Recent evidence suggests that tumor necrosis factor alpha (TNF) and angiotensin II (ANGII) induce oxidative stress contribute to cardiovascular disease progression. Here, we examined whether an interaction between TNF and ANGII contributes to altered cardiac mitochondrial biogenesis and ATP production to cause cardiac damage in rats. Rats received intraperitoneal injections of TNF (30 µg/kg), TNF + losartan , or vehicle for 5 days. Left ventricular (LV) function was measured using echocardiography. Rats were sacrificed and LV tissues removed for gene expression, electron paramagnetic resonance and mitochondrial assays. TNF administration significantly increased expression of the NADPH oxidase subunit, gp91phox, and the angiotensin type 1 receptor (AT-1R) and decreased eNOS in the LV of rats. Rats that received TNF only had increased production rates of superoxide, peroxynitrite and total reactive oxygen species (ROS) in the cytosol and increased production rates of superoxide and hydrogen peroxide in mitochondria. Decreased activities of mitochondrial complexes I, II, and III and mitochondrial genes were observed in rats given TNF. In addition, TNF administration also resulted in a decrease in fractional shortening and an increase in Tei index, suggesting diastolic dysfunction. TNF administration with concomitant LOS treatment attenuated mitochondrial damage, restored cardiac function, and decreased expression of AT1-R and NADPH oxidase subunits. Mitochondrial biogenesis and function is severely impaired by TNF as evidenced by downregulation of mitochondrial genes and increased free radical production, and may contribute to cardiac damage. These defects are independent of the downregulation of mitochondrial gene expression, suggesting novel mechanisms for mitochondrial dysfunction in rats given TNF. The renin-angiotensin system (RAS) plays an important role in the pathophysiology of cardiovascular disease. Over-activation of the local tissue RAS is one of the most common pathophysiological mechanisms contributing to the progression of cardiovascular diseases, and is also an important target for current pharmaceutical therapies. Approximately 50 million Americans suffer from hypertension; several etiological agents, including obesity and lifestyle changes, have been attributed to the progression of this disease and its effects on target organs. Hence, one of the main objectives in treating hypertension is end organ protection. Several Clinical trials using calcium channel blockers and RAS antagonists have been effective A growing body of evidence suggests that the main effector peptide of the RAS, angiotensin II (ANGII), induces inflammatory molecules and contributes to the pathophysiology of cardiovascular disease 2•−), hydrogen peroxide (H2O2), and peroxynitrite (OONO−). This overproduction of mitochondrial ROS contributes to cardiac dysfunction. In addition to ROS formation, reduced expression of mitochondrial genes and proteins can contribute to mitochondrial dysfunction.Cardiac function is highly dependent on mitochondrial ATP supply; hence, the heart is especially susceptible to the disruption of mitochondrial energy production. Mitochondrial reactive oxygen species (ROS) are involved in cell signaling, but are also potential mediators of oxidative stress. Oxidative stress mediated by activation of the ANGII type-1 receptor (AT-1R) plays a crucial role in the progression of cardiovascular diseases The objective of the study was to determine whether increased ROS production, induced by TNF and ANGII, which is associated with ATP deficit, could directly cause mitochondrial oxidative damage, leading to depletion/reduction of mitochondrial genes and proteins, dysfunction of the mitochondrial electron transport system, and consequently, cardiac damage. Results from this study will help us elucidate the role played by TNF in inducing oxidative stress by examining the superoxide producing machinery in the heart, and its contribution to cardiac dysfunction. In this context, the purpose of this study was to investigate whether an AT-1R blocker (losartan) could attenuate the functional and structural changes that occur in cardiac mitochondria upon TNF induction. Identifying the links between TNF, ANGII, and oxidative stress at the mitochondrial level in contributing to cardiac damage may lead to a better understanding of the progression of cardiovascular disease and, ultimately, lead to new and effective treatment strategies.All experimental procedures were in compliance with all applicable principles set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals . This study was approved by the Institutional Animal Care and Use Committee of the Louisiana State University School of Veterinary Medicine .ad libitum. Rats were divided into 3 groups. One group of rats received TNF at a dose of 30 µg/kg, ip, the second group received normal saline vehicle, and group 3 received TNF + losartan , for 5 days. Rats were sacrificed by carbon dioxide inhalation, and left ventricle (LV) samples were collected for gene expression and measurement of oxidative stress markers. Mitochondria were isolated by differential centrifugation for functional studies. Electron paramagnetic resonance (EPR) spectroscopy was used to measure free radical production in the cytosolic and mitochondrial fractions. The structural integrity of mitochondrial membranes was measured using swelling assay and transmission electron microscopy (TEM) analysis.Studies were conducted using adult male Sprague–Dawley rats  obtained from Harlan . Animals were housed in temperature-(23±2°C) and light-controlled (12 h light/dark cycle) animal quarters; standard rat chow and water were provided Blood pressure were measured noninvasively using a Coda 6 Blood Pressure System , which utilizes a tail-cuff occlusion method and volume pressure recording (VPR) sensor technology. In this system, unanesthtized rats from each group were warmed to an ambient temperature of 30°C by placing them in a holding device mounted on a thermostatically controlled warming plate. Rats were allowed to habituate to this procedure for 3 days prior to each experiment. Blood pressure values were averaged from at least six consecutive cycles per day obtained from each rat.Cardiac function was measured as described previously 2•−, 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) was used for measurement of tissue peroxynitrite (OONO−); and 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethyl-piperidine (PPH) was used for mitochondrial O2•− and hydrogen peroxide (H2O2) studies 2•− measurement; or CPH (500 µM) for 30 min for OONO− measurement. Aliquots of incubated probe media were then taken in 50-µl disposable glass capillary tubes (Noxygen Science Transfer and Diagnostics) for determination of LV ROS, O2•−, or OONO− production. All EPR measurements were performed using an EMX ESR eScan BenchTop spectrometer and super-high quality factor microwave cavity .Total LV ROS and superoxide production rates were measured using EPR as described previously Total RNA extraction, cDNA synthesis, and real-time RT-PCR were performed as previously described LV mitochondria were isolated by differential centrifugation of heart homogenates as described previously Protein expression in mitochondria was analyzed by western blotting as previously described 2•− and H2O2 production in mitochondria were measured using EPR as described previously 2•− production. Catalase (50 U/ml) was added to measure H2O2 formation. PPH allows the detection of extracellular and extra mitochondrial production of O2•−2•− to produce a stable PP• nitroxide radical which can be detected with EPR 2•− production and H2O2 production were determined by EPR under the same settings as were used for measurement of mitochondrial O2•− and H2O2 production.Mitochondrial O2) containing 1 mM EGTA. Then, the oxygen spin label NOX-13.1-OS (5 µM), CMH (200 µM), and one of the following substrates were added to the mitochondrial suspension: 20 mM glutamate (complex I), 5 mM succinate (complex II), or 5 mM pyruvate (complex III). After adequate mixing, the samples were taken into capillary tube and mitochondrial complex I, II and III were measured using EPR. Activity of each mitochondrial complex was quantified by EPR under the same settings described previously Measurements of mitochondrial complex I, II and III enzymatic activities were performed as previously described ATP production rates and ADP/ATP ratios were quantified in isolated mitochondria using a commercially available kit  as described previously . Data were analyzed by ANOVA, followed by Bonferroni’s multiple comparison tests. In all cases, p<0.05 was considered statistically significant.All data are expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism version 5.00 for WindowsBlood pressure measurements were obtained for all experimental animals during the 5 day treatment period. Administration of TNF with or without LOS did not have any effect on blood pressure parameters .When compared to control animals, TNF treated rats had progressive increases in LV end-diastolic dimension (LVD), LV end-systolic dimension (LVS) and TEI index and decreases in fractional shortening (FS %) measurements . Additio2•− and OONO− production rates in LV tissue, as determined by EPR, were all significantly higher in LV tissues of TNF treated rats than in control and TNF+LOS groups . This assertion is supported by several observations. Increased superoxide production, induced by TNF interacting with ANGII, contributes to mitochondrial dysfunction by depleting mitochondrial genes and proteins and decreasing respiratory complex activity, thereby influencing ATP synthesis and ultimately resulting in cardiac dysfunction. Administration of the ANGII type 1 receptor (AT-1R) antagonist losartan (LOS), to TNF-treated animals attenuates TNF-induced oxidative stress by modulating free radical production and increasing mitochondrial gene expression, which leads to a normalization of both mitochondrial complex activity and ATP synthesis, and thereby prevents cardiac dysfunction.In our study, TNF treatment resulted in significant damage to the mitochondrial membrane and was accompanied by increased mitochondrial OOur echocardiographic findings suggest that TNF decreases FS% and increases Tei index, both of which are indicative of diastolic dysfunction. We also observed increases in left ventricular diastolic (LVD) and systolic (LVS) dimensions, which indicate decreased left ventricular contractile function. Treatment with LOS improved left ventricular contractile function in our study; this could be due to reductions in cytokines and oxidative stress and possible increases in mitochondrial biogenesis gene expression and respiratory chain function. This is supported by our observation that LOS treatment decreased free radical generation both in the cytosol and mitochondria. Mitochondrial dysfunction may play an important pathogenic role in the progression of cardiac dysfunction. In the present study, we report markedly reduced LV tissue levels of specific mitochondrial enzyme activities for respiratory complexes I, II and III, enzymes critical to the generation of ATP. Furthermore, the reduced myocardial complex I,II and complex III levels correlated with the increased LV tissue TNF-α protein levels, suggesting an association between increased TNF-α and mitochondrial dysfunction. In the diabetic rat heart, mitochondrial dysfunction (decreased ATP synthesis rate and decreased State 3 respiration) was found to accompany diastolic dysfunction The roles of TNF and ANGII as potent inducers of oxidative stress in a number of cell types, including cardiac myocytes, are well established Here, we report that AT-1R activation plays an important role in peroxynitrite production, and that AT-1R blockade reduces peroxynitrite production. Although LOS produces its beneficial effects via inhibition of AT-1R, the exact molecular mechanism contributing to these effects is still unclear. We have shown that TNF markedly decreased both endothelial nitric oxide synthase (eNOS) expression and mitochondrial biogenesis in TNF-treated rats. Thus, our findings are in agreement with those of earlier publications demonstrating that ANGII regulates NOS expression TNF administration also upregulated the expression of inducible NOS (iNOS). This is probably because the iNOS gene is regulated by the transcription factor, NF-κB, which is known to respond to as well as induce, TNF production Increased iNOS expression and peroxynitrite production formation  have been observed in rats after TNF treatment. Higher NO production via iNOS has been shown to be associated with the pathogenesis of cardiac dysfunction and heart failure. We did detect an increase in iNOS mRNA and peroxynitrite formation in the hearts of rats treated with TNF, and these increases in iNOS expression, ROS production, superoxide generation, and peroxynitrite formation were accompanied by a marked loss of mitochondrial genes, whereas these changes were attenuated in TNF +losartan treatment with preservation of cardiac function. In the present study, we have also shown that TNF- α markedly decreased both eNOS expression and mitochondrial biogenesis in LV of TNF treated rats. Downregulation of eNOS seems to be the major molecular mechanism by which TNF-α affects mitochondrial biogenesis in these models; its effects on mitochondria can be reversed by treatment with losartan. These results suggest that TNF-α plays a relevant role in decreasing eNOS expression and mitochondrial biogenesis in metabolically active LV tissues of TNF treated animals.Mitochondrial permeability transition pores (MPTP) are multiprotein complexes consisting of the adenine nucleotide translocase (ANT), voltage dependent anion channel (VDAC), cytochrome C, cyclophilin D, and several other ancillary proteins. The exact components that constitute the MPTP remain a subject of debate, although it is generally accepted that ANT and cytochrome C are major pore components Cardiac function is an energy driven process. In this study, we measured mitochondrial genes in the LV in TNF treated rats using real time RT-PCR. PGC1α is a coactivator of nuclear transcription factors, including PPARγ, PPARα, and nuclear respiratory factor 1 (NRF-1) and these genes are known to enhance mitochondrial activity. PGC-1α is abundantly expressed in the heart, and is known to 1) activate most genes of mitochondrial function and biogenesis, and 2) stimulate both fatty acid oxidation and oxidative respiration in cardiac tissue


In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4+ and CD8+ T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process  VP2 also represents the major determinant of host range and virus-host interactions. The virion contains a single-strand DNA genome of approximately 5 kb and is encapsulated by a non-enveloped icosahedral particle Canine parvovirus (CPV), which belongs to the genus CPV is a highly contagious infectious virus of dogs and can spread to all tissues through the blood. The major consequences of CPV infection are fatal myocarditis in 2–3 week old pups and hemorrhagic enteritis in vitro, and the immunogenicity of the particles in mice and dogs were investigated.Virus-like particles (VLPs) have been generated and used as vaccine candidates for a variety of viruses due to their ability to induce humoral and cellular immune responses Esherichia coli DH10Bac/BmNPV (Bombyx mori nuclear polyhydrosis virus) and BmN cells were provided by Jiangshu University of Science and Technology, China. BmN cells, originating from insect ovaries, were cultured in TC-100 insect cell culture medium  with the addition of 10% fetal bovine serum  at 27°C.Silkworms (JY1) were supplied by the Sericultural Research Institute, Chinese Academy of Agricultural Science and were reared on mulberry leaves under standard conditions at 27°C BamHI and HindIII sites of the pFastBac1 to generate a recombinant transfer plasmid pFastBac1-VP2. Then pFastBac1-VP2 was transformed into E. coli DH10Bac/BmNPV competent cells. The E. coli DH10Bac cells containing the recombinant bacmid were propagated in Luria Bertani medium containing 50 µg/ml kanamycin, 7 µg/ml gentamicin, 10 µg/ml tetracycline, 100 µg/ml X-gal and 40 µg/ml IPTG . The recombinant bacmids were extracted and characterized by PCR. BmN cells at 1×106 cells/well were transfected with 2∼3 µg recombinant bacmid DNA using lipofectamine 2000 transfection reagent  according to the manufacturer's instructions. The supernatant containing recombinant baculovirus was harvested from the BmN cells 96 h post-transfection. Titers of the baculovirus were determined by a plaque assay.The VP2 gene of CPV was amplified and cloned into the 5 pfu of the recombinant baculovirus were injected into silkworm pupae with a needle (26 gauge) and syringe. The infected pupae were collected on day 5, ground in bicarbonate buffer  and centrifuged at 9000×g for 15 min at 4°C. The supernatants were collected and stored at −20°C for protein expression analysis (Western blot) and VLP purification by ultracentrifugation.About 4×10VP2 protein expression was confirmed by Western blot. The supernatants of samples, which were purified as described above, were mixed with loading buffer and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene fluoride membranes (Invitrogen) and blocked with 1% BSA (Invitrogen) for 2 h. After washing in PBST (PBS plus 0.05% Tween-20) the membranes were incubated with a 1∶300 diluted monoclonal mouse anti-CPV antibody (produced in-house) for 2 h at 3°C. The membranes were washed again, and following a 2 h incubation with a horseradish peroxidase-labeled goat IgG anti-mouse antibody  the membranes were visualized using DAB  solution .6 cells/ml) were grown at 27°C in TC-100 medium supplemented with 10% FBS and inoculated with the recombinant baculovirus Bac-VP2 (1×105 pfu). At 2 days post infection, the 6 well culture plates were fixed with cold acetone at −20°C for 2 h, washed with PBST, and then incubated with an in-house mouse anti-CPV monoclonal antibody (diluted 1∶150 in PBST) and Evans Blue (diluted 1∶500 in PBST) at 37°C for 2 h. After washing, the plates were stained with FITC-labeled goat anti-mouse IgG (1∶150 diluted in PBST) at 37°C for 2 h. Following three more washes, the plates were examined under a fluorescent microscope.BmN cells , and then subjected to sucrose density gradient centrifugation at 35,000 rpm (210053× g) in an SW41 Ti rotor (Beckman) for 2.5 h at 4°C. The gradients were fractionated, and fractions that contained VLPs (confirmed by Western blot) were pooled and centrifuged for 2 h at 41,000 rpm  in an SW41 Ti rotor to remove the sucrose. The precipitate was resuspended in PBS, and VLPs were visualized by negative-stain electron microscopy.Purification of CPV-VLPs. The supernatants of samples described above (see 2.3) were precipitated by a 30% saturated solution of ammonium sulfate (pH 7.0). The protein pellet was recovered by centrifugation at 10,000× Silkworm pupae were harvested at 5 days post-infection and homogenized by grinding in 4 ml of phosphate-buffered saline  on ice for 5 min per gram of silkworm pupae. Homogenates were immediately titered by the hemagglutination (HA) test to calculate the hemagglutination unit (HAU) they contained. The raw homogenates were administrated to dogs via the oral route.4Cl). Single cell suspensions were then prepared to concentrations of 106 cells/ml in PBS containing 2% FBS and 0.1% NaN3. The cells were stained with antibodies (BD Bioscience) against CD3, CD4 and CD8 for 30 min at 4°C. Single or double staining was performed on ice. Data on CD4 and CD8 proliferation were acquired with a LSR-II flow cytometer (BD Bioscience) and analyzed using FlowJo software (Tree Star).BALB/c mice (n = 9) were inoculated intramuscularly with 50 µg of purified VLPs. A control group of nine mice were immunized with 50 µg of total protein isolated from silkworm pupae that were not infected with the recombinant baculovirus. Mice (n = 3) were sacrificed and their spleens and lymph nodes were collected at days 3, 6 and 9. Red blood cells were lysed using an ammonium chloride lysis solution (150 mM NH506 cells/ml) was added and incubated for 4 or 5 days in order to assess the cytopathic effect (CPE). The highest dilution of sera showing complete inhibition of CPE was taken as the neutralization titer.Mice (n = 6) were immunized intramuscularly with 70 µg of CPV-VLPs and boosted again with the same dose two weeks later. Three mice were bled at random on the second, fourth, sixth and eighth week post immunization. Neutralizing antibody titers were determined for CPV. The mouse sera were inactivated at 56°C for 30 min, and then diluted serially (2-fold) and mixed with equal volumes of CPV virus containing 100 TCID4 HAU of VLPs. The other group (n = 3) were treated as negative controls and were orally administrated 2 ml of raw homogenates of normal silkworm pupae. Two weeks later, dogs were boosted with the same doses and via the same route. Blood samples were collected from the neck vein pre-immunization and weekly up to 10 weeks post immunization.In total six dogs were randomly separated into two groups, one group (n = 3) were orally administrated with 2 ml of raw homogenates containing 4×10Serum samples were inactivated at 56°C for 30 min, then serially diluted two-fold (25 µl of serum) in phosphate-buffered saline  in V-96-well plates. Subsequently 25 µl of 8 HAU of CPV were added. The samples were then incubated at 37°C for 1 h after which 50 µl of 1% (v/v) pig erythrocytes were added and the plates were incubated at 4°C for 1 h. Titers were expressed as the reciprocal of the highest serum dilution that completely inhibited hemagglutination. Geometric means were also calculated.HA tests were carried out at room temperature using 1% (v/v) of pig erythrocytes containing 0.1% (w/v) of BSA. Serially two-fold dilutions using 25 µl of homogenates were made in PBS  in V-96-well plates. Then 25 µl of PBS was added to each well for 1 h at 37°C. Subsequently 50 µl of 1% (v/v) pig erythrocytes were added and the plates were incubated at 4°C for 1 h. The HA titer was determined as the highest dilution of homogenates showing hemagglutination.In total six dogs age between 12 and 15 weeks were used in this study. They were supplied by commercial dog farm named Kylin and housed in animal facilities with free access to water and food. No CPV vaccine had been administrated before.All housing conditions and experimental procedures (mice and dogs) were approved by Animal Care and Use Committee of the Chinese People's Liberation Army (No:SYXK2009-045) and were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain.The expression of VP2 in BmN cells was confirmed by immunofluorescence using a mouse anti-CPV monoclonal antibody and FITC-labeled goat anti-mouse IgG at two days post-infection with the recombinant baculovirus . The sch9. In contrast the normal silkworm pupae homogenates did not show hemagglutination.To investigate whether parvovirus-like particles could be formed correctly, the infected samples of BmN cells and pupae were analyzed by electron microscopy. The micrographs clearly showed that the VP2 protein expressed in the cells and pupae self-assembled into parvovirus-like particles with diameters of about 25 nm . VLPs we+ T cells proliferated in spleens and lymph nodes at 3 days post immunization compared to the mock infected group , this system can express homologous and heterologous proteins in the silkworm pupae at high levels. The products of VP2 self-assemble into parvovirus-like particles in both insect cells and silkworm pupae. The particles were no distinguishable from wild-type virus in size or morphology, but do completely lack the DNA or RNA genome of the virus. The VLPs also showed hemagglutinating activity when incubated with piglet erythrocytes which is seen for CPV. Taken together, these data indicate that the VLPs produced in the silkworm pupae were similar to the wild-type CPV virion and retained their receptor binding characteristics.+ and CD8+ T cell responses were quantitatively analyzed by flow cytometry. Our results showed that VLPs induced higher CD4+ and CD8+ T cell responses in mice. The immunogenic ability of VLPs is based on their morphological properties (e.g. size between 20–100 nm and shape), allowing them to be taken up by antigen presenting cells for processing, presentation and promotion of DCs maturation and migration, which are essential for the stimulation of the host's innate immune responses The immunogenicity of VLPs has been well documented by previous studies. For example Ebola virus VLPs were shown to activate mouse bone marrow derived dendritic cells (DCs) as measured by the increased surface expression of CD40, CD80, CD86 and MHC class I and II, as well as the increased secretion of IL-6, IL-10, MIP-1α and TNF-α VLPs are also able to stimulate B-cell-mediated immune responses. A previous study reported that a single immunization with 0.7 µg of porcine parvovirus (PPV) VLPs yielded complete protection in target animals against infectious PPV isolates 4 HAU VLPs without adjuvant via the oral route, all dogs developed a system immune response against the VLPs which demonstrated that the VLPs are immunogenic when presented to the gut mucosal immune system. By week four following the first immunization, the serum HI titer was >1∶80 which was sufficient to protect all the immunized dogs from CPV infection. Even without an adjuvant, the VLPs elicited a strong humoral immune response that might be explained by the fact that the VLPs are of a size that can be taken up by DCs and so may possess a self-adjuvanting characteristic. For oral vaccination, gastric acid and enzymatic digestion are major concerns, since they may interfere with vaccine absorption. Our study demonstrated that CPV-VLPs can withstand gastric acid and remain immunogenic.Analogously, after two doses of 4×10With regard to the use of VLPs as oral vaccine, this strategy does not require a protein purification process. The silkworm pupae homogenates were safe when used in dogs and the baculovirus does not need to be inactivated. Although the VLPs are immunogenic, the appropriate doses of VLPs and the level of IgA at mucosal secretions  as well as the cytokines should be investigated further. The immune response might be improved if the VLPs possessed a specifically tailored epitope that could target DCs to enhance their ability of antigen processing. In short, to make these VLPs into a feasible and practical oral vaccine, additional research is required.In summary, VLPs represent a new type of subunit vaccine that are able to stimulate efficient cellular and humoral immune responses against viruses. Our study describes the high immunogenicity of parvovirus-like particles produced in silkworm pupae using a Bac-to-Bac system. These results suggest that, following further studies, CPV-VLPs might be a safe, convenient and effective vaccine for preventing diseases associated with CPV. The use of a baculovirus expression vector system to produce VLPs is attractive. Advantages of this system are low-cost, convenience, and the capacity to produce large and multiple proteins in addition to possessing co-translational and post-translational modifications present in mammalian cell-based systems, including protein glycosylation and phosphorylation and processing


Rheumatoid arthritis (RA) is an inflammatory disease of the joint characterized by chronic synovitis causing pain, swelling and loss of function due to destruction of cartilage and bone. The complex series of pathological events occurring in RA is largely regulated via excessive production of pro-inflammatory cytokines, the most prominent being tumor necrosis factor (TNF). The objective of this work was to elucidate possible involvement of group IVA cytosolic phospholipase A2 (cPLA2α) in TNF-induced regulation of synovitis and joint destructive effectors in RA, to evaluate the potential of cPLA2α as a future therapeutic target.The involvement of cPLA2α in tumor necrosis factor (TNF)-induced intracellular signaling cascades in synoviocytes  was analyzed by arachidonic acid (AA) release assay, synoviocyte enzyme activity assay, gene expression analysis by real-time PCR and ELISA immunoassay for the detection of prostaglandin E2 (PGE2), interleukin 8 (IL8) and stromelysin-1 (MMP3), respectively. Inhibitors of cPLA2α enzyme activity  significantly reduced TNF-induced cellular release of AA, PGE2, IL8 and MMP3. This reduction was evident both at transcriptional, protein or metabolite levels. Interestingly, cPLA2α inhibition affected several key points of the arachidonyl cascade; AA-release, cyclooxygenase-2 (COX2) expression and PGE2 production. Furthermore, the results suggest that cPLA2α is subject to transcriptional auto-regulation as inhibition of cPLA2α resulted in reduced PLA2G4A gene expression in TNF-stimulated synoviocytes. cPLA2α appears to be an important regulator of central effectors of inflammation and joint destruction, namely MMP3, IL8, COX2, and PGE2. Decreased transcription of the PLA2G4A and COX2 genes in response to cPLA2α enzyme inhibition further suggest a self-reinforcing effect of cPLA2α inhibition in response to TNF. Collectively, these results support that cPLA2α is an attractive therapeutic target candidate as its inhibition reduces the production of multiple key pro-inflammatory factors involved in RA pathogenesis.  Rheumatoid arthritis (RA) is an auto-immune and systemic inflammatory disease affecting 0.5-1% of the population, worldwide. In RA, chronic synovitis causes pain, swelling and loss of joint function due to degradation of cartilage and bone erosion . A key msn-2 ester bond of membrane glycerophospholipids. The arachidonyl specific group IVA cytosolic PLA2 enzyme (cPLA2α) encoded by the PLA2G4A gene is a major contributor to the elevated levels of AA in inflammation -arachidonic acid ([3H]-AA), and liquid scintillation cocktail Ultima Gold were from NEN Perkin Elmer . Leupeptin and pepstatin were from Roche Molecular Biochemicals . M-MLV reverse transcriptase, dNTPs and DTT were from Invitrogen . Random hexamer primers and RNAsin were from Promega . DNAse- and RNAse-free water was from VWR . RNeasy® minikit was from Qiagen , ELISA kits for PGE2, IL8 and MMP3 were from Cayman Chemicals , Bender Medsystems  and RayBiotech , respectively. AVX002 was provided by Avexxin AS  and synthesized by Synthetica AS . All other reagents were from Sigma-Aldrich .Recombinant human TNF was from R&D systems . Arachidonyl trifluoromethyl ketone  was from Enzo Life Sciences . PBS was from Oxoid . -AA (0.4 μCi/mL) in serum-free DMEM before experimental treatment. [3H]-AA release was analyzed in triplicates as previously described [3H]-AA in supernatants relative to total [3H]-AA incorporated into the cells. IC50 values for inhibitors were calculated as mean ± SD of at least 3 independent experiments.Cells were labeled for 18 hrs with [escribed . The resSW982 synoviocytes were serum starved over-night before stimulation with TNF . Cells were lysed and 200 μg of total protein was analyzed for cPLA2α activity as described –27. Brom5´-GACATACTCCAAACCTTTCCAC-3´ and 3´-CTTCTCCACAACCCTCTGC-5´, MMP3, 5´-TGATGAACAATGGACAAAGGATAC -3´ and 3´-CTGTGAGTGAGTGATAGAGTGG-5´, COX2, 5´-GGGGATCAGGGATGAACTTT -3´ and 3´-TGGCTACAAAAGCTGGGAAG -5´, PLA2G4A, 5´-CATGCCCAGACCTACGATTT -3´ and 3´-CCCAATATGGCTACCACAGG -5´, GAPDH, 5´-CATCAAGAAGGTGGTGAAGCAG-3´ and 3´-TGTAGCCAAATTCGTTGTCATACC-5´. Cq values for each amplification curve were calculated by the Mx3000P software (Stratagene). Fold changes in mRNA expression and statistical analysis were calculated by the REST 2009 software [Total RNA was isolated using RNeasy® minikit (Qiagen) according to kit protocol. RNA concentrations and integrity was monitored by NanoDrop spectrophotometric measurement  and total RNA (1 µg) was reverse transcribed as described in . Specifisoftware  with measoftware  using GAELISA analyses were performed according to their respective kit protocols. The read-out for all ELISAs was carried out with a Multiscan plate reader (Ascent Labsystems). The corresponding Ascent software for Multiscan, Version 2.4.1 was used to obtain the data. Mean estimated IC50 value for PGE2 production was calculated from 3 independent experiments.For AA release and ELISA analysis, statistical analyses were performed in SPSS Statistics 20 using one-way ANOVA at 95% confidence level in conjunction with Tukey HSD test. For real-time PCR data, statistical analysis was performed by the REST 2009 software . DiffereMMP3, IL8 and PGE2 are important regulators of inflammation and joint destruction in RA ,30,31. M As we aimed to investigate involvement of cPLA2α in regulating TNF-induced expression of these metabolites, we further characterized the PLA2G4A gene expression in synoviocytes. We found the PLA2G4A transcript to be expressed in untreated cells, and further induced 4.8 ± 1.1-fold by TNF , an induction that was significantly reduced by 50% following AVX002 treatment (p ≤ 0.03, 14C-AA from phospatidylcholine visualized by TLC chromatography (To validate the inhibitory effect of AVX002 on cPLA2α in our model system, we investigated the potency of AVX002 to restrain cPLA2α enzyme activity in cell lysates from untreated and TNF-stimulated cells. Lysates of TNF-stimulated cells displayed an 80% increase in activity of cPLA2α compared to untreated cells as indicated by increased hydrolysis and release of tography . This sttography . Furthertography . The capTo evaluate a potential role for cPLA2α in regulating destruction of cartilage in RA, we analyzed the effects of AVX002 on MMP3 expression. Indeed, AVX002 dose-dependently reduced TNF-induced MMP3 transcription with a maximum inhibition of 69% at 5 μM (p ≤ 0.01, Next, the effect of AVX002 in regulating expression of the known neutrophil attractant IL8 was investigated by QPCR and ELISA immunoassay. AVX002 dose-dependently reduced TNF-induced up-regulation of IL8 transcription, with a maximum inhibition of 63% at 5 μM (p ≤ 0.03, In RA, chronic inflammation and joint destruction is driven by excessive production of pro-inflammatory cytokines, chemokines and eicosanoids. In this study, by applying the chemical cPLA2α inhibitors AVX002 and ATK, we demonstrate that cPLA2α may be an important effector of TNF in intracellular signaling directly related to synovitis. in vitro [Our results demonstrate that AVX002 efficiently and persistently reduces AA-release and hence the availability of substrate for pro-inflammatory eicosanoid production in synoviocytes . We havein vitro  and thatin vitro –41. BaseThe important role of PGE2 in propagating inflammation and pain is commonly recognized as reflected by the effective symptom relief of pain and stiffness by NSAID treatment in RA patients ,13. The As degradation of cartilage and bone are major hallmarks of RA, disruption of these destructive processes represents a central therapeutic objective. MMP3, with its wide range of substrate specificity and ability to activate other MMPs, is essential in RA cartilage degradation  and corrGiven the complexity of TNF signaling networks, focusing on an intracellular therapeutic target downstream the TNF receptor may show reduced adverse effects compared to TNF-blocking therapy as many key host defense mechanisms are not targeted. Accordingly, a modulation of cPLA2α enzyme activity by specific cPLA2α inhibitors and subsequently normalizing downstream signaling may represent an alternative or supplement to current therapeutic strategies for RA treatment. Indeed, cPLA2α is expressed in RA synovium , and hasTaken together, we demonstrate that cPLA2α may have an important role in regulating TNF-induced intracellular signaling in synoviocytes. Hence, our results suggest that cPLA2α may be involved in both inflammatory, angiogenic and tissue destructive processes and may hence be a promising therapeutic target to reduce inflammation and discomfort, pain, reduced functionality and mobility associated with RA.


Objective. Discuss the changes and roles of PAF in the reproductive tract infection by observing the expression of platelet activating factor (PAF) and tumor necrosis factor α (TNF-α) in seminal plasma of patients with leukocytospermia. Methods. The seminal plasma was obtained from 22 cases of leukocytospermia and 15 cases of normal males; the peroxidase dyeing method was adopted for seminal plasma white blood count; the ELISA was adopted to test PAF and TNF-α concentration in seminal plasma. Result. PAF concentration (2.14 ± 0.43 ng/mL) of leukocytospermia group was significantly lower than the normal group  while TNF-α (5.51 ± 1.46 ng/mL) was significantly higher than that of normal group (3.48 ± 1.08 ng/mL). There was negative correlation between PAF and TNF-α , ; the same situation existed in PAF and WBC ; but TNF-α was positively correlated to WBC . Conclusion. (1) Low expression of PAF and high expression of TNF-α in leukocytospermia affect the sperm motility, which is one of the reasons that leads to infertility. (2) Lower expression of PAF has its particularity during the reproductive tract infection. An increase of white blood cells in seminal plasma can lead to the decrease of sperm quality and the increasing number of inflammatory cytokines such as interleukin IL-6 is the main factor involved in the process . AccordiMale patients age from 23 to 40 years old, who came to the Urology Surgical Department of our hospital from April to September 2011, were enrolled. According to WHO directives, 22 cases with leukocytospermia and 15 normal males were chosen. All the patients did not have any urinary, reproductive, blood, or endocrine diseases. The patients who have ever suffered from mumps, cryptorchidism and sexual dysfunction and ever accepted radiotherapy or chemotherapy were excluded. All the indexes were in the normal range according to seminal plasma regular analysis. The study was admitted by the local ethical committee, and written informed consent was obtained from all participants.Volunteers should accept abstinence for seven days firstly. Then, hands and penis should be washed with soap before the specimen collection. The semen should be obtained in one time by using masturbation method and be placed into disposable sterile dry plastic container with cover. The container was put into a water-bath box in 37°C constant temperature. After the full liquefaction of semen plasma, the computer-assisted semen analysis (CASA) carried out a routine analysis. The rest of the seminal plasma was collected and accepted centrifugal test for 10 minutes at 4000 r/min, the upper layer of seminal plasma was placed into Eppendorf tube and stored at −20°C for reservation. Density count of seminal plasma white blood cell in seminal plasma refer to peroxidase dyeing method, recommended by the WHO.α) enzyme league immune kit were bought from Jiahui Biotechnology Company (RD import repacking), Beijing Weili WJY9000 sperm analyzer, low temperature centrifuge , 37°C constant temperature water-bath box, full-wavelength enzyme standard instrument (Finland Leibo Company). Human platelet activation factor (PAF) enzyme league immune kit and human tumor necrosis factor  statistical software was used for processing the data. Normal characteristic test and homogeneity test of variance would be carried out before intragroup comparison of PAF and TNF-P < 0.01, while the difference in sperm density and liquefaction time was not obvious, P > 0.01 . WBC count in leukocytospermia group was (1.33 ± 0.18) × 106/mL, and TNF-α concentration was (5.51 ± 1.46 ng/mL), which was significantly higher than that of normal group WBC (0.43 ± 0.19) × 106/mL and TNF-α (3.48 ± 1.08 ng/mL), and the difference was statistically significant (P < 0.01) (PAF concentration in leukocytospermia group was (2.14 ± 0.43 ng/mL), which was significantly lower than the normal group (6.21 ± 1.38 ng/mL); the difference was statistically significant ( < 0.01) . r = 0.452, P < 0.01) and sperm vigor . In this group, PAF has no significant correlation with sperm density and liquefaction time. And what's more, it has positive correlation with sperm survival rate (α has no significant correlation with sperm density and liquefaction time. (P > 0.01). While it has negative correlation with sperm survival rate  and sperm vigor  (TNF- < 0.01) .r = −0.62,  P < 0.01) ; PAF concentration had significantly negative correlation to TNF-α   , however < 0.01) .9/L, and it is mainly caused by the reproductive tract infection. The white blood cells in seminal plasma can produce many inflammatory cell factors. With the affection of these cell factors, the sperm quality, density and its vigor get lower; sperm capacitation and acrosomal reaction would be affected as well, which lead to male infertility.The reproductive tract infection is one of the common causes of clinical infertility. Leukocytospermia refers to the increase of white blood cell (WBC) density in seminal plasma which is more than 1 × 10−10 and 1 × 10−13 mol/L, the sperm's motility is prominently increased after 120 min culture time [−9 mol/L) can induce AR better [−3 M with 120 min of cultivating time, at the same time, ATP concentration improves obviously as well. We also found that the mitochondrial function of the sperm through PAF processing remains unchanged, and the integrity of sperm has not been damaged either [PAF is a kind of cell factors found in recent years, and it is involved in various physiological and pathological processes. PAF has close relationship with procreation, fetal development, and delivery . Sperm mure time ; PAF conure time , therebyure time . AR% is R better . The semd either . At the α) is a kind of important cell factor involved in the inflammation reaction progress. It is mainly produced by macrophages, and widely involved in inflammation response, immune response, endotoxin sex shock, and other pathologic process. At the same time, TNF-α is a kind of cell factor, which has close relations with sterility occurrence and development. A certain amount of TNF-α can significantly inhibit the activity of sperm acrosomal enzyme and acrosomal reaction [α has certain influence on sperm mitochondrial function, it can interfere sperm energy metabolism and lower sperm movement ability. It may also promote the sperm apoptosis through the mitochondria apoptosis way, which causes infertility [α also can reduce the amount of nitrate in normal sperm and affects NO synthesis, which is necessary for sperm movement [α can mutually cooperate with each other to promote the progress of inflammatory response [α? The mechanism is still unclear.Alpha subtype of tumor necrosis factor . The existing research results showed that PAF is favorable for sperm function activities, while TNF-α could inhibit sperm function activities. So we believe that, under condition of reproductive tract infection, the organism increase the composition of cell factors which can inhibit sperm activity on one hand, such as TNF-α, and decrease the composition of cell factors which can promote sperm on the other hand, such as PAF, thereby affect sperm vigor. This is one of the reasons why leukocytospermia cause infertility. PAF and TNF-α are considered as inflammation media and would increase in inflammatory diseases. However PAF level is lowered under the state of reproductive tract infection, this phenomenon is of great significance and worth being studied.ELISA method was adopted to observe the expression of PAF and TNF-What is the fundamental cause of the PAF level reduction in leukocytospermia? This point need to be further studied. It has important significance to reveal the mechanism of reproductive tract infection and infertility. The adjustment of PAF is affected by several factors, wherein it is related to platelet activating factor acetylhydrolase (PAF-AH). PAF-AH is specific to PAF, its concentration is negatively correlated with PAF. PAF-AH level has significant negative correlation with sperm motility in panda seminal plasma and has negative correlation with the state of sperm movement too . The conα in leukocytospermia affects the sperm quality, both of which contributed to sterility. The reason why PAF shows a low expression during reproductive tract infection is still not clear now, and it is of important significance, which deserves further study.In conclusion, low expression of PAF or high expression of TNF-


Apoptosis and cell shedding was quantified using immunohistochemistry for active caspase-3, and gut-to-circulation permeability was assessed by measuring plasma fluorescence following fluorescein-isothiocyanate–dextran gavage. LPS, at doses ≥0.125 mg/kg body weight, induced rapid villus IEC apoptosis, with peak cell shedding occurring at 1.5 hours after treatment. This coincided with significant villus shortening, fluid exudation into the gut lumen and diarrhea. A significant increase in gut-to-circulation permeability was observed at 5 hours. TNFR1 was essential for LPS-induced IEC apoptosis and shedding, and the fate of the IECs was also dependent on NFκB, with signaling via NFκB1 favoring cell survival and via NFκB2 favoring apoptosis. This model will enable investigation of the importance and regulation of pathological IEC apoptosis and cell shedding in various diseases.The gut barrier, composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, prevents the entrance of harmful microorganisms, antigens and toxins from the gut lumen into the blood. Small intestinal homeostasis is normally maintained by the rate of shedding of senescent enterocytes from the villus tip exactly matching the rate of generation of new cells in the crypt. However, in various localized and systemic inflammatory conditions, intestinal homeostasis can be disturbed as a result of increased IEC shedding. Such pathological IEC shedding can cause transient gaps to develop in the epithelial barrier and result in increased intestinal permeability. Although pathological IEC shedding has been implicated in the pathogenesis of conditions such as inflammatory bowel disease, our understanding of the underlying mechanisms remains limited. We have therefore developed a murine model to study this phenomenon, because IEC shedding in this species is morphologically analogous to humans. IEC shedding was induced by systemic lipopolysaccharide (LPS) administration in wild-type C57BL/6 mice, and in mice deficient in TNF-receptor 1 ( It allows absorption of nutrients from the intestinal lumen into the circulation, while preventing the entry of injurious microorganisms, toxins and antigens. In the small intestine, IECs are generated in the crypt, migrate up the villus and are shed at the villus tip . In miceectively . During ectively .In various inflammatory conditions, however, the loss of IECs from the villus exceeds the rate of epithelial generation in the crypt. This process, which we have termed ‘pathological IEC shedding’ remains poorly understood. Such pathological IEC shedding might represent the earliest intestinal injury in a variety of intestinal diseases and is likely to have important consequences, potentially resulting in gap formation in the epithelium, permeability defects and villus shortening (villus atrophy). Indeed, increased numbers of shedding IECs with corresponding focal permeability defects and epithelial gaps have been observed in inflammatory bowel disease (IBD), including both Crohn’s disease (CD) and ulcerative colitis (UC) . It has Clinical issueEpithelial cell loss and defects in the epithelial barrier are common early events in the pathogenesis of acute intestinal or diarrheal diseases as well as chronic intestinal inflammatory disorders, such as Crohn’s disease and ulcerative colitis. Increased intestinal epithelial cell (IEC) shedding, which is potentially associated with increased gut permeability, is thought to be a key contributor to these initial injuries. However, the molecular events underlying IEC shedding and disruption of intestinal barrier integrity during inflammatory disease states remain poorly understood. A robust animal model would greatly facilitate in-depth investigation of these processes. Mice exhibit a morphologically analogous form of small intestinal villus epithelial cell shedding to that observed in humans and have the potential to allow important mechanistic insights because of the availability of established transgenic and conditional mutant models.ResultsUtilizing the inflammatory response induced by systemic administration of lipopolysaccharide (LPS) in mice, the authors developed a new model for the investigation of IEC shedding. A threshold dose of ≥0.125 mg LPS/kg body weight delivered by intraperitoneal injection induced rapid and dynamic villus IEC apoptosis and shedding, which peaked at 1.5 hours post-administration. This coincided with significant villus shortening, fluid exudation into the gut and the onset of diarrhea. Activation of caspase-3 occurred concomitantly with IEC shedding in virtually all affected cells, suggesting that apoptosis is triggered within the epithelium prior to cells being shed. By examining the responses to LPS in transgenic mice, the authors showed that TNFR1 is essential for the induction of apoptosis and shedding. Furthermore, the fate of IECs is dependent on NFκB signaling, with signaling by NFκB1 favoring cell survival and signaling via NFκB2 favoring apoptosis.Implications and future directionsSystemic LPS administration in mice provides an effective and reliable trigger of pathological IEC shedding that parallels that observed in human intestinal diseases. The authors’ detailed characterization of affected mice confirms that villus epithelial apoptosis and cell shedding occurs with acute fluid exudation into the gut, leading to diarrhea. Furthermore, they provide evidence that TNFR1 and NFκB2-dominant signaling pathways are important in IEC apoptosis and shedding. By conducting an in-depth investigation of the kinetics, dose response and signaling mechanisms underlying these processes, they have established a novel and robust model that will facilitate further investigation of epithelial gap formation and barrier dysfunction in the inflamed intestinal epithelium. Further studies using this model are likely to provide insights into disease pathogenesis and support the development of more targeted treatments for inflammatory disorders.in vivo, making such experiments expensive and possibly yielding artefactual results. We have also found TNF to be an inconsistent stimulus of IEC shedding. We therefore sought to find a simple, inexpensive, rapid, reproducible and pathologically relevant stimulus to investigate the process of IEC shedding in detail in an animal model.Although recombinant tumor necrosis factor (TNF) has been previously shown to induce pathological intestinal villus epithelial cell shedding in mice , the exoLipopolysaccharide (LPS) is an integral component of Gram-negative bacteria and is a potent activator of the innate immune system. It represents a pathogen-associated molecular pattern (PAMP) recognized by Toll-like receptor 4 (TLR4) , which iWe have therefore examined in detail the earliest phase of LPS-induced murine gut injury. We demonstrate that intraperitoneally administered LPS is a simple, rapid and consistent stimulus of villus IEC apoptosis and shedding in the murine small intestine and that this occurs several hours prior to the onset of crypt apoptosis. This early response coincides with fluid effusion into the small intestinal lumen and diarrhea. We have subsequently characterized the dose response and the kinetics of this highly dynamic phenomenon. Using knockout mouse models, we have found that TNFR1-mediated signaling is essential for these events, with an NFκB2-dominant response favoring apoptosis. These data provide interesting insights into the control of IEC homeostasis in inflammatory disease, because the NFκB2 pathway has not previously been linked to IEC apoptosis and shedding.To establish the time-dependent intestinal effects of LPS, we administered 10 mg phenol-extracted LPS (PE-LPS)/kg body weight by intraperitoneal (i.p.) injection to adult female wild-type (WT) mice, and euthanized them after 1, 1.5, 2, 3, 4 and 6 hours. Diarrhea was observed from 1.5–2 hours. At necropsy, there was serosal pallor of the small intestine , which esupplementary material Fig. S1). These observations suggest that LPS causes rapid and specific small intestinal villus epithelial injury, and that peak shedding correlates with the onset of clinical diarrhea.We performed histopathological examination of hematoxylin and eosin (H&E)-stained sections to characterize intestinal injury. At 1.5 hours  there waP<0.01: ANOVA). The reduction in villus height was still evident in treated versus non-treated mice through to 6 hours post-LPS . A similar trend was also observed in both the jejunum and ileum, but differences did not reach statistical significance.Because villus shortening is commonly utilized as a measure of small intestinal damage, we measured villus heights after LPS administration. In the duodenum at 1.5 hours after LPS administration, mean villus height was reduced by 29%  to 260.5P<0.05: Kruskal-Wallis) , reaching significance at 3 hours, at which point a 40% decrease was observed  . This coP<0.05: Kruskal-Wallis) in plasma fluorescence, at 76.3±21.7 fluorescent units  by oral gavage, followed by 10 mg LPS/kg body weight. At 5 hours post-LPS (5 hours FD4), there was a fivefold increase  for active caspase-3. Large numbers of villus IECs exhibited positive immunolabeling as early as 1 hour, with almost universal immunolabeling of shed cells seen within the small intestinal lumen . Villus P<0.05: Kruskal-Wallis). Comparable IEC apoptosis and cell shedding were also observed at 1.5 hours after LPS treatment in the jejunum and ileum . We therefore concluded that LPS caused dynamic villus IEC apoptosis and shedding, and that this occurred relatively uniformly throughout the small intestine. The almost universal positive labeling of IECs undergoing shedding additionally suggests that activation of the terminal pathway of apoptosis occurs prior to shedding in this model, rather than being triggered by detachment as occurs during the process of anoikis. Interestingly, crypt IEC apoptosis as interpreted by active caspase-3 IHC did not show a comparable magnitude of increase to that observed in villi at 1.5 hours  increased the number of apoptotic and/or shedding IECs with similar distribution along the villus axis in the duodenum, jejunum and ileum . ApoptosP<0.05: ANOVA)  reduction in villus height [348.1±17.1 μm versus 399.0±35.5 μm in vehicle-treated control mice ] but wit: ANOVA) . LPS dosTo assess whether the LPS purification and/or extraction method altered IEC apoptosis and shedding, we administered high-purity ion-exchange chromatography extracted LPS  to WT mice for 1.5 hours. This preparation caused similar villus shortening to 10 mg PE-LPS/kg body weight  . IEC apo−/−Tlr4 mice would exhibit LPS-induced small intestinal injury, to exclude the possibility of alternative mechanisms. IE-LPS (10 mg/kg body weight) caused negligible change in villus height in −/−Tlr4 mice  −/−Myd88 mice, which lack the TLR signaling adapter molecule Myd88 in IECs, to systemic administration of 10 mg PE-LPS/kg body weight. These mice showed very comparable amounts of apoptosis and shedding (14.1±1.1% IECs) to their WT and heterozygous counterparts   of 27% in treated versus untreated  compared with 5% shortening in treated versus untreated WT mice   compared with WT mice. Together, these results suggest that LPS-induced intestinal injury is dependent on NFκB2, whereas NFκB1 might be necessary to suppress IEC apoptosis and shedding.NFκB is a major transcriptional regulator downstream of TLR4. We therefore administered PE-LPS to  WT mice . This ge WT mice . In cont-Wallis) , and red-Wallis) . Interes WT mice , and sig-Wallis)  comparedTnf and Cd40 showed marked upregulation, and we therefore analyzed these two proapoptotic genes by quantitative PCR (qPCR) using triplicate samples from individual animals (Tnf mRNA (P<0.05: randomization test). qPCR also showed a non-significant increase of +2.1 for Cd40. These data, in conjunction with histopathological findings and activation of caspase-3, suggest that apoptosis is the predominant form of cell death occurring in LPS-induced small intestinal injury.Because activation of caspase-3 does not categorically confirm that cell death has occurred by apoptosis, we performed an array analysis of 89 genes associated with various cell death pathways in PE-LPS-treated compared with untreated WT animals. We found that LPS predominantly altered expression of genes associated with apoptosis, rather than those associated with autophagy or necrosis . Tnf and animals . This shsupplementary material Fig. S3) than caused by LPS.Because TNF is a key mediator of endotoxic shock, and was markedly upregulated at the mRNA level in our array, we tested whether TNF would cause comparable enteric injury to LPS. At 1.5 hours, TNF  caused equivalent duodenal villus shortening  to that Tnf mRNA, suggest that TNF is central in the pathogenesis of LPS-induced small intestinal injury.These data, together with significant induction of intestinal −/−Tnfr1 mice were administered 10 mg PE-LPS/kg body weight for 1.5 hours, there was no villus shortening  or TNFR2 (p75) were required to cause LPS-induced gut injury. When ortening  and signs height . These f−/−Nfĸb1 and −/−Nfĸb2 mice. −/−Nfĸb1 mice were highly sensitive to TNF  and exhibited a significant reduction in villus height compared with TNF-treated WT mice . Conversely, −/−Nfĸb2 mice were resistant to the TNF-induced reduction in villus height (340.1±15.1 μm) and IEC apoptosis and shedding (2.5±0.7%) compared with similarly treated WT animals  and the adapter molecules CD14  and MD-2Tnf mRNA abundance in small intestinal epithelial enriched extracts 1.5 hours after LPS administration. The significance of TNF as the crucial mediator in our model was further demonstrated by the fact that TNF administration caused very comparable IEC apoptosis and shedding as did LPS, consistent with previous results  under appropriate UK Home Office licenses or with approval and monitoring by the Children’s Hospital Los Angeles Institutional Animal Care and Use Committee.Wild-type (WT) C57BL/6 mice supplied by Charles River , and transgenic strains on a C57BL/6 background, including −/− mice  and Tlr4−/− mice   or ion-exchange chromatography (IE-LPS)  was diluted in sterile phosphate-buffered saline (PBS) and administered to mice by intraperitoneal (i.p.) injection.LPS from Murine recombinant TNF  was diluted in sterile water and administered by i.p. injection to mice at 0.33 mg/kg body weight.en bloc. The intestinal lumina were flushed with PBS and immediately fixed in 10% neutral buffered formalin with selected organ samples. After 24 hours fixation, tissue was routinely processed and embedded in paraffin wax. Tissue sections (3–5 μm thickness) were prepared and stained either with H&E or used for immunohistochemistry (IHC).Following euthanasia, the intestinal tract was dissected Tissue sections were treated with 1% hydrogen peroxide in methanol to block endogenous peroxidases, followed by heat-induced antigen retrieval in 0.01 M citrate acid buffer (pH 6) and incubation with a rabbit polyclonal anti-active-caspase-3 antibody . Peroxidase-labeled anti-rabbit EnVision™  and 3,3′-diaminobenzidine were used for visualization.normal’ if there was no or weak diffuse non-specific brown staining and cells had a basally located basophilic nucleus;apoptotic’ if there was defined positive staining that was confined to cytoplasmic or nuclear borders when compared with any background staining of neighboring IECs;shedding’ if there was defined positive staining that was confined to cytoplasmic or nuclear borders and in addition there was apical elevation of the cytoplasmic membrane, and/or an apically positioned nucleus.For quantification of apoptotic and shedding IECs, individual epithelial cells were counted from the base of the villus (above crypt level) to the mid-point of the villus tip in 18–20 well-orientated hemivilli at 400× magnification . Data are then represented as percentage of villus length, 0% therefore representing the villus base and 100% representing the villus tip.To allow comparison of cell positional data, villi were adjusted to a fixed length of 100 cells by using WincryptsImageJ  was used® F200 plate reader  from blood collected post-mortem at 5 hours after gavage in order to allow FD4 to be distributed throughout the intestinal tract. Mice were euthanized via a rising CO2 concentration and blood taken by cardiac puncture. To assess the effect of LPS on permeability, it was administered at set time points prior to the end of the experiment, i.e. at 1.5 hours, 3 hours and at the same time as FD4 for the 5-hour time point. Plasma was separated from heparinized whole blood by centrifugation at 5000 rpm for 2 minutes in a minicentrifuge.Fluorescein-isothiocyanate-conjugated dextran  was diluted to 22 mg/ml in PBS and administered at 20 ml/kg body weight by oral gavage ± i.p. injection of LPS. Plasma fluorescence was measured by a TECAN Infinite2 reverse-transcription kit . An 89 gene Cell-Death Pathway Finder array (SABiosciences) was performed on a Roche LightCycler®480 followed by validation with replicate samples using TaqMan® gene expression assays for β-actin (Mm01205647_g1), TNF (Mm00443260_g1) and CD40 . Cycling conditions were performed as per the manufacturer’s instructions.Small intestinal extracts were isolated with chelation buffer solution as previously described , and RNA© . Normally distributed data were assessed by ANOVA with Holm-Sidak post-hoc test, and non-parametric data were analyzed by ANOVA on ranks  with Dunn’s post-hoc test. P<0.05 was considered significant. REST© software was used for comparison of qPCR data from individualized samples by randomization test as previously described (n numbers indicate the total number of mice studied.Data represent mean ± s.e.m. Comparisons were made between treatment groups and controls using SigmaPlot 12escribed . n numbe


In addition, we highlight opposite evidence showing protective effects of these two main cytokines when conditions of duration, amount of expression or state of activation of the target or neighboring cells are changed. Furthermore, we discuss these results in the frame of previous disappointing results from anti-TNF-α clinical trials against Multiple Sclerosis, another neurodegenerative disease with a clear neuroinflammatory component. In conclusion, we hypothesize that the available evidence suggests that the duration and dose of IL-1β or TNF-α expression is crucial to predict their functional effect on the SN. Since these parameters are not amenable for measurement in the SN of PD patients, we call for an in-depth analysis to identify downstream mediators that could be common to the toxic (and not the protective) effects of these cytokines in the SN. This strategy could spare the possible neuroprotective effect of these cytokines operative in the patient at the time of treatment, increasing the probability of efficacy in a clinical setting. Alternatively, receptor-specific agonists or antagonists could also provide a way to circumvent undesired effects of general anti-inflammatory or specific anti-IL-1β or TNF-α therapies against PD.Neuroinflammation has received increased attention as a target for putative neuroprotective therapies in Parkinson’s Disease (PD). Two prototypic pro-inflammatory cytokines interleukin-1β (IL-1) and tumor necrosis factor-α (TNF) have been implicated as main effectors of the functional consequences of neuroinflammation on neurodegeneration in PD models. In this review, we describe that the functional interaction between these cytokines in the brain differs from the periphery  and present data showing predominantly a toxic effect of these cytokines when expressed at high doses and for a sustained period of time in the  Parkinson’s Disease (PD) is a neurodegenerative disorder affecting over 1% of the population above 65 years of age . Its carAlthough a load of valuable information has been obtained during the last years, the etiology of not more than 5% of PD cases has been revealed to be caused by genetic defects . EnvironIn parallel to an intense effort to unravel the etiology of PD, increasing evidence is now available on the physiopathology of this illness. It is foreseen that the understanding of the pathological process of neurodegeneration and the subsequent identification of the molecules mediating this process will allow the detection of new therapeutic targets. These novel targets should enable the design of disease-modifying therapies, slowing down or halting disease progression. The involvement of neuroinflammation in the pathogenesis of PD in patients is supported by several reports that anti-inflammatory medication use is associated with a reduction in the risk of PD . MoreoveAn ubiquitous physiopathological feature of the degenerating SN in animal models and PD patients is the presence of robust microglial activation in that brain region. Not so long ago, microglial activation has been regarded as a physiological consequence of neuronal death, whose function was solely to remove neuronal debris. This vision was wrong. Microglial activation can lead to toxic but also protective effects on the on-going neurodegenerating neurons of the SN  and the parameters that will affect their final effect on the dopaminergic neurons.Most of the available evidence leads to conclude that IL-1β and TNF-α are toxic for the dopaminergic neurons of the SN. However, completely opposite results have also been obtained, showing IL-1β and TNF-α-derived neuroprotective effects. Several groups including ours, have found that the final effect of these cytokines on dopaminergic cell viability depends on many variables such as concentration, duration of expression and state of activation of the target or neighboring cells , 2006.Before presenting and discussing the data that lead to these conclusions, a general overview of each cytokine is presented, necessary for the full understanding of the biological effects of these cytokines on neurodegeneration.IL-1β was first described by around 1943 as an agent coming from rabbit peritoneal cells. Its first biological property to be discovered was to produce fever, and therefore was termed endogenous pyrogen . IL-1β iThere are multiple steps of regulation of IL-1β production and activity, including transcription, translation, cleavage, and cellular secretion . In macrNeurons have been shown to be particularly susceptible to IL-1β-mediated toxicity. For example, Purkinje neurons suffer apoptosis when microglia releases IL-1β and TNF-α . IL-1β pIL-1β is a pro-inflammatory cytokine with pleiotropic biological actions in the periphery and the brain. IL-1β has been found in the cerebrospinal fluid and post-mortem striata of PD patients . In addiγ; As stated above, the final functional outcome of IL-1β on dopaminergic cell loss will depend on variables such as concentration, duration of expression, and timing among stimuli. In terms of the influence of the duration and levels of IL-1β expression, it is irrefutable that the sustained expression of IL-1β in the SN at pro-inflammatory levels causes irreversible and pronounced dopaminergic neuronal loss in the SN . This coper se in the SN can exacerbate on-going neurodegeneration via IL-1β production from primed microglial cells.Concerning the state in which a dopaminergic neuron perceives IL-1β signaling, it is important to notice that during neuronal cell loss in PD, and also in prion and AD models, the surrounding microglial cells are activated to a particular state defined as “primed” or “atypical” . In thisImportantly, in clinical terms, nigral neurodegeneration elicited by two different treatments can be exacerbated from the periphery by eliciting a sustained, succeeding intravenous inflammatory stimulus , 2010. Iper se, but in the presence of a second challenge with 6-OHDA in the striatum, the death of TH neurons was higher in the animals which received the pro-inflammatory and neurodegenerative stimuli  and TNFR2  bind membrane-integrated TNF (memTNF) as well as sTNF . TNFR1 iOf interest, TNFRs can have counteracting functions, at least in neuronal tissues, as recently demonstrated in a murine model of retinal ischemia, where TNFR1 apparently aggravated tissue destruction, whereas TNFR2 was protective via activation of PKB/Akt. It has been proposed that TNFR1 or 2 specific agonists and antagonists could mediate specific protective effects in PD . In otheDuring chronic or acute neuroinflammation high levels of sTNF are found in the cerebrospinal fluid and post-mortem brains of PD patients as well as in animals treated with dopaminergic neurotoxins as MPTP and 6-OHDA used to model nigral degeneration in non-human primates and rodents . High exMany genetically engineered mouse models were designed in order to elucidate the contribution of TNF-α in neurodegeneration. It should be noticed that this models have limitations and they may not exactly reflect what happens in the brain of PD patients. Despite of these restrictions these models had proven to be very useful unraveling the role of TNF-α in this disease.The effects of TNF-α on the nigrostriatal pathway were investigated by administration of MPTP in knock-out mice lacking TNF-α, or TNFRs. First reports showed that TNF-α was upregulated in the striatum after MPTP administration . Null miThese conflicting results can be attributed to several methodological issues. The doses of MPTP used in these studies were different [15 mg/kg on 8 consecutive days in Similar contradictory results have been reported in rats injected with the neurotoxin 6-OHDA in the striatum. In vitro, TNF-α administration or expression has been shown to be toxic to dopaminergic neurons  in the SN, In experiments where TNF-α was expressed chronically, toxic effects of TNF-α were clearly observed. An early report by However, if lower levels of TNF-α were expressed in the SN, a transient neuroprotective effect against 6-OHDA toxicity in the SN and in the striatum was observed . In deveThe cholinergic anti-inflammatory pathway should also be taken into account in order to understand the contribution of TNF-α in PD. This pathway regulates the innate immune response after injury or infection. After an insult, TNF-α (and other cytokines) are produced by the cells of the innate immune system, augmenting and prolonging the inflammatory response by activating other cells to release many cytokines . This initial beneficial response can be detrimental if it lasts for a long time or it is exacerbated. As a way to control this response, TNF-α stimulates the afferent branch of the vagus nerve which conveys this signal to the CNS. In turn, the motor branch of the vagus nerve releases acetylcholine in the periphery. In particular, this neurotransmitter interacts with the alpha7 subunit of the nicotinic acetylcholine receptor expressed in macrophages, and other cytokine-releasing cells. This interaction activates intracellular signal transduction which inhibits release of pro-inflammatory cytokines, in particular TNF-α. This negative feedback circuit regulates the inflammatory response, maintaining homeostasis , reviewe in vitro, in a LPS model.These findings may have therapeutic consequences. Vagal stimulation has proven effective for the treatment of epileptic seizures refractory to pharmacological treatments in adult patients  and possThe neuroimmunology of the brain parenchyma differs in many aspects from the immune response elicit systemically . As stated above, IL-1β and TNF-α effects in the brain could be distinctly separated from each other, since they are not mutually inducible in the brain parenchyma. However, the influence of these variables on their net functional effect seems to be similar in general terms. In the healthy SN , the acute administration or expression of both cytokines seems to have no dramatic effect on dopaminergic neurons in the SN, unless expressed at supra-physiological levels . In other words, sustained and high IL-1β and TNF-α expression will univocally lead to neurodegeneration, while acute and low expression will lead to no or neuroprotective effects.In conclusion, as expected for molecules with pleiotropic functions, the net, univocal effects of IL-1β or TNF-α on dopaminergic neuronal viability l levels . On the l levels .In termsl levels  whereas l levels . The oveFigure 1B; It is also clear that, in the degenerating SN, when microglial cells are primed, acute and sub-toxic pro-inflammatory stimuli irreversibly leads to increased neurodegeneration (Hence, if these variables are affecting IL-1β and TNF-α effects on dopaminergic viability, how can this information be translated to future treatments to PD patients where these variables are uncontrollable and not measurable?in vitro and in vivo demonstrating toxic effects of TNF-α, clinical trials using anti-TNF-α molecules were approved and started (Indeed, a similar scenario has occurred in the MS field. MS is an autoimmune demyelinating disease whose inflammatory component is much more characterized and typical than in PD. In models of MS, TNF-α accelerated the onset of disease and increased apoptosis of oligodendrocytes . Based o started . Unfortu started . Later i started  and indu started . Also, T started . TherefoThus, how can we learn from the MS field and design clinical trials with a higher probability of achieving therapeutic efficacy?Based on the evidence discuss in this review, we are certain that general anti-IL-1β or TNF-α therapies against PD have low probability of success. In a similar way, general anti-inflammatory treatments may inhibit toxic but also beneficial effects of TNF-α, IL-1β, or other cytokines.One possibility to increase the success rate of future clinical trials could be to use receptor-specific agonists or antagonists to circumvent undesired effects of general anti-inflammatory or specific anti-IL-1β or TNF-α therapies against PD. This option has been suggested especially for TNF-α, where the different biological effects of TNFR1 and TNFR2 have been more exhaustively studied . In addiFinally, we believe that there are common and specific downstream mediators of the toxic effects of IL-1β and TNF on dopaminergic neurons in the SN. The identification of these IL-1β- or TNF-derived molecules with univocal toxic effects should open the possibility to inhibit IL-1β or TNF-α-detrimental effects while sparing their neuroprotective properties, increasing the probability of efficacy in a clinical setting. A similar, but reverse rational can be applied to neuroprotective-only molecules. Fortunately, numerous animal models and experimental set-ups have been generated in the past 10 years to be able to identify these candidate molecules.Undoubtedly, neuroinflammation is no longer regarded as the expected consequence of neuronal demise leading to the clearance of cell debris, but a phenomenon dramatically affecting PD progression and therefore a target for neuroprotective or disease-modifying therapies. The actual challenge is to reach the clinic with compounds univocally targeting the detrimental effects of neuroinflammation, sparing possible beneficial effects of these pleiotropic molecules.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.


Isolated limb perfusion with TNF-α and melphalan is used with remarkable efficiency to treat unresectable limb sarcomas. Here we tested the ability of TNF-α to directly induce apoptosis of sarcoma cells. In addition, we investigated the impact of p53 in the regulation of such effect.ex vivo with TNF-α were processed for TUNEL staining. It revealed substantial endothelial cell apoptosis and levels of tumour cell apoptosis that varied from low to high. In order to investigate the role of p53 in TNF-α-induced cell death, human sarcoma cell lines (n = 9) with different TP53 and MDM2 status were studied for their sensitivity to TNF-α. WtTP53 cell lines were sensitive to TNF-α unless MDM2 was over-expressed. However, MutTP53 and NullTP53 cell lines were resistant. TP53 suppression in WtTP53 cell lines abrogated TNF-α sensitivity and TP53 overexpression in NullTP53 cell lines restored it. The use of small molecules that restore p53 activity, such as CP-31398 or Nutlin-3a, in association with TNF-α, potentiated the cell death of respectively MutTP53 and WtTP53/AmplMDM2. In particular, CP-31398 was able to induce p53 as well as some of its apoptotic target genes in MutTP53 cells. In WtTP53/AmplMDM2 cells, Nutlin-3a effects were associated with a decrease of TNF-α-induced NF-κB-DNA binding and correlated with a differential regulation of pro- and anti-apoptotic genes such as TP53BP2, GADD45, TGF-β1 and FAIM.We first analysed the ability of TNF-α to induce apoptosis in freshly isolated tumour cells. For this purpose, sarcoma tumours (n = 8) treated More effective therapeutic approaches are critically needed for the treatment of unresectable limb sarcomas. Our results show that restoring p53 activity in sarcoma cells correlated with increased sensitivity to TNF-α, suggesting that this strategy may be an important determinant of TNF-α-based sarcomas treatment. Sarcomas are rare, poorly understood cancers, refractory to standard therapies in vivo with the induction of endothelial cell apoptosis in vitro with the specific suppression of αVβ3-mediated endothelial cell adhesion While the effect of melphalan, as a DNA-damaging alkylating agent, is clearly established, the effect of TNF-α remains controversial even though a TNF-α selective anti-vascular mechanism has been established TNF-α has been demonstrated to be either anti-proliferative, growth enhancing, or ineffective on transformed cell lines TP53 status was reported in vitroin vivo in sarcoma patients treated with TNF-α and melphalan TP53 is mutated in more than 20% or non functional in up to 60% of STS  MDM2 gene amplification (AmplMDM2). Importantly, TP53 mutations or inactivations were found to correlate with a poorer outcome in sarcoma patients In this regard, the dependence of TNF-α-induced cell death on in vitro and inhibit tumour growth in animal models It has been reported that pharmacological and/or genetic restoration of the wild-type p53 pathway induce tumour cell death In the present study, we demonstrate that TNF-α induces cell death not only in tumour vasculature but also in sarcoma tumour cells harboring wild type p53, and that resistance to TNF-α could be linked to lack of p53 function in this type of tumour. Furthermore, we demonstrate that combining TNF-α with drugs that restore the p53 pathway enhances global apoptotic effects. These findings suggest that this combination therapy approach could be used to treat highly resistant sarcoma tumours.The Institutional Review Board of the Gustave Roussy Institute (Commission Scientifique des Essais Thérapeutiques) specifically approved the study and all patients included in the study signed an informed consent.Mouse anti-CD31 monoclonal antibody conjugated with Alexa Fluor 488 was purchased from BD Pharmingen; mouse anti-p53 (DO-1) and anti-TNFR1 antibodies from Santa Cruz Biotechnology ; anti-p21 and anti-MDM2 from Calbiochem ; rabbit anti-BAX polyclonal antibodies from Cell Signaling Technology ; mouse anti-GAPDH monoclonal antibody from Millipore  and mouse anti-actin monoclonal antibody from Sigma-Aldrich . Human recombinant TNF-α (Beromun) was obtained from Boehringer Ingelheim . Nutlin-3a was kindly provided by Dr L. Vassilev  and CP-31398 by Dr L. Kopelovich . TO-PRO3 was purchased from Invitrogen .2, then washed and maintained in the same conditions for 72 h in fresh culture medium without TNF-α. Successively, cultured slices were washed in PBS and fixed by immersion in cold methanol for 10 min at −20°C. TUNEL reaction was performed using in situ Cell Death Detection Kit  according to manufacturer’s instructions. Next, the slices were incubated overnight at 4°C with mouse anti-CD31 antibody conjugated with Alexa Fluor 488 and subsequently for 1 h with TO-PRO3. Slices were finally mounted on glass slides using fluoromount-G . Stacks of confocal slices were collected with a LSM 510 laser scanning confocal microscope  using 20X/0.75 Plan-Apochromat objective. The excitation wavelengths were 488 nm for Alexa 488, 543 nm for the TUNEL positive nuclei revealed by the Cell Death Detection Kit, and 633 nm for TO-PRO3. Images were acquired using BP505–530, BP560–615 and LP 650 emission filters respectively. Z-projection of stacks was done using LSM Image Examiner software . Cell counting was done by ImageJ software using the cell counter plugin and the percentage of apoptotic endothelial and tumour cells was determined.Pieces of fresh sarcoma tumours were obtained after the surgical treatment of eight patients with resectable STS of the retroperitoneum. Tumours were immediately embedded in 6% low-melting point agarose . 200 µm thick slices were cut at room temperature with Leica VT 1000 S Microtome . The slices were incubated in culture medium (RPMI containing 10% FCS) with or without 50 ng/ml of TNF-α for 90 min at 37°C and 5% CORhabdomyosarcoma cell line (KYM-1) HT1080 cells were grown in DMEM (4.5 g/ml glucose) with 10% FCS, and the remaining cell lines in RPMI supplemented with 10% FBS and 1% sodium pyruvate . To study TNF-α effects, cells were seeded at 25% confluency, and one day later they were either treated with 50 ng/ml TNF-α alone or in combination with 7.5 µg/ml CP-31398 or 5 µM Nutlin-3a for the indicated times. For cell irradiation, we administered 15 Gy using the MXR−225/22 X-ray machine  at a dose rate of 1 Gy/min.TP53 gene was performed by Sanger sequencing based on PCR product and did not include generation of new data as the reference sequence used for primer design and for alignment was already published and available in RefSeq database as NM_000546.4 for TP53 . PCR amplification of 9 amplicons covering exons 2 to 11 (corresponding to the whole coding sequence), including at least 20 bases in flanking introns was performed. Detailed protocol and primer sequences are available on demand. Purified DNA was sequenced using BigDye® Terminator Cycle Sequencing Kit . Sequencing reactions were analyzed on 48-capillary 3730 DNA Analyzer® in both sense and antisense directions. All mutations detected were controlled with independent amplification at least one time. Sequence reading and alignment were performed with SeqScape® software .DNA from cell lines was extracted from cell pellets with QIAamp Blood Tissue Kit  according to manufacturer’s instructions. Resequencing of 6) and propidium iodide labeling  after 72 h treatment with TNF-α alone or in combination with CP-31398 or Nutlin-3a. The samples were analyzed on a FACSCalibur flow cytometer and data were processed using CellQuest software .Cell death was assessed by 3.3-dihexyloxacarbocyanine iodide , and a protease inhibitor mixture . Equivalent protein extracts (30 or 50 µg) were resolved by SDS–PAGE, transferred to a nitrocellulose membrane , and incubated overnight with appropriate primary antibodies at 4°C. The labeling was visualized using horseradish peroxidase-conjugated secondary antibodies and by enhanced chemiluminescence .Cells were harvested using EDTA 1 mM, washed twice with PBS, and incubated with anti-TNFR1 or isotype control antibodies  for 30 min. at 4°C, followed by incubation with phycoerythrin-conjugated goat anti-mouse antibody . The samples were analyzed on a FACSCalibur flow cytometer and data were processed using CellQuest software .HT1080 transfection was performed using Lipofectamine2000 Transfection Reagent , according to the manufacturer’s instructions. Expression vectors encoding shRNA designed for specific silencing of p53 or luciferase were purchased from InvivoGen. Stably transfected HT1080clones were selected with 500 µg/ml Zeocin . MFH95 cells, MFH100, and MFH152 cells were transfected by electroporation in an Easyject electroporation system  using the p53 encoding plasmid pC53-SN3 for MFH-95.Harvested cells were gently fixed in 70% cold-ethanol, and incubated over night at −20°C. 70% ethanol was then removed by centrifugation, and after PBS-washing, pelleted cells were incubated at 37°C for 1 hour in a mixture containing 0.5 ml PBX1x, 10 µl of 10 mg/ml RNAse A, and 25 µl of 1 mg/ml propidium iodide solution. Cell cycle analysis was then performed by flow cytometry.For analysis of NF-κB DNA binding activity, 15 µg of nuclear extracts were incubated with the radioactive labelled HIV-LTR tandem κB oligonucleotide as κB probe For apoptotic gene expression analysis, total RNA was extracted after 16 h of treatment using Trizol  following manufacturer’s instructions, and reverse transcription was performed using a High Capacity cDNA Reverse Transcription Kit . For quantification of apoptosis related genes, real time PCR (RT-PCR) was performed using Platinum SYBR green qPCR SuperMix-UDG w/ROX  and the 7900 HT Fast Real-time PCR System . Apoptosis dedicated arrays were elaborated by the PETC platform of INSERM U576 and INSERM U876. The relative expression level of 91 apoptosis-related genes was normalized using four different house-keeping genes . For each sample, cycle threshold (Ct) values for the housekeeping genes were determined for normalization purposes, and delta Ct (ΔCt) between the mean of target-gene values and housekeeping-genes values was calculated. Relative expression levels of target gene mRNAs between control cells and Nutlin-3a or Nutlin-3a + TNF-α-treated cells were calculated and expressed as fold change over control. Values represent the mean of duplicates and are representative of two independent experiments.Results are expressed as mean ± standard deviation (SD) of values obtained in at least three independent experiments. Differences between samples were analysed using the 1- way ANOVA. When it was statistically significant, post-hoc analysis used Student t-test with Bonferroni corrections. Differences were considered statistically significant for p value inferior to 0.05/number of comparisons. All calculations were performed using the Microsoft Office Excel statistical analysis software 2003.ex vivo tumour culture assay, recapitulating conditions that might occur in vivo during ILP, was performed. Slices from eight retroperitoneal sarcomas prepared immediately after surgery  were treated with or without TNF-α for 90 min, washed and then maintained in fresh medium without TNF-α for 72 h before TUNEL assay and subsequent labelling with anti-CD31 antibody and TO-PRO3. Double staining of endothelial cells with anti-CD31 antibody (green) and TUNEL (red) showed co-localization of both colours (yellow), indicating that TNF-α induces apoptosis of endothelial cells. Moreover, co-localization of nuclear staining  and TUNEL (red), resulting in purple, also indicates that TNF-α induces apoptosis of tumour cells. Untreated slices showed no or few TUNEL-positive nuclei, excluding the possibility that spontaneous apoptosis was induced by our experimental conditions . In MFH100 and MFH152 cells, TP53 had respectively a homozygous 524 G>A (R175H) and 503 A>C (H168P) missense mutation (MutTP53). In MFH95 and LMS148 cells, a TP53 deletion was observed since no amplification of TP53 exons 2 to 11 could be obtained (NullTP53). Moreover, Western blot analysis showed that T449, T778 and T1000 cells over-expressed MDM2 while MFH100, MFH152, MFH95 and LMS148 cells displayed non-functional p53 pathway based on the absence of p53 increase following irradiation  had wild type TP53 gene  and propidium iodide (PI) labeling after 72 h of TNF-α treatment. WtTP53/WtMDM2 cell lines (KYM-1 and HT1080) displayed high levels of apoptotic cells after TNF-α treatment (40% and 82% respectively) . To verify if the resistance to TNF-α was not due to a low expression level of the TNF receptor type 1 (TNF-R1), we assessed the surface expression of TNF-R1 by flow cytometry  and sensitivity to TNF-α-induced apoptosis.As ctively)  whereas ytometry . Most ofWtTP53/WtMDM2 TNF-α sensitive HT1080 cell line by stable transfection of a vector encoding a short hairpin RNA (shRNA) targeting p53 (shRNA-p53) or luciferase mRNA as a control (shRNA-luc)  . SilenciRNA-luc) . MoreoveNullTP53 TNF-α resistant MFH95 cell line using pC53-SN3 expression plasmid. We obtained an efficient p53 expression associated with a concomitant induction of p21 . These results show that in MutTP53 cell lines, restoration of wild-type p53 activity can enhance susceptibility to TNF-α induced cell death.The small molecule CP-31398 was reported to stabilize the wild-type-associated epitope (mAb1620) of the p53 DNA-binding domain, thus conferring a wild-type conformation to mutant p53 and rescuing p53 functions reatment . FurtherCP-31398 . ResultsWtTP53/AmplMDM2 sarcoma cell lines  were incubated with Nutlin-3a for 16 h. Western blot analysis showed an increase of p53 protein level leading to p21 expression and an increase of BAX expression, suggesting a restoration of p53 transcriptional activity after treatment  and anti-apoptotic genes (TGF-β1 and FAIM) display a different pattern of transcript induction and repression in sensitized cell lines when the combination of Nutlin-3a and TNF-α is used. Further studies are thus needed to determine the molecular mechanisms associated with the potentiating pro-apoptotic effect of TNF-α combined with Nutlin-3a, and to determine whether NF-κB-DNA binding repression by Nutlin-3a is involved in the observed potentiation of apoptosis when both TNF-α and Nutlin-3a are used. Indeed, this may have important implications since the use of both CP-31398 and Nutlin-3a, which act on various pathways, may be useful in overcoming drug resistance and thus could result in a more efficient therapeutic response.It has been reported that in p53-mutated cell lines, NF-κB was induced at higher levels following TNF-α treatment MutTP53 and WtTP53/AmplMDM2 cell lines, restores sensitivity to TNF-α. This is of importance in the clinical setting where the use of TNF-α administered locally and in combination with other drugs that restore the p53 pathway may contribute to improved efficacy of such therapy. These findings thus provide a new strategy for treating highly refractory STS, in which p53 is frequently mutated or inactivated.In conclusion, our data indicate that activating p53 with CP-31398 or Nutlin-3a, in respectively