plagiorchis elegans was variable and never reached 100%. Reasons for this are speculative, however, the low prevalence of Neorickettsia observed in some of our samples (11 to 52%) differs from the high prevalence of other, related bacterial endosymbionts, e.g. Wolbachia in Wolbachia-dependent filariid nematodes, where the prevalence among progeny is universally 100%. This suggests that, unlike the Wolbachia-filaria relationship, the Neorickettsia-digenean relationship is not obligatory mutualism. Our study represents the first quantitative estimate of the Neorickettsia transmission through the asexual phase of the digenean life cycle.Prevalence of  Neorickettsia  are intracellular endosymbionts of digeneans. Neorickettsiae are presumably maintained throughout the digenean life cycle by vertical transmission during the sexual and asexual reproductive phases of the parasite. Digeneans are endoparasitic flatworms with complex life cycles involving asexual reproduction in mollusks (=first intermediate host) and sexual reproduction in vertebrates (=definitive host)  Figure . In somelineages ,7.Neorickettsia risticii is the most widespread member of the genus in the United States and worldwide  and Neorickettsia risticii [GenBank: KF556679].PCR amplicons of both L. stagnalis collected in all three ponds, 240 shed digenean cercariae  in digeneans, although not the highest prevalence of digeneans in snails .We used 5 es Table . Fifty sNeorickettsia through the complex digenean life cycle does not fit easily into the classical categories of vertical transmission. Due to the presence of both sexual and asexual stages in digenean life cycles it is clear that the vertical transmission of Neorickettsia through the digenean life cycle is not wholly dependent on transovarian transmission as in the case of some other bacterial pathogens/symbionts of invertebrates.Transmission of Neorickettsia-infected digeneans , the same snail species , and the same body of water  other genera associated with either ticks or other arthropods  or filarial nematodes (Wolbachia). In the case of Ehrlichia and Anaplasma, vertical transmission plays little role in their circulation [Wolbachia infects the ovaries and other tissues of many species of arthropods and filarial nematodes and is transmitted exclusively by vertical transmission [Neorickettsia are unique among the members of the family because it can be transmitted both vertically and horizontally. In this respect, the patterns of transmission exhibited by Neorickettsia are more similar to those observed among pathogenic spotted fever group Rickettsia  [Transmission in the digenean-culation ,30. In csmission -33. Thustsiales) .Neorickettsia, Wolbachia is maintained through vertical transmission, however, there are key differences between the 2 genera of bacteria. Wolbachia and the filariae have a mutualistic relationship [et al.[Wolbachia, are dependent on the presence of these endosymbionts. Landmann et al. [Wolbachia pass it on to 100% of eggs. The exact nature of the interrelationships between Neorickettsia and their digenean hosts is not known. Currently, there is no evidence suggesting a mutualistic relationship between digeneans and Neorickettsia. In our study, the majority of P. elegans did not harbor Neorickettsia, which proves that these digeneans are not dependent on their neorickettsial endosymbionts. Presently available data are insufficient to classify the endosymbiotic relationships of Neorickettsia with digeneans as either parasitic or commensal.Like tionship -37. Ferrp [et al. supposedn et al.  showed tNeorickettsia through asexual stages of the P. elegans life cycle is lower than 100% (Figure Neorickettsia ranged from 11 to 91%. Even in a case where 100% of screened daughter sporocysts harbored Neorickettsia, only 90% of the cercarial progeny were infected. These findings are in contrast with the situation in some other bacterial endosymbionts, such as Wolbachia in Wolbachia-dependent filariid nematodes, where the vertical transmission rates are 100%.Our data demonstrate that the transmission efficiency of % Figure . In cercThe authors declare that they have no competing interests.SEG participated in study design, collected snails, analyzed the data, carried out experiments, created figures, and drafted the manuscript. VVT participated in study design, collected snails, analyzed the data, and helped draft the manuscript. JAV participated in study design, analyzed the data, and helped draft the manuscript. All authors read and approved the final version of the manuscript.