The expression of tumor necrosis factor-α (TNF-α) mRNA was observed by using real-time polymerase chain reaction.Partial intestinal obstruction was induced surgically in male imprinting control region (ICR) mice. ICC networks were studied by Immunohistochemistry. Electrical activity was recorded by intracellular recording techniques. The expression of ICC phenotype marker c-kit receptor tyrosine kinase (c-kit), membrane binding stem cell factor (mSCF), the endogenous HPartial intestinal obstruction resulted in ICC networks were disrupted above obstruction 14 days after the operation. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed and their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized. The expression of c-kit and mSCF was significantly decreased, also suggesting the disruption of the ICC network. The expression of TNF-α was significantly increased in the tunica muscularis of the obstructed intestine. Treatment of cultured intestinal smooth muscle cells with TNF-α caused dramatic down regulation of mSCF. The expression of CBS and CSE was significantly decreased in the tunica muscularis of the obstructed intestine. Intraperitoneal injection (i.p) of DL-propargylglycine, an irreversible inhibitor of CSE, and aminooxyacetic acid, an inhibitor of CBS, elevated the expression of TNF-α mRNA in the tunica muscularis of the ileum. Obstruction-induced over expression of TNF-α was significantly improved by supplementation of NaHS, but not the expressions of mSCF and c-kit.2S biosynthesis is related to over expression of TNF-α in obstructed small intestine. TNF-α-mediated mSCF down-regulation is not the only reason of partial intestinal obstruction-induced loss of ICC.The down regulation of endogenous H Several congenital or acquired digestive tract motility disorders are associated with localized intestinal obstruction. Partial obstruction of the intestine has been shown to induce distention and hypertrophy of the gastrointestinal (GI) smooth muscles above the site of occlusion in both clinical situations and animal models In the gastrointestinal tract, the distribution of ICCs throughout the musculature is associated with nerve structures. ICCs generate a periodic depolarization at a characteristic frequency that is called the slow wave or pacemaker activity. This involves rhythmic oscillations of intracellular calcium and activation of membrane ion channels 2S) is a pungent gas that is formed endogenously in mammalian tissues by the H2S biosynthesis enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) 2S biosynthesis has been found in the gastrointestinal tract 2S exerts complex effects on inflammation. Administration of sodium hydrosulfide (NaHS), a “fast releasing” H2S donor, provoked an inflammatory reaction in mice 2S over a period of hours both in vitro and in vivo. GYY4137 exhibits anti-inflammatory activity as evidenced by a reduction in the lipopolysaccharide (LPS)-induced increase in plasma proinflammatory cytokines , nitrite/nitrate, C-reactive protein, and L-selectin in conscious rats 2S exerts complex and at times opposing effects on inflammation in whole animals 2S in the inflammatory cytokine TNF-α expression in the obstructed intestine.Hydrogen sulfide  mice  were obtained from the Experimental Animal Center of Shanghai Jiaotong University School of Medicine. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Science and Technology Commission of P.R.C. . The protocol was approved by the Committee on the Ethics of Animal Experiments of Shanghai Jiaotong University School of Medicine (Permit Number: Hu 686-2009). All surgery was performed under ether, and all efforts were made to minimize suffering. A total of 57 mice were used in our study. Sixteen mice were used in the obstructed model research and were divided into control group and obstructed group. 16 mice were used to study the role of endogenous H2 1.2; NaHCO3 23.8; KH2PO4 1.2; glucose 11.0; CaCl2 2.4. The segment was opened by cutting lengthwise, washed with Krebs, and the mucosa and submucosa removed by sharp dissection Partial intestinal obstruction was induced by surgically placing a ring of silicon tube around the ileum 30–50 mm oral to the ileocecal sphincter to cause distension of the intestine above the site of obstruction according to the methods described by Chang et al 2S in obstruction-induced intestinal inflammation, we used DL-propargylglycine , an irreversible inhibitor of CSE, and aminooxyacetic acid , an inhibitor of CBS, to inhibit H2S biosynthesis. The mixture solution of PAG and AOA was administered via intraperitoneal injection (i.p) twice a day for four days. The control mice were injected with same volume of phosphate-buffered saline (PBS).To investigate the role of endogenous H2S on expressions of TNF-α mRNA, mSCF and c-kit proteins in obstructed intestinal smooth muscle tissues, obstructed mice model was established. The solution of NaHS (10 umol/kg) To observe the effect of supplementation of HSmooth muscle strips were stretched to 110% of their resting length, fixed with ice-cold acetone , and processed as whole mounts. Following fixation, preparations were washed for 60 min in phosphate-buffered saline . Tissues were then incubated in 10% goat serum containing 0.3% Triton X-100 for 1 hour at room temperature to reduce nonspecific antibody binding 2 and 5% CO2. Cells were impaled with KCl-filled glass microelectrodes with resistances of 50–90 MΩ. Electrical responses were recorded and amplified through a high input impedance amplifier . Experiments were performed in the presence of nifedipine  in the perfusion solution to reduce contraction and facilitate cell impalement. Slow waves in mouse intestine have been previously shown to be unaffected by nifedipine Strips of smooth muscle (8 mm×4 mm) were cut parallel to the longitudinal axis of the intestine, oral to the site of occlusion. The smooth muscles were placed in a recording chamber with the submucosal aspect of the muscle facing upwards at 37°C in an atmosphere of 95% OTunica muscularis or cultured intestinal smooth muscle cells were lysed in RIPA buffer . Protein concentration was determined using the Bradford method. The lysate  was subjected to electrophoretic separation with 10% SDS–PAGE and transferred to nitrocellulose membranes . The membrane was incubated with 5% milk to reduce nonspecific antibody binding. Western blot was performed using antibodies directed against c-Kit , mSCF , CBS , CSE , and GAPDH . Alkaline phosphatase (AP) conjugated secondary antibodies and BCIP/NBT alkaline phosphatase color development kit  were used to detect protein bands. The images were captured by Adobe Photoshop and analyzed with Quantity One image software TTGGTGGTTTGTGAGTGTGAG - 3′, (228 bp). Amplification was performed in parallel samples using glyceraldehyde-3-phosphate dehydrogenase  as a control. Briefly, total RNA was isolated from smooth muscle tissue as recommended by the manufacturer of TRIzol Reagent. RNA concentration was determined by reading absorbance at 260/280nm and was adjusted to 0.4 µg/µL. Reverse transcription was performed according to the manufacturer’s instructions. cDNA samples were used for PCR using specific primers for TNF-α. The following conditions were used for PCR amplification: 95°C for 4 min; 95°C for 30 sec; followed by 38 cycles at 58°C for 1 min; 72°C for 30 sec; 72°C for 7 min. The PCR products were separated on a 2% agarose gel. Possible contamination of DNA was tested by skipping the reverse transcription step prior to PCR. Detectable fluorescent bands were visualized by an ultraviolet transilluminator (Bio-Rad) and the area was measured using Quantity One image software The mRNA expression of TNF-α in intestinal smooth muscle tissue was detected by real-time polymerase chain reaction (RT-PCR). Specific primers for mouse TNF-α were 5′ -GACGTGGAACTGGCAGAAGAG- 3′ and 5′ - 2 culture flasks for culture in DMEM containing 10% heat-inactivated fetal bovine serum (FBS), 2 mmol/L glutamine, 100 mmol/L HEPES, 100 U/mL penicillin, 100 mg/mL streptomycin, and incubated at 37°C in a humidified incubator containing 5% CO2. ISMCs between passages 5–7 were used and displayed typical spindle shape morphology and a “hill-and-valley” pattern of growth. ISMCs were characterized by immunohistochemical staining for smooth muscle-specific α-actin . DAPI was used for nuclear staining Intestinal smooth muscle cell (ISMC) culture was prepared from mouse intestine using an explant technique 6 cells/well and incubated in 1 mL of serum-supplemented DMEM  for 48 hours. Thereafter, the cell layers were washed with PBS and incubated for 24 hours in 1 mL of serum-free DMEM. After 24 hours, the cells were washed with PBS and incubated for 24 hours in 1 mL of serum-free DMEM containing 10 ng/mL of TNF-α  ISMCs were seeded into 6-well plates at a density of 1×10t-test was used to determine statistical significance. P<0.05 was considered statistically significant.Data were expressed as means ± SE. Student’s Partial ileal obstruction causes intestinal distention and thickening of smooth muscle layer  and the Previous studies demonstrated that ICCs were lost from the intestinal region immediately oral to the partial mechanical obstruction Won et al To determine whether partial obstruction-induced decrease in mSCF expression was mediated by up-regulation of TNF-α, ISMCs were treated with TNF-α. Cultured ISMCs displayed typical spindle-shaped morphology and a “hill-and-valley” pattern of growth -a. ISMCs2S has been suggested to be proinflammatory, H2S donating compounds have been shown to induce inflammation 2S production suppress inflammation 2S appears to be anti-inflammatory. The anti-inflammatory effect of H2S may be indirect, either due to its tissue-protective effect or hypothermic effect. However, H2S may also have direct anti-infllammatory effect by acting directly on the immune system 2S played a role. The expression of enzymes for H2S biosynthesis, CSE and CBS, in the obstructed ileum was determined using Western blot. Representative immunoblots for CSE, CBS and GAPDH from control and obstructed groups are shown in H2S biosynthesis leads to the increase of TNF-α mRNA in obstructed intestine, the inhibitors of CSE and CBS, PAG and AOA, were administrated via intraperitoneal injection. Our results demonstrated that inhibiting H2S biosynthesis by AOA and PAG enhanced the expression of TNF-α mRNA in small intestinal tissue  in obstruction-induced over expression of TNF-α mRNA and down expressions of mSCF and c-kit proteins. The results demonstrated that supplementation of exogenous H2S significantly improved obstruction-induced over expression of TNF-α mRNA, however, did not significantly enhanced the expressions of mSCF and c-kit proteins. Previous study confirmed that TNF-α involved in inflammation-induced loss of ICC 2S biosynthesis might be involved in obstruction-induced loss of ICC via up-regulation of TNF-α expression, with the latter suppression of mSCF expression in intestinal smooth muscle. However, obstruction is very complicated pathophysiologic process so over expression is not one and only reason of loss ICC in obstructed intestinal smooth muscle tissue. Therefore, in despite of supplementation of exogenous H2S significantly improved obstruction-induced over expression of TNF-α but not significantly restored the expressions of mSCF and c-kit proteins.Although we demonstrated that TNF-α reduced the expression of mSCF, the molecular mechanisms underlying this process need further investigation. H2S biosynthesis in obstructed small intestine. However, over expression of TNF-α is not one and only reason of loss ICC in obstructed small intestine, many factors involved in obstruction-induced down expressions of mSCF and c-kit proteins.In summary, chronic partial mechanical obstruction of the small intestine induced a smooth muscle remodeling response to obstructive injury, for example, hyperplasia and hypertrophy, and was accompanied by electrical slow wave dysfunction. Obstruction-induced loss of ICCs may be related to mSCF down-regulation mediated by TNF-α; TNF-α up-regluation is possibly induced by down regulation of endogenous HFigure S1Morphological changes and H&E stained in control and obstructed ileum. The open abdomen and dissection of the entire gastrointestinal tract of a control mouse (A-a). An arrow points to the portion of intestine bearing an opened ring of silicon tubing. 14 days after the creation of a partial obstruction (B-a). An arrow points to the portion of obstructed intestine bearing a ring of silicon tube and surgical thread. Cross-section of the ileum in control (A-b) and obstructed (B-b) mouse tissue stained with hematoxylin-eosin (H&E). In the obstructed intestine, there was a marked increase in the diameter of the ileum (C-a) and a marked increase in the thickness of both the longitudinal and circular muscle layers (C-b). Levels of significance compared to controls are indicated by asterisks .(TIF)Click here for additional data file.


While the serum levels of IL-6 and IL-1 beta dropped rapidly following the initiation of therapy, the concentrations of TNF-alpha and sTNFR-II steadily increased to a plateau. Although significant clinical efficacy was observed, etanercept had to be discontinued when after 12 weeks of therapy the patient was found to have pneumocystis pneumonia.We report a rheumatoid arthritis patient who was treated with etanercept. Serum levels of tumor-necrosis-factor- (TNF-)  Consequently, several publications have focused on the serum and/or synovial fluid levels of various inflammatory cytokines in patients with RA, psoriatic arthritis, and ankylosing spondylitis, with the clinical utility of such measurements being advocated [alpha inhibitors have been found to induce a rapid and sustained attenuation of disease activity in patients with RA. We describe here a patient with RA treated with a TNF-alpha inhibitor, etanercept for 12 weeks before discontinuation of therapy for pneumocystis pneumonia (PCP). The serum concentrations of TNF-alpha, sTNFR-I and -II, IL-6, and IL-1 beta were measured before, during, and after etanercept therapy. Rheumatoid arthritis (RA) is an autoimmune inflammatory disease in which cytokines, such as tumor-necrosis-factor- (TNF-) dvocated . Recentl3 , hemoglobin 10.7 mg/dL, platelets 48.7 × 104/μL, erythrocyte sedimentation rate (ESR) 75 mm/h, C-reactive protein (CRP) 3.58 mg/dL, rheumatoid factor (RF) 219.6 IU/mL, and anticyclic citrullinated peptide antibody (ACPA) 189 IU/mL. Serum β-D-glucan was negative. The purified protein derivative skin test for tuberculosis was negative. A 54-year-old Japanese man who had been diagnosed with RA for 8 years according to the 1987 American Rheumatism Association classification criteria for RA was admitted with polyarthritis. He complained of pain and swelling in the wrists, fingers, and knees and had difficulty walking without assistance. He claimed to have morning stiffness lasting about 3 hours. Past medical history included a left total knee arthroplasty complicated by a traumatic fracture of the left distal femur postoperatively and a cerebral infarction with residual right hemiparesis. His medication regimen included sulfasalazine (1000 mg/day), bucillamine (300 mg/day), mizoribine (150 mg/day), and prednisolone (10 mg/day). The Steinbrocker stage was IV and functional class was 4. He was unable to stand unassisted due to bilateral knee pain. Swelling and tenderness were present in the proximal interphalangeal and metacarpophalangeal joints of both hands and fingers, as well as in the wrists and knees. Right-sided muscle weakness was observed. Laboratory findings were as follows: white blood cell count 8800/mmalpha, TNFR-I and -II, IL-6, and IL-1 beta concentrations were measured using commercial ELISA kits.Treatment was initiated with subcutaneous injections of etanercept 25 mg twice a week. Blood samples were collected before the first dose (at baseline), 24 hours after the first injection (day 1), daily until day 8, and weekly thereafter. Specimens were centrifuged at 3000 rpm and the sera stored at −80°C until the assay was performed. TNF-After initiating etanercept, the patient's clinical condition improved markedly, with swelling and tenderness of the wrists and finger joints significantly better. He was able to stand with the aid of crutches despite residual hemiparesis. The duration of morning stiffness dropped to below 30 minutes. The 28-joint count Disease Activity Score (DAS28) dropped significantly within the first 4 weeks and continued to decrease , achieving the European League Against Rheumatism (EULAR) moderate response criteria. The CRP dropped to below 0.30 mg/mL within the first 2 weeks of therapy. Subsequently, the oral immunosuppressive regimen of sulfasalazine, bucillamine, and mizoribine was discontinued after initiation of treatment. Prednisolone was reduced to 5 mg/day.β-D-glucan was found to be positive, and sputum polymerase chain reaction (PCR) was positive for pneumocystis ; the sTNFR-I, -II, and IL-1 beta values were not found to be significantly elevated  and 6-7 weeks (range: 300 ng/mL to 700 ng/mL), respectively. The sTNFR-I and IL-1-beta levels remained relatively unchanged . At week 12, concurrent to the patient developing clinical symptoms of PCP, the levels of IL-6, CRP, and TNF-alpha showed an acute peak, which tapered downward over the following 3 weeks with the institution of an appropriate antifungal regimen  not on biologics were significantly higher than in healthy controls (n = 80) (7.28 ± 4.16 ng/mL versus 4.40 ± 0.88 ng/mL) [Etanercept is a recombinant fusion protein consisting of two p75 receptors and the Fc domain of the human IgG1 that binds to both soluble and cell-bound TNF-alpha . Althougalpha , it is nalpha concentrations were elevated within 24 hours of etanercept therapy in the present patient and remained high until the cessation of etanercept treatment, which supports previous reports that TNF-alpha concentrations are elevated during etanercept therapy [alpha by the possibility that the assay might be measuring TNF-alpha that is bound to etanercept. Etanercept has an approximately 50-fold greater affinity for human recombinant TNF-alpha in a binding inhibition assay and is approximately 1000 times more efficient than monomeric soluble TNFR. In addition, etanercept has a 5- to 8-fold longer plasma half-life compared with naturally occurring soluble TNFR. [alpha is known to be immunoreactive although without biological activity [alpha as measured by the ELISA was elevated during the course of treatment, disease activity was well inhibited. The levels of CRP were clearly lower during the course of treatment. TNF- therapy . We coulle TNFR. , 8. Etanactivity . In this Mori et al. reported that etanercept lowered the TNFR-I, IL-6, and IL-1 beta serum concentrations in juvenile idiopathic arthritis patients. The concentrations of IL-6 at weeks 2, 4, 8, and 12 of IL-1 beta at week 4 and of TNFR-I at week 12 were significantly lower than before treatment . Our datalpha increased by more than 300 pg/mL. Previously reported was a patient with high concentrations of TNF-alpha on etanercept therapy with development of gastric outlet obstruction, suggesting that serum TNF-alpha may rise in the early phase of the pneumonia, gastrointestinal obstruction, and other infectious/inflammatory complications [alpha itself accounted for the majority of the elevated concentration of TNF-alpha at the time of pneumonia in this case. On the other hand, TNF-alpha concentrations were steady after the start of etanercept therapy until the cessation of the drug. This phenomenon might demonstrate that the TNF-alpha levels reflected the proper drug dosage in the course of etanercept treatment. In general, therapies using biological agents have been highly effective in the treatment of RA. However, cases with serious adverse events have also been reported. Biologics may mask the clinical symptoms of infection because they diminish the production of acute inflammatory proteins . In the ications . Furtheralpha and TNFR-II concentrations measured by ELISA might consist of the TNF-etanercept complex and even etanercept itself, these concentrations could reflect the microenvironments in the course of etanercept therapy. Monitoring TNF-alpha levels might provide much information in terms of avoiding serious adverse events and evaluating clinical efficacy. In conclusion, although TNF-


Camellia sinensis) and cowslip (Echium amoenum) are used in Iranian traditional medicine. The aim of this study was to conduct the survey on the ability of Iranian black tea and cowslip extracts on secretion of tumor necrosis factor-alpha  by non-infected and infected mouse macrophages. A macrophage infection model with Legionella pneumophila and enzyme linked immunosorbent assay (ELISA) technique was used in this study. Research showed that the concentrations of TNF-alpha in non-infected and infected macrophage culture supernatant treated with various concentrations of Iranian black tea and cowslip extracts was significantly higher than the control. Various concentrations of cowslip  had significantly a great effect on the induction of TNF-alpha secretion in comparison with the Iranian black tea extract (P < 0.0001). In conclusion, we demonstrated the ability of Iranian black tea and cowslip on the induction of TNF-alpha, which can exert an anti-L. pneumophila activity on macrophages at a low dose. However, further studies to elucidate the mechanism(s) of the induction of macrophages by Iranian black tea and cowslip as well as their potential inhibitory effects on the growth of infected cells and their possible antitumor effects should be carried out in future works.Many species of tea ( Echium amoenum) and tea (Camellia sinensis) are used in Iranian traditional medicine. Cowslip grows in many geographical regions of Iran. This plant contains mucilage, carbohydrates, flavonoids, anthocyanin, Na+ and K+ ions, tannins, phenols, and some alkaloids that have tranquilizer, anti-anxiety and diuretic effects. Tea has been known traditionally as a healthy drink with some beneficial effects on health. Its effects on health had not been demonstrated by well-controlled laboratory studies until the 1970s , which constitute seven forms, including epigallocatechin gallate (EGCg). EGCg is a major catechin compound in tea extract and is also the most active form among the tea catechins in a variety of biological activities . For insL. pneumophila  was dissolved in 1 L of distilled water and the mixture decocted for 5 min. After filtering, the extract was dried using water bath. Each 100 g of dried plant yielded about 15 g of dried extract powder , 15. TheMethodsMacrophage cell culture 5 cells/mL for 2h in 5% CO2 at 37°C. The resulting monolayer cells were washed with Hanks’ balanced salt solution , supplied with 10% FCS-RPMI 1640 medium without antibiotics, and then used for experiments (The J774 A.1 murine (BALB/c Mouse) macrophage cell line, purchased from the cell bank of Pasteur Institute of Iran, was used in this study. The cells were maintained in RPMI 1640 medium  containing 10% heat-inactivated fetal calf serum . The J774 A.1 cells were allowed to adhere to 24-well tissue culture plates at a concentration of 5×10Bacteria L. pneumophila M138 was obtained as a gift from Dr. Mojtaba Moosavian . The bacteria were cultured on BCYE Medium (Sigma) for 3 days at 37°C. The bacterial suspensions were prepared in pyrogen-free saline, and the concentrations of bacteria were determined by spectrophotometry (otometry .Macrophage infection L. pneumophila (infectivity ratio of 10 bacteria per cell) for 30 min, washed to remove non-phagocytized bacteria, and incubated in RPMI 1640 medium containing 10% FCS with no antibiotics. The cultures were then incubated for up to 48 h at 37 °C in 5% CO2 (The macrophage monolayers were infected with n 5% CO2 .Macrophage cell culture treatment with extracts 2.The macrophage cultures (non-infected and infected with bacteria) were treated with various concentrations  of Iranian black tea and cowslip extracts for up to 24 h at 37°C in 5% COELISA techniqueThe concentrations of TNF-alpha in the culture supernatants of macrophage cultures were determined by Sandwich ELISA, using matched antibody pairs for ELISA . Various concentrations were calculated, using the standard curve constructed for each plate.Statistical analysisP ≤ 0.05.For performing descriptive statistics on raw data and statistical analyses, independent sample T-test and Tukey post-test were used . Statistical significance was taken at Direct effect of iranian black tea extract on TNF-alpha production by non-infected macrophagesP<0.0001). The data showed statistically significant differences between the effect of different concentrations of cowslip extract on the production of TNF-alpha.Compared to the control group, various concentrations of Iranian black tea extract  significantly induced secretion of TNF-alpha from non-infected macrophages . The data obtained from this study showed no statistically significant difference between the effect of 5 and 50 μg/mL concentrations on the production of TNF-alpha (P < 0.05).Compared to the control group, various concentrations of Iranian black tea extract  significantly induced secretion of TNF-alpha from the infected macrophages . The data showed statistically significant differences between the effect of different concentrations of cowslip extract on the production of TNF-alpha  (P < 0.001).Compared to the control group, various concentrations of cowslip  significantly induced secretion of TNF-alpha from non-infected macrophages . The data showed statistically significant differences between the effect of different concentrations of cowslip extract on the production of TNF-alpha  (P < 0.001). Compared to control group, various concentrations of cowslip  significantly induced secretion of TNF-alpha from infected macrophages (P <0.0001). In other words, these two extracts induced production of TNF-alpha compared to the control groups.Briefly, impact of various concentrations of Iranian black tea and cowslip present within non-infected and infected groups on the induction of TNF-alpha were statistically significant .  Various concentrations of cowslip  had a significantly greater effect on the induction of TNF-alpha secretion in comparison with the Iranian black tea extract .  There were significant differences between the of 0.5 and 5 μg/mL (but not 50 μg/mL) concentrations of cowslip and Iranian black tea on induction of TNF-alpha secretion. In other words, compared to the Iranian black tea extract, the cowslip extract had a greater effect on the production of TNF-alpha  of the induction of macrophages by Iranian black tea and cowslip, their potential inhibitory effects on the growth of infected cells and their possible antitumor effects should be carried over in the future.In conclusion, in this study we demonstrated the ability of Iranian black tea and cowslip to induce TNF-alpha, which can exert an anti-


TNF-α is a key pathogenic factor in inflammatory arthritis. Rapid and transient signaling and functional responses of cells to TNF-α, such as activation of NF-γB and MAPKs, are well known. These signaling mechanisms are widely assumed to be functional in cells chronically exposed to TNF-α and to mediate the pathogenic effects of TNF-α in chronic inflammation. We investigated the responses of primary macrophages to TNF-α over the course of several days and compared patterns of signaling and gene expression to RA synovial macrophages. The acute inflammatory response to TNF-α subsided after several hours and was followed by an IFN response characterized by sustained expression of STAT1 and downstream target genes. TNF-α-mediated induction of an IFN response was mediated by IFN-β and was sensitive to inhibition by Jak inhibitors. Concomitantly TNF-α induced a state of macrophage resistance to the homeostatic cytokines IL-10 and IL-27. Microarray analysis demonstrated that sustained TNF-α signaling induced expression of novel genes not appreciated to be 'TNF-inducible', but are highly expressed in RA synovial macrophages. Induction of an IFN response and abrogation of homeostatic cytokine signaling was also observed in RA synovial macrophages and likely contributes to the pathogenic actions of TNF-α during arthritis.TNFAIP3 (encodes A20) or by cytokines that suppress A20 expression or antagonize its function. These data suggest that augmenting homeostatic functions and signals and thereby rebalancing the pro- versus anti-inflammatory profile of TNF-α may represent an efficacious alternative therapeutic approach to suppress chronic inflammation.Subsequently and surprisingly, TNF-α induced a tolerant state in macrophages, with diminished cytokine production on lipopolysaccharide (LPS) challenge and protection from LPS-induced lethality. TNF-α-induced cross-tolerization was mediated by coordinate action of two inhibitory mechanisms, suppression of LPS-induced signaling and chromatin remodeling. Mechanistically, TNF-α-induced cross-tolerance was distinguished from TLR-induced tolerance by strong dependence on the nuclear kinase GSK3, which suppressed chromatin accessibility and promoted rapid termination of NF-γB signaling by augmenting negative feedback by A20 and IγBα. These results reveal an unexpected homeostatic function of TNF-α and provide a GSK3-mediated mechanism for preventing prolonged and excessive inflammation. This homeostatic mechanism may be compromised during RA synovitis, possibly by hypomorphic alleles of Overall, the data reveal novel signals and functions of TNF-α and that are likely operative during chronic inflammation and RA synovitis. Targeted inhibition of these non-traditional functional components of the TNF-α response may be efficacious in alleviating chronic inflammation while preserving acute TNF-α responses and host defense against infections.


One of the most important decisions animals have to make is how to respond to an attack from a potential predator. The response must be prompt and appropriate to ensure survival. Invertebrates have been important models in studying the underlying neurobiology of the escape response due to their accessible nervous systems and easily quantifiable behavioral output. Moreover, invertebrates provide opportunities for investigating these processes at a level of analysis not available in most other organisms. Recently, there has been a renewed focus in understanding how value-based calculations are made on the level of the nervous system, i.e., when decisions are made under conflicting circumstances, and the most desirable choice must be selected by weighing the costs and benefits for each behavioral choice. This article reviews samples from the current literature on anti-predator decision making in invertebrates, from single neurons to complex behaviors. Recent progress in understanding the mechanisms underlying value-based behavioral decisions is also discussed. Successful avoidance of a predatory attack is essential for survival and future reproductive success. Failure to detect a predator before an attack initiation, failure to fight off an attack, or failure to respond to an attack with an immediate escape, can be deadly. Many aspects of nervous system function must be optimized to control anti-predator behavior, including careful sensory assessment of threat stimuli, which sometimes involves multimodal integration, rapid transmission of this information within neural structures, and finally, fast and accurate motor activation. Importantly, predator avoidance is often produced under conflicting circumstances. Many daily activities that are essential for survival, such as feeding, mate search, or habitat selection, can increase visibility and thus vulnerability to predation. Animals trying to satisfy important needs while avoiding predation face a trade-off, e.g., between eating and the risk of being eaten. Thus, the selection of the most desirable behavior requires careful calculation of costs and benefits associated with different behavioral options. For example, foraging animals must accurately measure predation risk and weigh this risk against current nutritional state. Such cost-benefit analyses are made by the nervous system through the integration of external sensory signals with current internal states, and these decisions ideally lead to behavioral choices that optimize an animal’s fitness.Invertebrates are superbly suited to measure both the behavior and neural mechanisms underlying predator avoidance. In many invertebrates, an accessible nervous system with described neural escape circuits controls discrete escape behaviors. Thus, the link between neural machinery and behavioral expression is often identifiable and quantifiable. More recently, economic decision making, i.e., costs-benefit calculations under predatory risk, has been measured and described in a number of invertebrate species. This has opened up exciting new avenues for gaining a better understanding of complex “neuroeconomic” processes at a level of analysis not feasible in vertebrates.The first section of this review summarizes some of the foremost examples of anti-predator behavior and underlying neural circuitry found in four different arthropods. Both the specializations and shared features of these nervous systems that allow these animals to escape immediate predatory threats are discussed. The second part focuses on economic decisions made by invertebrates in situations where the risk of predation must be carefully weighed against other vitally important needs. Finally, we suggest some important future directions for the further identification of neural mechanisms underlying behavioral decisions.While predators can provide direct cues such as visual or mechanosensory signals that alert prey to the presence of a predator, indirect cues, such as odors, also allow the assessment of a potential predatory threat. However, indirect cues are frequently more ambiguous and seldom provide information on the degree or immediacy of the danger posed. And indirect cues that signal the presence of a predator  can divert attention from other vital activities or suppress these activities altogether. Different risk assessment behaviors, apprehension, and vigilance, are responses to indirect predator cues commonly described in vertebrate animals ,” frequently the largest axons in an animal’s nerve cord, which can be readily identified by their size, location, or morphology. These characteristics allow for rapid identification and often make these neurons accessible to a wide range of cell biological and electrophysiological studies.Because of their simplicity and clear function, these circuits have been excellent models for the study of the neural basis of behavior. Recent work, however, has uncovered a surprising degree of flexibility not previously recognized in these “simple,” “reflexive” systems. High-speed video recordings have exposed a previously unappreciated level of complexity to arthropod escape behaviors that has made researchers question the structure and even identity of the underlying circuits that were originally assumed to be responsible for escape  is sufficient to produce an escape motion that thrusts the animal upward and away from the point of caudal stimulation  are the only stimulus to reliably produce GF-mediated escape behavior, and then only in white-eyed fruit fly mutants, what role, if any, that the GF system plays in actual escape behavior of wild-type fruit flies is now unclear. Although stimuli that reliably recruit the GF system in wild-type flies are unknown, it seems unlikely that the GF system is simply the vestige of a lost escape circuit. While the newly identified looming sensitive pathway might be tuned to a selective set of stimulus features, the GF system could still serve as a robust, broadly tuned escape circuit capable of producing rapid escape behavior when more selective systems fail  which receive excitatory inputs from lobula giant movement detector (LGMD) neurons that are responsive to looming stimuli. With a one-to-one relationship with the LGMDs, the DCMDs produce action potentials in response to looming stimuli, with their firing rate increasing as the looming object gets closer. Thus, the DCMDs were originally thought to play a major role in jump production, sometimes compared to the giant fibers in crayfish and fruit flies that control their fast escape maneuvers Burrows, . HoweverLocusts also produce an avoidance behavior during flight. When looming stimuli are presented, flying locusts produce a gliding dive similar to the dives used by other insects to evade aerial predators. After DCMD neurons are activated by a looming stimulus, they produce short-latency excitatory postsynaptic potentials (EPSPs) in a motor neuron that raises the wing into the gliding posture. Stimuli that evoked high-frequency firing in the DCMDs also reliably elicited the gliding response, and the behavior was less frequently observed when high-frequency DCMD spikes were absent  with current internal drives , thus trading off the costs and benefits of different alternatives before deciding which one to choose. For example, a hungry animal is more likely to choose a behavioral option that involves risks because the value placed on foraging is greater than the value placed on other alternatives such as hiding. If the benefit of finding a meal outweighs the estimated cost of being attacked by a predator, the decision is to forage. If the value placed on foraging is low because the animal is satiated, other behavioral options become more valuable and behavioral output will shift toward less risky activities. The literature on value-based decision making, especially with a focus on prey behavior in predator-prey interactions, is extensive and covers a wide range of organisms (e.g., Ydenberg and Dill, The relatively new field of “neuroeconomics” is concerned with the neural underpinnings of value-based decision making in humans and other non-human primates Schall,  and therWhen juvenile crayfish are exposed to fast-moving shadows while foraging in an artificial stream environment, they respond by choosing one of two behavioral actions: they either freeze in place and remain motionless for several seconds before resuming foraging or they produce a tail-flip mediated by the MG neuron that propels the animal backward and away from the approaching shadow and the expected food source (Liden and Herberholz, Recently, Liden et al.  used theThe marine snail has been a fruitful model for studying the neural mechanisms underlying decision making and behavioral choice. Using a “competing behaviors” paradigm, early work suggested that different incompatible behaviors were organized in a hierarchical model, each controlled by command-like neurons that produced one behavior while inhibiting others. For example, when the sea slug was feeding, avoidance withdrawal in response to a tactile stimulus was suppressed (Kovac and Davis, Recent work, however, has shown that sea slugs base their decisions on cost-benefit computations (Gillette et al., Recent work in the arthropods discussed suggests that the escape behavior of all may be more complex and varied than has generally been assumed. Quantitative ethograms that divide complex escape maneuvers into a sequence of simpler events can help identify variability within each system. Moreover, combining ethograms with measures of neural structure or neural activity can elucidate the link between discrete motor actions within a series of behavioral events and the corresponding underlying neural mechanisms (Harley et al., Based on the high-speed video analysis of the behavior of fruit flies and locusts, a reexamination of the “simple” escape behavior of other arthropods is warranted. Perhaps an analysis at a temporal resolution comparable to that of the speed of production of these behaviors will uncover a degree of flexibility and control not previously appreciated in these animals as well. For example, while the escape tail-flip and freezing behavior of the crayfish in response to visual stimuli have been assumed to be two distinct behaviors, which has been supported by video analysis at 250 fps (Liden et al., While this new appreciation for the complexity of arthropod escape behavior has reinvigorated work on giant fibers and escape behavior, it raises two significant issues. First, if the giant fiber systems previously assumed to underlie observed escape behaviors are not in fact necessary or sufficient for the production of these behaviors, what circuits are? While Fotowat et al.  have madIt is likely other arthropod models will have a similar redundancy in escape circuitry as has been described in the crayfish. Thus, a comprehensive understanding of decision making during predator avoidance will have to wait until all pathways and not just parts of some are fully characterized Figure . While tAs such, future work should focus on completing the picture of currently known circuits, where often substantial sensory or motor elements remain poorly characterized, as well as identifying unknown but hinted at command or non-command circuits. This hunt for currently uncharacterized circuits might be aided by the possible similarity to and knowledge of already characterized systems found in related species Figure . For insin vivo recordings in freely behaving animals (Fotowat et al., Due to the assumption that giant fiber systems were a singular system responsible for the production of all escape behaviors, there is currently much confusion as to what discrete escape behavior is subserved by what specific circuit. Since it now appears that there are likely many circuits that produce a range of escape behaviors, the spectrum of these behaviors and the stimulus conditions that lead to their display will need to be carefully cataloged and behavioral assays developed that can differentiate them. However, without the ability to simultaneously record both escape behaviors and neural activity, it will be difficult to ascribe a discrete escape sequence or subcomponent of escape behavior to a particular circuit or set of neurons. For this, the use of telemetry that allows for There is a notable lack of neuroethological studies focused on escape mechanisms produced under natural conditions. While staged encounters with natural predators in the laboratory provide some insight into the interplay between neural function and ecologically relevant escape behavior, these studies are sparse. Field studies on the other hand are often focused on ecology and behavior and not designed to investigate neural processes. Occasionally, data sets obtained separately in the field and laboratory allow for a comparative view and for correlating firing patterns of individual neurons and natural escape behavior (e.g., Hemmi and Tomsic, Finally, the neuromodulation of escape behavior by monoamines such as octopamine, serotonin and dopamine is worth further exploration. Although a number of the escape circuits discussed have been shown to be responsive to the application or removal of monoamines (Glanzman and Krasne, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ectodomain of matrix protein 2 (M2e) is highly conserved among influenza A viruses and can be a promising candidate antigen for a broadly cross-protective vaccine. In this study, a tetrameric M2e (tM2e) and a truncated form of flagellin (tFliC) were coincorporated into virus-like particles (VLPs) to enhance its immunogenicity. Our data showed that the majority of M2e in VLPs was presented as tetramers by introducing a foreign tetramerization motif GCN4. Intranasal immunization with tM2e VLPs significantly enhanced the levels of serum IgG and IgG subclasses compared to soluble M2e (sM2e) in mice. tM2e VLPs also induced higher M2e-specific T-cell and mucosal antibody responses, conferring complete protection against homologous influenza virus infection. The immunogenicity of tM2e VLPs was further enhanced by coincorporation of the membrane-anchored tFliC (tM2e chimeric VLPs) or coadministration with tFliC VLPs as a mixture, but not the soluble flagellin, inducing strong humoral and cellular immune responses conferring cross-protection against lethal challenge with heterotypic influenza viruses. These results support the development of tM2e chimeric VLPs as universal vaccines and warrant further investigation. Influenza A virus (IAV) is a negative sense single-stranded RNA virus responsible for annual seasonal epidemics worldwide and, occasionally, pandemics caused by emerging novel subtypes/strains derived by reassortment with avian or porcine viruses , 2. CurrThe ectodomain of the influenza A M2 protein (M2e) is highly conserved among influenza A viruses and is considered to be a promising target for inducing cross-protection against different influenza A virus subtypes . Some M2Toll-like-receptor- (TLR-) based immune adjuvants can induce efficient mucosal adjuvant activity . The bacThe mice were housed and treated according to Emory University  guidelines and all animal studies were approved by the Emory University Institutional Animal Care and Use Committee (IACUC).Spodoptera frugiperda Sf9 cells  were maintained in suspension in serum-free SF900 II medium  at 27°C in spinner flasks at a speed of 80 to 100 rpm. Madin-Darby canine kidney (MDCK) cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum . Mouse-adapted influenza A/Philippine/2/82 (H3N2) and A/PR/8/34 (H1N1) were prepared as lung homogenates from intranasally infected mice and were used for challenge studies. The M2e peptides were synthesized at GenScript  as shown in A GCN4 sequence-stabilized tetrameric M2e (tM2e) construct was generated as described  but withμL sample buffer  and heated at 95°C for 5 min before being analyzed by sodium dodecyl sulfate-polyacyrlamide gel electrophoresis (SDS-PAGE).The presence of membrane-anchored tM2e on cell surfaces was determined by a cell surface immune-assay. Two days postinfection with tM2e rBV and mock rBV (rBV expressing human immunodeficiency virus Gag) in 6-well plates, Sf9 cells were washed with phosphate-buffered saline (PBS) at 4°C and then incubated with 1 mL of NHS-SS-biotin dissolved in PBS (0.5 mg/mL) for 30 min at 4°C. Biotinylation was quenched by adding pre-cooled PBS containing 0.1 N glycine. After three washes with precooled PBS, the cells were then lysed with lysis buffer  and precipitated with avidin resins  at 4°C overnight. After precipitation, resins were washed twice with lysis buffer plus 0.4% SDS and then mixed with 15 To produce VLPs containing influenza M1 and tM2e (tM2e/M1 VLPs) and truncated flagellin-containing chimeric VLPs (tM2e/tFliC/M1 cVLPs), Sf9 cells were coinfected with rBVs expressing M1 and tM2e, tFliC, tM2e and M1, or tFliC and M1, respectively. The multiplicities of infection (MOIs) of rBVs expressing tM2e, M1 were adjusted to 4 : 2 to produce tM2e VLPs, and the MOIs of rBVs expressing tFliC, tM2e, M1 were adjusted to 1 : 4 : 2 to produce chimeric tFliC-containing tM2e VLPs (cVLPs). The quality of the purified VLPs was determined by Western blotting analysis and immunoelectron microscopic observation. The sterility was determined by inoculating LB medium and incubating the culture at 37°C for 48 h. At 60 hr postinfection, Sf9 cells were washed with 0.3 M HEPES containing 0.02 M lysine and were blocked in 0.2 M HEPES-1.5% bovine serum albumin (BSA) for 60 min at 4°C. After blocking, cells were incubated with mouse anti-M2e antibody  at a 1 : 100 dilution at 4°C for 60 min. After washing, a 10 nm gold-conjugated goat anti-mouse antibody  was added at a dilution of 1 : 100 for another 60 min. Cells were then washed and fixed with 1% glutaraldehyde in PBS for 30 min, incubated with 1% osmium tetroxide for 1 hour, and then stained with tannic acid. The samples were dehydrated and embedded in EMBED 812 . Samples were then stained with uranyl acetate and lead citrate and examined with a Philips  CM 10 electron microscope at the Robert P. Apkarian Integrated Electron Microscopy Core of Emory University.μg soluble tM2e protein, tM2e VLPs (tM2e VLP), tM2e/tFliC VLPs, a mixture of tM2e VLPs and tFliC VLPs, a mixture of tM2e VLPs and soluble flagellin protein with 0.5 μg M2e content, or 0.5 μg soluble flagellin protein only at 4-week intervals. Each group included 18 mice. Four weeks after the final immunization, mice were challenged with a lethal dose of A/PR/8/34 ) or A/Philippines/82 influenza virus (5 LD50). Mice were monitored daily to record body weight changes and mortality using 25% loss in body weight as the Institutional Animal Care and Use Committee (IACUC) endpoint.For animal experiments, 6–8 weeks old female BALB/c mice  were intranasally (i.n.) immunized three times with 0.5 μm filter and stored at −80°C for further assays. Lymphocytes from lung and spleen samples were collected from mice sacrificed 4 weeks after the final boost and were used for ELISPOT as described previously [Blood samples were collected at one week before immunization for preimmune sera and 2 weeks after each immunization for immune sera by retro-orbital plexus puncture. Sera were collected by a brief spin (5000 rpm for 5 min) after clotting (about 2 hours at room temperature). Mucosal samples were collected before challenge and 4 days after challenge. Nasal washes were collected by lavaging mouse nostrils repetitively with 1 mL of PBS containing 0.05% Tween 20 (PBST). For lung washes, individual mouse lungs were lavaged repetitively with 1 mL PBST. After a brief centrifugation (8000 rpm) for 10 min, supernatants were filtered through a 0.22 eviously .μg/mL) as coating antigens. The highest dilution which gives an OD450 of at least twice the standard deviation of that of the naïve group at the same dilution was designated as the Ab endpoint titer. To evaluate the cross-reactivity to variant M2e-sequence peptides, immune sera were tested for binding to M2e peptides derived from Phi/82, PR/8, CA/09, and VIET/04, respectively, by ELISA. The optical density at 450 nm was used to compare the level of reactivity.M2e specific antibody (Ab) titers in immune sera were determined by ELISA as described previously , 11 usinμg/mL of puromycin , 5 μM of amantadine  and 10% FBS  at 37°C in 5% CO2. Confluent M2-expressing MDCK monolayer cells were fixed by 0.05% glutaraldehyde and 10% buffered formalin  for 30 min at room temperature and were used to determine Ab levels binding to M2 expressed on cell surfaces by ELISA as described [Antibodies recognizing native M2 protein were determined by using cell surface ELISA. M2-expressing MDCK cells were maintained in DMEM media with 7.5 escribed . γ) and interleukin 4 (IL-4) secreting T cells from immunized mouse splenocytes or lung cells were evaluated using ELISpot kits  according to the manufacturer's instructions [γ or IL-4 Abs was used to coat Multiscreen 96-well filtration plates . A volume of 100 μL freshly isolated splenocytes (1 × 107 cells/mL) were added to each well and stimulated with M2e peptide (10 μg/mL). The plates were incubated for 40 h at 37°C with 5% CO2. The development and counting of ELISpot were performed following the manufacturer's procedures.Interferon gamma (INF-ructions . Anti-mo Whole lungs were collected at day 4 postinfection (p.i.) and ground using cell strainers . Lung homogenates were centrifuged at 1000 RPM for 10 min to remove tissue debris. MDCK cell-based plaque assay was used for lung virus titration as described previously.t-test. The analyses were done by using GraphPad Prism version 5.00 for Windows . P values of less than 0.05 (P < 0.05) were considered to be statistically significant. Comparisons among vaccinated groups were performed using a two-tailed Student's μg total protein) and tM2e/tFliC VLPs  were loaded and detected. Amount of M2e protein incorporated in M2e VLP was calculated by spot densitometry analysis using serial diluted rM2e protein as a standard as shown in To improve the immunogenicity of M2e, a modified tetrameric M2e (tM2e) construct was designed as showed in P < 0.05)  . The flagellin-containing VLPs (G3 and G4) elicited higher levels of IgG2a  compared to tM2e VLPs (G2) . Immune sera also showed similar binding reactivity to native M2 expressed on MDCK cell surfaces, suggesting that antibodies induced by tM2e VLPs can recognize conformational epitopes on native M2. These data show that tM2e VLPs can elicit strong humoral responses and that membrane-anchored truncated flagellin shows a strong adjuvant effect.To determine if i.n. immunization with tM2e VLPs could induce enhanced humoral responses, immune sera were evaluated for antigen-specific IgG titers using ELISA with M2e peptides as coating antigens, or cell surface ELISA using M2-expressing MDCK cells. As shown in  < 0.05) . The lev < 0.05) . FlagellP < 0.05). These results demonstrate that the assembly of the tM2e antigen into VLPs can significantly enhance the immunogenicity of tM2e and can induce M2e-specific antibodies with some cross-reactivity to variant M2e sequences. These results also show that when M2e and flagellin are combined together in VLPs (G3) or tM2e VLPs and flagellin VLPs are mixed together (G4), systemic responses were enhanced more than tM2e VLPs mixed with soluble flagellin protein after immunization (G5), which indicate that the membrane-anchored truncated flagellin shows a stronger adjuvant effect than the soluble form in induction of serum IgG responses. Although M2e is highly conserved in influenza A viruses, some amino acid substitutions occur in different strains and subtypes. To evaluate the cross-reactivity of M2e-specific Abs induced by tM2e VLPs, sera from mice immunized i.n. with tM2e VLPs were assayed using ELISA with variant M2e peptides as coating antigens. As shown in P < 0.05). Furthermore, groups G3 and G4 have significantly higher levels of IgG and IgA compared to G2 which received only tM2e VLPs. After challenges with Phi/82 or PR8 virus, the lung wash IgG and IgA levels increased, as well as nasal wash IgA, and there was no significant IgG change between prechallenge and postchallenge nasal washes (P < 0.05). These data demonstrate that tM2e VLPs stimulate M2e-specific mucosal Ab responses in mice, and incorporating the membrane-anchored flagellin in VLPs as adjutants can further stimulate mucosal antibody secretion.A strong mucosal antibody response is associated with the prevention of viral entry in the upper respiratory tract of mice . To evall washes . Similarγ and IL-4 secreting cells in both the spleens and lungs of immunized mice were evaluated by cytokine ELISpot as described previously [γ secreting cell populations in the spleens and lungs after stimulation with M2e peptide or tM2e VLPs compared with mice immunized with soluble tM2e . A higher frequency of IL-4 secreting cells was also detected in the spleens of immunized mice in G2, G3, G4, or G5 compared to that of mice vaccinated with soluble tM2e (P < 0.05). However, only G2, G3, and G4 had higher frequencies of IL-4 secreting cells in the lungs after stimulation with tM2e VLPs and only background levels of cytokine secreting cells were detected after M2e peptide stimulation . In contrast, mice immunized with tM2e/tFliC VLPs or tM2e VLP + tFliC VLPs had significant lower virus titers in their lungs after Phi/82 or PR/8 challenge (P < 0.05) and exhibited reduced morbidity compared with mice in the tM2e protein group. All mice in G3 and G4 survived the lethal challenge with homologous Phi/82 H3N2 virus or heterosubtypic PR/8 H1N1 virus. These results indicate that i.n. immunization of mice with tM2e VLPs with flagellin as an adjuvant significantly reduces virus titers in the lungs and completely protects mice against severe disease and death from lethal infection with the homologous virus or heterosubtypic virus.As shown in M2e is considered to be a promising target for inducing cross-protection because it is highly conserved among influenza A viruses . HoweverVLPs is an effectve platform to increase the immunogenicity of antigen . BesidesκB or cytosolic receptor NLRC4-mediated activation of the inflammasome [β and IL-18 to provide a sufficient second signal. The cytosolic innate signaling drives the T-cell proliferation necessary to generate antibodies to both flagellin itself and a coadministered antigen [κb [Flagellin can promote murine antibody responses by either membrane receptor TLR5-mediated activation of NF-ammasome . VLPs arammasome . Flagell antigen . Solubleigen [κb . In our igen [κb , 26. HowOur aim was to combine the advantage of VLPs as a vaccine platform and flagellin as an adjuvant to increase the immunogenicity of the conserved M2e and induce cross-protective immunity to homo-/heterologous influenza viral infections. From our challenge data we can see that even with high level of Ab titers, M2e VLPs induce limited protection from heterosubtypic virus infection. One possible reason is that so far only a single human CTL epitope has been identified for M2e inducing memory CTL activity , and M2eThe mucosal immune system is the first immunological barrier against viruses that invade the body via the mucosal surface . M2e-basAlthough M2e is relatively conserved in all influenza A viruses, variation of M2e sequences in different viruses may indicate the different protection efficacy of M2e vaccines to various virus infection. In our study, tM2e VLPs showed complete protection of mice from Phi/82 (H3N2) virus infection; it still cannot protect mice from PR/8 (H1N1) virus infection which may be associated with the 1-aa substitutions in the M2e of PR/8. Indeed, our ELISA binding assay illustrated that even a 1-aa variation could impact the binding avidity of M2e-specific Abs with synthesized peptides. However, it is noteworthy that tFlic-containing VLPs showed complete protection of mice from either Phi/82 (H3N2) virus infection or PR/8 (H1N1) virus infection. Therefore, it is worth to further investigate those strategies that could enhance the cross-protection of M2e vaccines, for instance, incorporating representative M2e sequences from different subtypes into one construct, or coexpressing with HA or NA antigens, as well as combining with better adjuvant strategy. M2e is one of the target antigens for a universal vaccine against influenza. Strategies like administration in naturally conformational form carried by VLPs and adjuvanted by bioactive factors demonstrate significant improvement of both immunogenicity and protective efficacy of M2e. Our results support the promising application of tM2e cVLP-based universal vaccines and warrant further investigation.The supplemental material provided includes:(1) Table 1 includes the endpoint IgG titer of each group using ELISA with M2e peptides as coating antigens.(2) Table 2 includes the endpoint IgG1 titer of each group using ELISA with M2e peptides as coating antigens.(3) Table 3 includes the endpoint IgG2a titer of each group using ELISA with M2e peptides as coating antigens.(4) Table 4 includes the endpoint IgG2a titer of each group using cell surface ELISA with M2-expressing MDCK cells.(5) Table 5 includes the P values of IgG titer between different groups when using ELISA with M2e peptides as coating antigens.(6) Table 6 includes the P values of IgG1 titer between different groups when using ELISA with M2e peptides as coating antigens.(7) Table 7 includes the P values of IgG2a titer between different groups when using ELISA with M2e peptides as coating antigens.(8) Table 8 includes the P values of IgG2a titer between different groups when using cell surface ELISA with M2-expressing MDCK cells.Click here for additional data file.


White matter injury (WMI) is the major antecedent of cerebral palsy in premature infants, and is often associated with maternal infection and the fetal inflammatory response. The current study explores the therapeutic potential of glutamate receptor blockade or cyclooxygenase-2 (COX-2) inhibition for inflammatory WMI.Using fetal ovine derived mixed glia cultures exposed to tumour necrosis factor-α (TNF-α) or lipopolysaccharide (LPS), the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl D-aspartate (NMDA) glutamate receptors and their contribution to inflammation mediated pre-oligodendrocyte (OL) death was evaluated. The functional significance of TNF-α and COX-2 signalling in glutamate release in association with TNF-α and LPS exposure was also assessed.AMPA and NMDA receptors were expressed in primary mixed glial cultures on developing OLs, the main cell-type present in fetal white matter at a period of high risk for WMI. We show that glutamate receptor expression and configuration are regulated by TNF-α and LPS exposure, but AMPA and NMDA blockade, either alone or in combination, did not reduce pre-OL death. Furthermore, we demonstrate that glutamate and prostaglandin E2 (PGE2) release following TNF-α or LPS are mediated by a TNF-α-COX-2 dependent mechanism.Overall, these findings suggest that glial-localised glutamate receptors likely play a limited role in OL demise associated with chronic inflammation, but supports the COX-2 pathway as a potential therapeutic target for infection/inflammatory-mediated WMI. Maternal infection or infection of the placenta is recognised as one of the most important causes of preterm birth, and of cerebral white matter injury (WMI) in the prematurely born baby -4. Consi2+-permeable GluR4-subunit containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors on developing OLs [2+-permeability of the AMPA receptor, greater GluR2 expression could be protective against glutamate excitotoxicity.Animal models of hypoxic-ischemic WMI have demonstrated that activation of microglia, disturbances in pro-inflammatory cytokine production and release of glutamate are associated with injury via receptor-mediated excitotoxicity -8. In whping OLs ,16,17 anping OLs . In cont2+-permeable phenotype, and there is an overall increase in Ca2+-permeable AMPA-GluR subunits in pre-OLs compared to mature OLs correlating with greater vulnerability, both in vitro[in vivo[The developing ovine brain at day 90 of gestation shares a similar susceptibility to WMI to the 24- to 32-week human brain - a period when subcortical white matter is populated predominantly by pre-myelinating OLs . Immatur in vitro,23 and io[in vivo,24.Although excitotoxic injury after oxygen-glucose deprivation is well documented ,25, it iThe purpose of this study was therefore to examine the contribution of glutamate and its receptor-mediated toxicity to the selective loss of immature pre-OLs in TNF-α or lipopolysaccharide (LPS) induced injury in cultures of preterm fetal ovine primary mixed glia. First, we evaluated glial expression of AMPA and NMDA receptors and determined whether expression levels were responsive to TNF-α or LPS exposure leading to an unfavourable glutamate receptor phenotype. To establish whether this injury involved glutamate receptor activation, we confirmed the presence of AMPA and NMDA receptors on pre-OLs and assessed their role by treating with AMPA or NMDA antagonists, either alone or in combination. We further tested the potential for glia to release glutamate following TNF-α or LPS treatments and whether this was via a TNF-α-COX-2 prostaglandin-mediated mechanism.Glial cells were isolated from fetal  ovine forebrains, characterised by immunocytochemistry detection of cell-specific markers, and maintained in a chemically defined medium, as previously described . BrieflyF)quinoxaline) which blocks AMPA receptors and to a minor degree kainic acid (KA) receptors [Prior to treatment with TNF-α  or LPS  cultures were pre-incubated (30 min at 37°C) with one of five treatments: the AMPA antagonist, NBQX , and incubated with primary antibodies overnight at 4°C. GluR2, GluR4  or NR1  subunits were localised to O4-positive pre-OLs . Secondary Alexa488 conjugates  were incubated for 3 h at room temperature, washed and Hoechst stained before being visualised in TNE buffer. Hoechst 33342 was used as a fluorescent nucleic marker; pre-OL death evaluated by co-localisation of condensed and fragmented nuclei with O4 on immunohistochemistry. Cells were imaged with an inverted fluorescent microscope and composite images generated with Adobe Photoshop . Quantification was performed by counting positively stained cells on four random fields of view, over two wells per experimental replicate.Protein lysates were prepared using sodium dodecyl sulfate (SDS) lysis buffer and resuspended in lithium dodecyl sulfate (LDS) sample-buffer (Invitrogen Life Technologies). Samples were electrophoretically separated on 4% to 12% bis-tris precast gels (Invitrogen Life Technologies) and protein bands were electrically transferred to polyvinylidene difluoride membranes . Membranes were blocked for 1 h at room temperature in Tris-buffered saline-Tween  Tween-20) containing 5% (w/v) non-fat milk powder, then incubated overnight at 4°C with antibodies against GluR2 , GluR4  or NR1  in TBS-T supplemented with 5% BSA. Membranes were incubated for 2 h at room temperature with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies  in TBS-T supplemented with 5% non-fat milk powder. Bands were visualised on X-ray films using ECL-plus substrate (Thermo Scientific) as per the suppliers’ instructions. Between all incubation steps, membranes were washed extensively with TBS-T.Total RNA was isolated by washing cells in ice-cold PBS, followed by suspension in TRIzol  and then stored at -80°C. mRNA was extracted using the RNeasy Mini Kit , according to the manufacturer’s instructions. Total RNA  was incubated with random primers and deoxynucleotide mix and incubated at 65°C for 7 min then reverse-transcribed in superscript III (Invitrogen Life Technologies) according to the manufacturer’s protocol. GluR1-4 and NR1 transcript abundance was determined by real-time PCR using TaqMan Gene Expression Assays . Amplification of GluR1, R2, R3 R4 and NR1 gene transcripts were performed in triplicate on an ABI PRISM 7900HT Sequence Detector (Applied Biosystems) using standard cycling conditions recommended by the manufacturer . Singleplex amplification was performed with a total reaction volume of 10 μL, containing 5 μL TaqMan Universal PCR Master Mix (Applied Biosystems), 1 μL cDNA template, 250 nM probe, 900 nM forward and reverse primers and 2.75 μL diethylpyrocarbonate (DEPC)-treated water. Standard curves of the target gene and the house-keeping gene, 18S, were included in each plate, consisting of five-fold serial dilutions of cDNA synthesised from an LPS-treated fetal ovine cerebellum. Primer and probes sets for all target genes were designed using the Primer Express software (Applied Biosystems). Analysis was performed using the relative standard curve method and expressed as fold change expression relative to time-matched controls. GluR2 was also expressed relative to time-matched controls, normalised to the expression of all other AMPA subunits.Glutamate concentrations in media before and after treatment with TNF-α or LPS and with or without NS398 or the TNF-α antibody, were determined using the Amplex®Red glutamic acid/glutamate oxidase assay kit (Invitrogen Life Technologies) according to the manufacturer’s instructions. Briefly, the reaction buffer  was added to samples of culture media and glutamate standards and incubated in the dark for 30 min at 37°C. Fluorescence was measured at an excitation wavelength of 544 nm and an emission wavelength of 590 nm with a multi-mode plate reader . Relative fluorescent units (RFU) were converted based on the RFU measurement of the known standard concentrations and adjusted for total protein content in the media sample (determined by bicinchoninic acid (BCA) assay; Thermo Scientific). Samples were assessed in duplicate and the final concentration represented as mM of glutamate per mg of total protein.PGE2 concentrations in media were determined by direct radioimmunoassay, similar to that described previously . ConditiP <0.05.Data are presented as mean ± standard error of the mean (SEM). Statistical comparisons between groups were performed using two-way analysis of variance (ANOVA). Where significant differences were observed, Tukey’s post-hoc analysis was used for comparisons of the means unless otherwise stated. The significance level was set at P <0.05, P <0.01, P <0.05; Figure P <0.001, P <0.001, P <0.05; Figure P <0.001; Figure TNF-α significantly reduced pre-OL survival after 24, 48 and 96 h (P <0.05), while MK-801 was associated with a significant decline in pre-OL survival at all time-points assessed (P <0.05). Combined NBQX/MK-801 substantially reduced pre-OL survival at 24 and 72 h (P <0.05), however despite this decrease, no significant change in survival compared to controls was observed at 48 and 96 h.Pre-OL survival was also evaluated in cultures treated with inhibitors alone to assess potential toxic effects of glutamate receptor inhibition Figure C. NBQX aThese results suggest that AMPA and NMDA receptor activation may not be primary contributors to inflammation-induced pre-OL injury and raise the possibility that a lack of improved survival after exposure to NBQX and/or MK-801 may be due in part at least to a general adverse effect on survival in cultures of mixed glia.P <0.05; Figure P <0.05; Figure GluR2 mRNA expression was significantly reduced at 24 h  revealed a significant reduction at 48 and 72 h following TNF-α exposure .TNF-α induced a transient reduction of GluR4 mRNA expression after 24 h , respectively, by western blot.A marked transient increase in mRNA expression of the obligatory NMDA subunit, NR1, was induced by TNF-α after 48 h  but did not significantly protect after 96 h of LPS treatment.We evaluated the consequences of TNF-α and COX-2 blockade to determine if effectors downstream of TNF-α or PGE2 signalling contribute to inflammation-mediated OL death. TNF-α significantly reduced pre-OL survival after 24, 48 and 96 h (P <0.05). After 72 h, pre-OL survival in TNF-α and NS398 co-treated cultures was comparable to those in untreated controls. In LPS-exposed cultures co-treated with NS398 pre-OL survival was significantly improved at 24 and 48 h compared to LPS treatment alone (P <0.05).NS398 co-treatment in TNF-α exposed cultures resulted in pre-OL levels comparable to those after TNF-α alone in the first 48 h  in association with a delayed rise in extracellular glutamate concentrations suggests increased potential for formation of more Ca2+-permeable AMPA receptors in ovine glia, including pre-OLs and thus potentially more vulnerable to excitotoxicity [This is the first observation in ovine-derived fetal glia that GluR2 subunit mRNA and protein expression  are transiently reduced after TNF-α exposure and persistently after LPS. Furthermore, pre-OLs expressed the Catoxicity ,46.We confirmed that ovine pre-OLs express the NR1 subunit in primary mixed glial cultures. NR1 protein expression, however, was differentially expressed by TNF-α and LPS with an acute, transient increase after LPS exposure and a gradual increase by 96 h after TNF-α exposure. Others have shown that NMDA receptors are expressed by OLs and contribute to white matter pathology after hypoxia-ischemia, by triggering loss of processes and increasing apoptosis and generation of reactive species ,47,48. W2+-permeable glutamate receptors. Others suggest the major source of glutamate in white matter lesions is by reversal of sodium glutamate transporters [Data from the current study show that glial derived extracellular glutamate levels are substantially increased in response to both TNF-α and LPS. To the best of our knowledge this is the first report in mixed glial cultures and is potentially important given that as discussed above, there is the potential for excitotoxic injury via the activation of more Casporters  from presporters , howeversporters , likely sporters .in vitro excitotoxic model [The present studies show that the TNF-α and LPS-induced increase in glutamate concentrations are mediated via a TNF-α and COX-2 dependent mechanism. Our findings are consistent with TNF-α signalling via TNFR1 initiating production of PGE2, and TNF-α and COX-2 inhibition improved pre-OL survival 48 h after LPS exposure. A role for COX-2 has already been established in neuronal LPS-induced injury  and COX-ic model . Thus, tOther cytokines may also activate COX-2. For example, exogenous (IL-1β) induced the release of PGE2 via activation of nuclear factor-kappa B (NFκB) and mitogen-activated protein kinase (MAPK) cascades . HoweverIn contrast, our data show that treatment with NBQX and/or MK-801 following TNF-α or LPS exposure was not protective against pre-OL death, suggesting that AMPA and NMDA receptor activation do not make a major contribution to inflammation-induced pre-OL injury. Consistent with this, in near-term fetal sheep treatment with topiramate, an AMPA receptor antagonist, 5 h after severe cerebral ischemia did not protect white matter or grey matter . Indeed,2+-permeability over 4 days after exposure. Critically, we observed that the increase in extracellular glutamate concentrations occurred after the onset of loss of pre-OL cells. This strongly suggests that the increase in glutamate may have been a consequence of cell death, perhaps due to impaired energy dependent reuptake or to release from lysing cells, rather than the cause of injury.In summary, this study suggests that AMPA and NMDA glutamate receptor inhibition does not seem to be an effective therapy for inflammation-mediated pre-OL death. Glutamate levels and glutamate receptor conformation were altered by inflammatory stimuli, leading to a delayed rise in extracellular glutamate concentrations combined with a receptor phenotype that is favourable to CaWe conclude that our hypothesis that glutamate receptor-mediated toxicity contributes to WMI requires refinement to address the overall lack of effect of receptor inhibition. In contrast, the TNF-α-Cox-2 pathway should be further explored as a potentially viable therapeutic target, to protect pre-OLs from neuroinflammatory damage of the immature brain.AMPA: Alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate; COX-2: Cyclooxygenase 2; LPS: Lipopolysaccharide; NMDA: N-methyl D-aspartate; OL: Oligodendrocyte; PGE2: Prostaglandin E2; TBS-T: Tris-buffered saline-Tween; TNF-α: Tumour necrosis factor-α; WMI: White matter injury.The authors declare that they have no competing interests.LW-M, AJG, LB, MM and MF conceptualised the study. LW-M and MF designed the protocol and carried out the glial cultures, and molecular and protein expression; LW-M and MF analysed data. AJG and MF provided critical interpretation. LW-M, AJG, LB, MM and MF drafted the paper. All authors have read and approved the final version of the manuscript.


Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheime2+ signaling, PKCα/PKCβI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNA-mediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount of soluble RAGE in the mouse lung. Our findings suggest that pharmacological stimulation of RAGE shedding might open alternative treatment strategies for Alzheimeŕs disease and diabetes-induced inflammation.The multiligand  Receptor for Advanced Glycation End products (RAGE) is a type I transmembrane protein belonging to the immunoglobulin superfamily and is usually expressed at low levels in epithelial, neuronal and vascular cells. The lung is the sole organ having high expression of RAGE under normal conditions The RAGE has been shown to play a crucial role in chronic inflammatory diseases, late diabetic complications, atherosclerosis and Alzheimeŕs disease Numerous studies have shown that administration of soluble RAGE (sRAGE) can alleviate full-length RAGE-mediated harmful processes by trapping RAGE ligands and preventing RAGE signaling. For example the application of sRAGE slowed-down tumor growth and reduced the amount of metastases in mice As soluble RAGE alleviates pathophysiological processes mediated by full-length RAGE, the stimulation of RAGE shedding may be used as a therapeutic attempt in the treatment of diseases such as Alzheimer and diabetes mellitus. The aim of our study was to investigate whether full-length RAGE is proteolytically converted into soluble RAGE following activation of G protein-coupled receptors (GPCRs). To answer this question, we investigated GPCRs stimulating various main signaling networks: the V2 vasopressin coupled to adenlylyl cyclase in vivoThe neuropeptide PACAP exhibits anti-inflammatory and neuroprotective properties primarily mediated through the PAC1 receptor For investigation whether the proteolytic machinery responsible for the shedding of RAGE is inducible via activation of G protein-coupled receptors, we generated cell lines co-expressing RAGE and a respective GPCR. The PAC1 receptor, the V2 vasopressin and the oxytocin receptor were included in the study because these GPCRs act through different major signaling pathways. In all three cases we found the shedding of RAGE to be inducible by agonist-induced GPCR activation . In cultSince ectodomain shedding is taking place at the cell surface, we quantified the amount of RAGE at that cellular compartment. For this purpose, cell surface proteins were labeled with a membrane-impermeable biotinylation reagent and after that shedding was induced for 2 hours with PACAP-27. Biotinylated proteins in cell culture supernatants and intact cells were isolated separately. Biotinylated secreted and full-length RAGE variants were quantified by Western blot analysis. In these experiments, PACAP-27 treatment increased the amount of secreted RAGE in the cell culture supernatant two-fold  and signD 500 nM vs. 0.5 nM) The PAC1 receptor is able to bind different PACAP ligands with similar high affinity. Treatment of PAC1/RAGE cells with 300 nM of either PACAP-27, PACAP-38 or acetylated PACAP-38 resulted in a strong and comparable induction of RAGE shedding . In addiSame experiments were performed on HEK/RAGE cells lacking OTR and V2R expression. On these cells, neither OT nor AVP were able to induce the release of RAGE (data not shown). Thus agonist-induction shedding of RAGE strictly requires the presence of respective GPCR.Next we analyzed whether the shedding of RAGE in PAC1/RAGE cells is dependent on the PACAP concentration. Cells were treated with different PACAP-27 hormone concentrations up to 3 µM for 4 h, and then the amount of shedded RAGE was determined by Western blot analysis . PACAP-2de novo gene expression.To analyze the kinetic of PACAP-induced RAGE shedding, we investigated shedding over a time period of six hours. Detectable amounts of sRAGE were noticed after 30 min of stimulation, and the amount of sRAGE continuously increased during the first three hours of 300 nM PACAP-27 treatment . Longer To estimate the potency of PACAP-induced RAGE shedding in our cellular system, the observed effects were compared to effects of phorbol ester-mediated RAGE shedding.Time-dependent treatment of PAC1/RAGE cells with PMA (1 µM) resulted in an increased shedding of RAGE. The kinetic of PMA-induced RAGE shedding was comparable with the PACAP-induced kinetic .in vitro assays with recombinant proteinases, the IC50 values for ADAM10 and MMP9 are 5.3 and 2.5 nM, respectively In a previous study we demonstrated that zinc-dependent metalloproteinases ADAM10 and MMP9  conduct RAGE shedding To confirm that ADAM10 and MMP9 act as sheddases on RAGE, we additionally performed siRNA-mediated knock-down experiments. Here we investigated the contribution of ADAMs 10 and 17 as well as of MMPs 2 and 9. Whereas the knock-down of ADAM10 and MMP9 resulted in a ∼ 50% reduction of constitutive RAGE shedding, the release of soluble RAGE was only slightly influenced after the knock-down of ADAM17 and not at all after knock-down of MMP2 . PACAP-iBinding of a PACAP agonist to the PAC1 receptor leads to activation of several intracellular signaling cascades including the adenylate cyclase system, stimulation of phospholipase C and the MAP kinase pathway As the name PACAP implies, it activates the adenylate cyclase/protein kinase A (PKA) pathway. Therefore the effects of H89 (5 µM), a cell-permeable inhibitor of PKA, and KT5720 (1 µM), a more specific cell-permeable inhibitor of PKA, on the stimulation of RAGE shedding were tested. Both inhibitors had no effect on the PACAP-induced shedding of RAGE .We also analyzed the effect of direct adenylate cyclase/PKA activation on RAGE shedding. For this purpose RAGE expressing cells were treated with either Forskolin (50 µM), an activator of the adenylate cylcase, or 8-Bromoadenosine 3′, 5′-cyclic monophosphate , an activator of PKA. Both compounds did not activate the shedding of RAGE  supportiPAC1 receptor activation also leads to stimulation of phospholipase C (PLC) which produces the second messenger molecules DAG and IP3 (Inositol trisphosphate) 50 = 20 mM). Both PKC inhibitors reduced the PACAP-induced shedding of RAGE: Treatment with Gö6976 and Gö6983 (both 1 µM) resulted in an approximate 60% and 50% inhibition of PACAP-stimulated RAGE shedding  and then the shedding process was induced for 2 h with PACAP-27. Compared to mock-treated cells, 2-APB decreased the PACAP-induced release of soluble RAGE about 40% ; Gö6983 selectively inhibits the PKC isozymes PKCα, PKCβ, PKCγ, PKCδ and PKCζ but it does not block PKCµ  and PACAP-initiated stimulation of CaMKII has been described 2+ signaling, PKCα/PKCβI and CaMKII play substantial roles in the activation of metalloproteinase-mediated RAGE shedding.Besides PKC, Ca2+ appears to be a dominant regulator of induced RAGE shedding, we analyzed whether agonistic activation of our cell lines leads to elevated intracellular Ca2+ levels. Depending on the coexpressed GPCR, RAGE expressing cells were treated with either PACAP-27, AVP or OT (each 300 nM). In all RAGE/GPCR coexpressing cells agonist treatment induced a strong intracellular Ca2+ signal as measured via calcium sensor Fura-2-AM  By using various inhibitors we identified several signaling pathways contributing to RAGE shedding. Next we analyzed whether these signaling pathways are contributing in parallel to the shedding of RAGE. Combinatorial treatment of cells with MAP kinase inhibitor and either PKC or CamKII inhibitor did not result in an enhanced inhibition of PACAP-induced RAGE shedding . When coin vivo. For this purpose, mice were treated for 3 months intranasally with either PACAP-38 or mock administration solution Finally we were interested whether PACAP-stimulated shedding is also evident In this report we provide evidence that metalloproteinase-mediated shedding of the type I membrane protein RAGE is inducible by activation of G protein-coupled receptors which are expected to be linked to different main signal transduction pathways. We demonstrate that RAGE shedding can be induced via activation of the V2 vasopressin, the oxytocin and the PAC1 receptor. The shedding of RAGE was induced 2 to 5-fold after activation of examined GPCR. Observed differences provide no information about the efficiency of individual GPCR-activated shedding induction, because cell lines expressing different amounts of GPCR and RAGE were compared. Nevertheless, our results clearly demonstrate that the RAGE shedding machinery is inducible via activation of various GPCRs. For the detailed analysis of involved signaling pathways we focused on the multi-branched signaling network of the PAC1 receptor since this also covers the more restricted signaling routes of V2R and OTR.2+ signaling, PKCα/PKCβI, CaMKII, PI3 kinase and MAP kinases are involved in the shedding activation process. A contribution of adenylate cyclase/PKA signaling in PACAP-induced RAGE proteolysis was not evident in our cellular system. In V2R/RAGE cells we could also not detect an involvement of adenylate cyclase/PKA signaling in AVP-induced RAGE shedding. In all our cell lines expressing different GPCRs  we observed agonist-induced intracellular calcium signaling. Therefore we conclude that Ca2+ seems to be an important regulator of the RAGE shedding machinery.By a biotinylation experiment, we demonstrated that PACAP-induced RAGE shedding takes place at the cell surface where a substantial amount of plasma membrane-located RAGE is proteolytically cleaved after activation of PAC1 receptors. Similar results have been published for PMA-induced RAGE shedding in vivo. So far induced shedding of RAGE was demonstrated only for unphysiological stimulators such as the phorbolester PMA and the calcium ionophore A23187 Analysis of lungs isolated from PACAP-treated mice further showed that the shedding of RAGE is also inducible in vivo. Thus, application of PACAP may also have therapeutic potential for the treatment of AD or inflammatory diabetic conditions in humans.In connection with the development of Alzheimeŕs disease it is important to consider that at least two aspects of metalloproteinase-mediated shedding processes are essential: (i) cleavage of APP by ADAM10 prevents generation of neurotoxic Aβ peptides; (ii) cleavage of full-length RAGE by ADAM10 prevents binding of the RAGE ligand Aβ to cell-bound RAGE in vitro assays with recombinant proteinases, GI254023X discriminates between ADAM17 (IC50 = 541 nM) and ADAM10 (IC50 = 5.3 nM)/MMP9 (IC50 = 2.5 nM) 50 values appear to be much higher: at 30 µM GI254023X has been reported to reduced PMA-induced shedding by only 20% In cells coexpressing RAGE and the PAC1 receptor, the shedding of RAGE was inducible by various PAC1 receptor agonists. Induction of RAGE shedding was dose-dependent and saturable. A similar effect has been observed for PACAP-induced shedding of APP Our comparative analysis of PACAP-induced RAGE shedding versus phorbol ester-induced shedding revealed a similar kinetic in the examined cellular system. Shedding increased continuously during the first three hours after its induction and reached a maximum after about four hours. Similar results for PMA-induced shedding have been described Pituitary adenylate cyclase-activating polypeptide) implies adenylate cyclase and subsequently protein kinase A (PKA) as targets of PACAP receptor activation. However, in our study we did not observe any effects of either adenylate cyclase/PKA activation or inhibition on the shedding of RAGE. PACAP-induced APP processing was also identified to occur independently from the adenylate cyclase/PKA system 2+ signaling. Inhibition of intracellular calcium release as well as blockade of calcium-dependent PKC isoforms or calcium/calmodulin-dependent protein kinase II (CaMKII) significantly reduced the PACAP-induced RAGE shedding. The importance of Ca2+ signaling in the induction of RAGE shedding is supported by our finding that activation of the oxytocin receptor leads to elevated sRAGE levels; this receptor is known to be connected to Ca2+ signaling The name PACAP (A role for the phosphatidylinositol 3-kinase (PI3-kinase) pathway has been demonstrated for the anti-apoptotic effect of PACAP 2+ levels are sufficient for induction of ADAM-mediated shedding processes, the activation of RAGE ectodomain shedding should be achievable via stimulation of different receptor classes including GPCRs, RTKs and ligand-controlled Ca2+ channels.Given that increased CaIt is well documented that shedding processes can be induced by quite distinct stimuli. For example, the catalytic activity of ADAM17, but not that of ADAM10, can rapidly and reversibly be induced by signaling pathways stimulated by thrombin, EGF, lysophosphatidic acid and TNFα. Activation of ADAM17 was independent from its cytoplasmic tail but required the presence of the transmembrane domain and led to the exposure of the ADAM17 catalytic site Independent from the missing links of cell and sheddase activation, our presented data provide evidence that pharmacological stimulation of RAGE shedding may open alternative treatment strategies for AD or RAGE-mediated diabetic complications in the future.The following antibodies were used: anti human RAGE N-terminal antibody Mab5328 (Millipore) detecting membrane-bound RAGE and rabbit serum Ab3260 detecting secreted RAGE Secondary anti-rabbit and anti-mouse peroxidase-coupled antibodies and the ECL detection reagent were from Pierce . The anti-rat POD-coupled secondary antibody was from Merck (Darmstadt). All peptide hormones were from Bachem. All other chemicals were either from Merck Biosciences or Sigma-Aldrich.RAGE: The untagged wild-type human RAGE expression vector pcDNA6-RAGE was generated by using the human RAGE cDNA, including its own translation termination codon. The human RAGE cDNA was subcloned from clone IRALp962E1737Q2 into EcoRI/XbaI sites of pcDNA6/V5-HisB. PAC1: The rat PACAP type I receptor, which has 92% amino acid identity compared to the human receptor, was used in the experiments. The PAC1 coding region was fused at the 5′end to a myc epitope coding sequence and was combined at the 3′end to an oligonucleotide adaptor coding for a Rho epitope in a pcDNA3 vector. Expression plasmids for the C-terminal HA-tagged bovine V2 vasopressin and human oxytocin receptor carrying a N-terminal c-myc and FLAG epitopes and a C-terminal GFP-tag are described elsewhere HEK Flp-In™ 293 cells (human embryonic kidney cells) (Invitrogen) were transfected with pcDNA3-PAC1 expression plasmid using Lipofectamine 2000 (Invitrogen) and selected via G418 to generate a stable cell line. Then one cell clone expressing the PAC1 receptor was transfected with pcDNA6-RAGE expression plasmid using Lipofectamine 2000 and selected via Blasticidin.HEK 293 cells (ATCC) stably expressing the oxytocin receptor (OTR) HEK 293 cells (ATCC) stably expressing the bovine vasopressin receptor (V2R) were generated by us by transfection of HEK 293 cells using plasmid pcDNA3-V2R-HA. A V2 vasopressin receptor-expressing cell clone was isolated by using G418 selection. Subsequently, this cell clone was transfected with plasmid pcDNA6-RAGE and a RAGE/V2R coexpressing cell clone was isolated via G418 and Blasticidin selection.Stealth RNAi duplexes were purchased from Invitrogen, and transfections were performed according the manufacturer’s protocol. The RAGE cleavage assay was performed 48 h after transfection.2, 1,5 mM CaCl2, 10 mM glucose). Aliquots of the suspension were added to calcium buffer and transferred into a cuvette that was placed into a thermostatically controlled (37°C) holder. The agonists  were applied to the cells after 100 s, and the changes of the [Ca2+]i were monitored spectrofluorimetrically ).Cells were incubated with 1,5 µM fura 2-AM in DMEM for 30 min at 37°C. Afterwards they were detached by using PBS/0,5 mM EDTA and centrifuged at 100 g for 5 min. Pellets were washed and resuspended in calcium buffer  was added. Experiments in the presence of inhibitors were performed by preincubating cells with the inhibitor in secretion medium for 1 h at 37°C and then PACAP hormones, arginine-vasopressin or oxytocin were added. After the appropriate incubation time, cell culture supernatants were collected centrifuged for 10 min at 660 g and proteins were precipitated with 10% trichloroacetic acid at 4°C. Proteins were analyzed by Western blotting. For detection of cellular proteins, the adherent cells on the 6 well plates were washed with PBS, then dissolved in reducing Laemmli buffer and assayed by Western blot analysis. For comparative and quantitative analysis, effects observed with solvent-treated cells were used as control and were set to 100%. As a further control, the effect of the inhibitors on RAGE shedding was also analyzed in the absence of hormones.Adherent cells were washed twice with PBS and then incubated for protein biotinylation with PBS containing 0.1 mM Sulfo-NHS-LC-Biotin (Pierce Biotechnology) for 30 min at room temperature. After washing with Tris-buffered saline (pH 7.4) the cells were incubated in secretion medium for 2 hours. The cells and supernatants were collected separately, and the supernatants were adjusted to 0.1% SDS. The cells were dissolved in 5% SDS, and then SDS was diluted to 0.1% by adding PBS. Biotinylated proteins were captured with NeutrAvidin Biotin-binding agarose (Pierce) for 2 h at 4°C. After that, the agarose beads were washed three times with PBS/0.1% SDS. Proteins were eluted from the binding agarose with reducing Laemmli buffer and analyzed by Western blotting. Actin could not be detected in isolated biotinylated protein fractions, thus demonstrating the integrity of the cells during the course of the experiment. As a control, actin could be detected in the cell lysate .Proteins of the cell culture supernatant or cell lysates were separated by 10% SDS-PAGE and blotted to nitrocellulose membranes . Membranes were probed with the appropriate primary and secondary antibodies and the labeled proteins were detected by chemiluminescence using the VersaDoc system  and quantified via the AIDA 4.25 software .2). Finally the gels were stained with Coomassie Blue. Clear areas within the stained gel result from digestion of gelatin by the gelatinase activity of either MMP9 or MMP2.Cells were washed twice with serum free DMEM, then serum free DMEM was added to the cells. After 18 h of incubation at 37°C, supernatants were collected and cleared from cells by centrifugation . For gelatin zymography 30 µl of serum-free cell culture supernatant was mixed with 10 µl of 4× NuPAGE LDS Sample Buffer (Invitrogen), and subsequently proteins were separated under non-reducing conditions in 10% SDS-polyacrylamide gels containing 0.1% gelatin. After electrophoresis gels were washed in 2.5% Triton X-100 at room temperature for 1 h and then incubated for 16 h at 37°C in reaction buffer  was prepared as described Mouse lung samples were divided into pieces in liquid nitrogen and ice cold homogenization buffer  per 10 ml) in the fourfold volume of the lung weight was added. Samples were homogenized in a tissue lyser  and the suspension was centrifuged for 2 h , then the supernatant was transferred in a new tube. The pellets containing insoluble lung material were resuspended in the fourfold volume of homogenization buffer with the tissue lyser  and afterwards centrifuged for 15 min . Then the supernatant was discarded and the remaining pellet was suspended in a twofold volume of TBS (with proteinase inhibitor tablet Complete Mini) using the tissue lyser. This suspension was used for analysis of membrane proteins.The protein contents of the soluble and membrane protein fractions were determined by the Bradford method. Proteins were analyzed by Western Blotting.