As such, the regulation and role of CD8+ T cell-derived TNF following viral infection is of great interest. Here, we show that the biphasic production of TNF by CD8+ T cells following in vitro stimulation corresponds to distinct patterns of epigenetic modifications. Further, we show that a global loss of TNF during IAV infection results in an augmentation of the peripheral virus-specific CD8+ T cell response. Subsequent adoptive transfer experiments demonstrated that this attenuation of the CD8+ T cell response was largely, but not exclusively, conferred by extrinsic TNF, with intrinsically-derived TNF making only modest contributions. In conclusion, TNF exerts an immunoregulatory role on CD8+ T cell responses following IAV infection, an effect that is largely mediated by extrinsically-derived TNF.TNF is a pro-inflammatory cytokine produced by both lymphoid and non-lymphoid cells. As a consequence of the widespread expression of its receptors (TNFR1 and 2), TNF plays a role in many important biological processes. In the context of influenza A virus (IAV) infection, TNF has variably been implicated in mediating immunopathology as well as suppression of the immune response. Although a number of cell types are able to produce TNF, the ability of CD8 Typically, cytokine production by antiviral CD8+ T cells occurs in an hierarchical fashion, with the majority producing IFNγ, and a subset of those producing TNF. Such ‘polyfunctionality’ within a T cell response is used to indicate an increased quality of response, and has been associated with heightened affinity of TCR-pMHCI recognition [CD8ytokines . One of ognition –4.+ T cell responses. TNF can be expressed as a membrane bound protein (mTNF) or cleaved and released as a soluble protein (sTNF) [+ and CD8+ T cells [+ T cells [+ T cells [+ T cell response to influenza in the lungs [+ T cell responses. However, studies with TNF knockout (Tnf-/-) mice are limited as this genotype causes defects in the follicular DC network as well as in B cell follicle and germinal centre formation [Tnf-/- mice have a profound defect in their immune architecture and cellular composition [Tnf-/- mice do not allow us to investigate the role of intrinsic TNF produced by CD8+ T cells and its role in the infection. Recently, Wortzman et al. [+ T cells, and demonstrated that TNF produced intrinsically by CD8+ T cells enhanced effector functions and regulated contraction of those cells via TNFR2 signaling.Tumor necrosis factor (TNF) can substantially influence antiviral CD8n (sTNF) . Followi T cells . TNF bin T cells . TNFR1 h T cells , suggesthe lungs . Studieshe lungs . Others he lungs . Moreoveormation . Consequposition . Therefon et al.  used an in vitro stimulation is dependent on co-stimulation and is associated with changes in histone post-translational modification (PTM) deposition at the Tnf gene locus. We also demonstrate that, following intranasal infection with influenza A virus (IAV), global TNF deficiency increased the magnitude of IAV-specific CD8+ T cell responses, as measured in the periphery, but did not significantly affect the recruitment of IAV-specific CD8+ T cells to the lungs. Moreover, this TNF-mediated attenuation of the IAV-specific CD8+ T cell response was found to be largely dependent on extrinsic TNF production, with only a moderate contribution by intrinsic CD8+ T cell-derived TNF. These data clearly indicate an immunoregulatory role for TNF during IAV infection, which occurs at the global, rather than local, level and is mediated predominantly by extrinsic TNF production.In our study, we show that the rapid and robust production of TNF after Tnf -/- and Tnf -/-/OT-I mice were bred and housed in specific pathogen-free conditions at the Biomedical Research Facility, Department of Microbiology and Immunology, The University of Melbourne . Tnf -/- mice [2 asphyxiation using a slow fill rate of 20% per volume per minute. All animal experimentation was conducted following the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes guidelines for housing and care of laboratory animals and performed in accordance with Institutional regulations after pertinent review and approval by the University of Melbourne Animal Ethics Committee , ovalbumin transgenic-I (OT-1), -/- mice  were obtommittee .+CD44lo T cells stimulated at 2 x 104 cells per well in mAb-coated 24-well plates for up to 72 hours (h) were incubated in the presence of 10 U/mL recombinant human IL-2 . The mAb were coated onto Nunc plates overnight in PBS at the following concentrations: anti-CD3ε (clone 145-2C11) at 10 μg/mL; anti-CD8α (clone 53–6.7) at 10 μg/mL; anti-CD11a (clone I21/7.7) at 5 μg/mL; anti-CD28 (clone 37.51) at 10 μg/mL [Sorted CD810 μg/mL . To perm10 μg/mL . Cells w6 cells total) were fixed in 1% formaldehyde, resuspended in ChIP lysis buffer  and sonicated to generate 200–1000 base pair fragments. Samples were then precleared using Protein A-agarose/salmon sperm DNA , split into 5 and incubated overnight with either 5 ug anti-H3K27me3, 3 ug anti-H3K4me3 or 4 ug anti-RNA polymerase II . A no antibody control and a total input positive control were also included. After washing, all samples  were incubated with Protein A-agarose/salmon sperm DNA with rotation for 1 hour followed by a series of washes in low salt, high salt, lithium chloride, and TE buffers. DNA was eluted before crosslink reversal with 0.2M NaCl at 66°C overnight, followed by protein digestion with proteinase K (Promega). Immunoprecipitated DNA was extracted by phenol: chloroform:isoamyl (25:24:1) extraction and resuspended in HPLC water. For analysis, real-time PCR was used to measure the levels of ChIP-DNA, such that resulting cycle threshold (Ct) values were converted to copy number (#copies = 105/2Ct-17) and samples were normalised to their corresponding total inputs with background subtraction (no-antibody control).Sort purified lymphocytes from LNs and spleen (~5 x 104 PFU of the HKx31 (H3N2) IAV strain in 30 μL of PBS [Mice were anesthetized by isofluorane inhalation and infected intranasally with 1x10L of PBS . Weights+CD44lo T cells (OT-I or Tnf -/-/OT-I) were sort purified  were taken for tetramer analysis at day 10 after infection.CD8purified  and 1x10bNP366 or KbPB1703) and APC-labeled MHCI tetramers (DbPA224 or DbPB1-F262) (University of Melbourne Tetramer Facility), stained with Fixable Live/Dead AquaBlue viability dye (Life Technologies), blocked with anti- CD16/32 mAb (clone 2.4G2), and stained with anti-CD3ε-PerCPCy5.5 , anti-CD8α-PacBlue  and anti-CD4-AF700 . Cells were acquired on a FACS Canto II flow cytometer (BD Biosciences), and data were analyzed by using FlowJo software (Treestar).Spleen or BAL from IAV-infected mice at day 10 were processed to single cell suspensions, red blood cells were lysed and the remaining leukocytes stained. We used PE-labeled MHCI tetramers (DAll experiments contained a minimum of 4 mice (4–10) and were repeated at least twice, with similar results. Each symbol represents either data from an individual sample or the mean of multiple samples. Error bars represent standard error of the mean (SEM). Mann-Whitney test with Bonferroni correction for multiple comparisons was used to compare multiple samples and statistically significant differences between groups are indicated as follows: ns = not significant (p>0.05), *p ≤ 0.05, or **p ≤ 0.01.+ T cells isolated from pooled lymph nodes of naïve WT mice. Cells were stimulated with anti-CD3 monoclonal antibody (mAb) in combination with co-stimulation from anti-CD28 mAb alone, anti-CD8/CD11a mAbs or anti-CD8/CD11a/CD28 mAbs. At 0, 5, 24, 48 and 72 h after stimulation, cells were harvested and the percentage of CD8+ T cells that produced TNF or IFNγ was assessed by flow cytometry . Total cell counts for a distinct but representative experiment were also determined  for histone modifications followed by quantitative PCR targeting the proximal promoter regions (PPRs) of Ifng and Tnf loci. We targeted histone 3 lysine 4 tri-methyl (H3K4me3) and H3K27me3 as these are associated with permissive or repressive chromatin structure and thereby transcriptional activity or inactivity, respectively [Tnf PPR at 5 h post-stimulation, corresponding to the peak frequency of TNF+ CD8+ T cells, and a subsequent removal (at 48 h) of the repressive H3K27me3 mark at this site . In contrast, the Ifng locus retained enrichment of the repressive H3K27me3 mark at 5 h but showed an increase in the relative enrichment of H3K4me3 at 48 h , corresponding with a substantial increase in the frequency of IFNγ+ cells at this time point . Binding of RNA polymerase II (pol II) is a further indication of transcriptional potential. Prior to stimulation the PPRs of Ifng and Tnf loci appeared poised for transcription, with both showing enrichment of pol II . Upon stimulation pol II binding was only retained at the Tnf PPR at 5 h, and was reduced but still present at 48 h. In contrast, pol II was lost from the Ifng PPR at 5 h, only becoming detectable again at 48 h, broadly corresponding to the relative kinetics of production of the two cytokines.The extremely rapid and robust production of TNF after CD8TNF mRNA . To deteectively , 21. The+ T cells after stimulation is, at least in part, controlled by the accessibility of local chromatin structure determined by deposition of key histone modifications at the PPR.Thus, these data suggest that the differential kinetics of TNF and IFNγ production by CD8+ T cell responses, we intranasally infected WT and TNF knockout mice (Tnf-/-) with the HKx31 (H3N2) strain of IAV . Global loss of TNF in influenza-infected mice led to an increased level of protein in cell-free bronchoalveolar lavage fluid (BAL) on d7 post-infection , typically used as an indication of loss of alveolar barrier integrity and accumulation of protein-rich fluid in the alveolar space [Fig 2C), or in viral clearance  between WT and Tnf-/- mice during primary influenza infection. We then analysed the endogenous IAV-specific CD8+ T cell response in the spleens and BAL of WT and Tnf-/- mice at day 10 (d 10) post-infection, which represents the peak of the acute response. To measure CD8+ T cell response magnitude, we used MHC class I tetramers, DbNP366, DbPA224, KbPB1703 and DbPB1-F262, to identify four influenza-specific populations. This analysis revealed significantly higher proportions  and a consistent trend toward increased absolute numbers  of IAV-specific CD8+ T cells in the spleen following infection of Tnf-/- mice. However, we found no noticeable difference in the proportions  or absolute numbers  of IAV-specific CD8+ T cells in the BAL of infected mice at d 10. Total splenic or BAL-derived CD8+ T cell numbers were not different between WT and Tnf-/- mice -specific TCR (OT-I) were transferred into either WT or Tnf-/- mice, followed by IAV infection  (gating strategy included as Tnf-/- mice that did not receive a transfer (No Tx) and we included WT OT-I→WT and Tnf-/- OT-I→Tnf-/- transfers to control for the effect of cell transfer . Both of these sets of controls were able to recapitulate our observations from Fig 3B) and numbers  of OVA-specific CD8+ T cells in the global absence of TNF in the spleen at d10 post-infection. In WT OT-I→Tnf-/- transfers, only responding CD8+ T cells can produce TNF and the OTI CD8+ T cell response was significantly higher than that observed when intrinsic and extrinsic TNF were present in WT OTI→WT transfers . This suggests that CD8+ T cell-derived TNF plays a modest role in attenuating the IAV-specific response. In Tnf-/- OT-I→WT transfers, CD8+ T cells selectively lack the capacity to produce TNF but the CD8+ T cell response magnitude was comparable to that observed in the complete absence of TNF in the Tnf-/- OT-I→Tnf-/- transfers . Finally, the loss of global, extrinsic or intrinsic TNF had no impact of the proportion of CD8+ T cells that were OVA-specific in the BAL . Again, this finding is consistent with + T cell- or extrinsically-derived TNF has no impact on local antigen-specific CD8+ T cell recruitment. Collectively, these data indicate that extrinsically-derived TNF has the dominant impact on antiviral CD8+ T cell responses by attenuating the magnitude in the secondary lymphoid organs, but CD8+ T cell-derived TNF can mediate a modest attenuation.A global absence of TNF affects lymphoid secondary structure and has a profound effect on a range of cell types and their function, especially given that the predominant sources of TNF are macrophages and dendritic cells , 23–25. Tnf-/- mice demonstrate that TNF is not needed for efficient clearance of virus from the lungs as it is not required for the antiviral activity of CD8+ T cells [+ T cells has also been shown to contribute to lung damage [+ T cell responses [+ T cell responses in the lungs [+ T cells can act in an autocrine fashion via TNFR2, to regulate the peak magnitude of the CD8+ T cell response and promote contraction [Tnf -/- mice.TNF is one of the major mediators of inflammation and its role during influenza infections has been described in several studies , 14, 26. T cells , 27, TNF T cells . With re T cells , 30. Theesponses , 32. In he lungs , and thi+ T cells in two waves; namely an immediate burst of TNF that peaks at ~5 h, followed by a more rapid accumulation of TNF production that continues to increase out to 72 h. Early TNF upregulation is consistent with an observation that TNF expression is necessary during priming of T cells in order to attenuate the subsequent effector response [+ T cell responses [+ T cell function [In this study, we showed, in accordance with previous studies , that TNesponses  and TNF esponses . Intrigufunction . In that+ T cells contain substantial levels of TNF, but not IFNγ, mRNA transcript [+ T cells after activation. It is difficult to compare these very early epigenetic changes and cytokine production characteristics immediately after in vitro stimulation with the events that occur in vivo. This is largely because in vivo cell division is initiated in very rare, antigen-specific T cell population 2–3 days after infection, which means cells cannot be readily detected in vivo until well beyond the timepoints analysed here. However, we have previously shown that OTI cells either activated in vivo by infection with HKx31-OVA or in vitro by a 5h peptide stimulation, produce TNF protein prior to IFNγ [Tnf and Ifng loci in naïve, effector (10 d) and memory (> 70 d) OTI cells has also been assessed [in vivo activated populations are not directly comparable to our analyses immediately after stimulation, naïve OTI cells exhibited elevated H3K27me3 deposition at the Ifng compared to Tnf locus, as well as detectable levels of H3K4me3 only at the Tnf, but not the Ifng, locus, consistent with our data and with rapid TNF production after stimulation.Apart from rapid epigenetic changes, we have also previously demonstrated that naïve OTI CD8anscript , which i to IFNγ , which sassessed . Althoug+ T cell responses was consistent with previous studies in several respects. Firstly, we found that TNF was not required for efficient clearance of virus from the lung [+ T cell response [+ T cell response magnitude, and thereafter to drive the contraction [+ T cell response.Our analysis of the impact of global loss of TNF on IAV-specific CD8the lung , 11, butthe lung , 11. Addresponse , 14. Coltraction  of the I+ T cell response magnitude in the lung [+ T cells in the spleen, but not in the lung. This suggests that TNF modulates the overall magnitude of the CD8+ T cell response but does not affect the recruitment of CD8+ T cells to the site of infection. This second discrepancy may be explained by the fact that previous studies investigated the role of TNF in response to relatively virulent strains of IAV, associated with high levels of virus replication and pulmonary inflammation. The HKx31 strain, in comparison, is known to induce a relatively mild infection when delivered intranasally. The requirement for TNF by the CD8+ T cell response has previously been shown to differ depending on the inflammatory context, with T cell responses to weak tumor antigens showing greater TNF dependence than a strong anti-LCMV response [Our study also contrasted with previous studies that had demonstrated that the loss of TNF increased the severity of influenza infection, as assessed by weight loss, and increased CD8the lung , 11. We response .+ T cell extrinsic versus intrinsic TNF during IAV infection demonstrates that CD8+ T cell-derived TNF mediates a modest attenuation of CD8+ T cell responses. This is supported by a previous study which concluded that intrinsic TNF potentiated the contraction of the CD8+ T cell response after viral clearance [+ T cell-derived TNF is not sufficient for full contraction of virus-specific CD8+ T cells and that extrinsically-derived TNF was largely responsible for this attenuation in the periphery. Thus, although intrinsically-derived TNF does not appear to play a critical role in this context of mild IAV infection, the fact that its role varies in a context-dependent manner means that an understanding of the kinetics and epigenetic control of TNF expression in CD8+ T cells is essential.Our dissection of the relative impact of CD8learance . Our stu+ T cell response following acute IAV infection, with extrinsically-derived TNF having the greatest impact on response magnitude and quality. This study also found that the effects of TNF were most apparent in the global peripheral response but had little effect on recruitment of CD8+ T cells to the site of infection.In conclusion, TNF is essential for attenuating the peak CD8S1 Fig(EPS)Click here for additional data file.S2 Fig(EPS)Click here for additional data file.S3 Fig(EPS)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Characteristics of these EV71 VLPs were studied using a variety of immunological and physicochemical techniques, which aimed to reveal that the purified EV71 VLPs have good morphology and structure consistent with natural EV71 empty capsids. Results of the amino acid analysis, SDS-PAGE, Western blotting and high-performance liquid chromatography confirmed the high purity of the EV71 VLPs. However the sedimentation coefficient of the VLPs showed that they were smaller than that of secreted EV71 VLPs purified by discontinuous cesium chloride density gradients, they were similar to the empty capsids of natural EV71 virions reported previously. Combined with the previous study that EV71 VLPs purified by a multistep chromatography process were able to elicit strong humoral immune responses in mice, our results further supported the conclusion that our EV71 VLPs had well-preserved molecular and structural characteristics. The EV71 VLPs produced from the baculovirus expression system and purified by a multistep chromatography process displayed key structural and immunological features, which would contribute to their efficacy as a HFMD vaccine.Human enterovirus 71 (EV71) is a major causative pathogen of hand, foot and mouth disease (HFMD) and has caused outbreaks with significant mortality among young children in the Asia-Pacific region in recent years. Towards developing a vaccine for this disease, we have expressed and purified EV71 virus-like particles (VLPs), which resemble the authentic virus in appearance, capsid structure and protein sequence, from insect cells (Sf9) using a multistep chromatography process. We demonstrated intracellular localization of the VLPs in host cells by  Human EV71, which is a non-enveloped, single positive-stranded RNA virus that is a member of the Enterovirus genus in the Picornaviridae family, has been identified as the major causative agent [Hand, foot and mouth disease (HFMD) is an infectious disease found in infants and young children worldwide and has become a major public health concern across the Asia-Pacific region since 1997. Although this illness is mild and self-limiting in most instances, cases caused by human enterovirus 71 (EV71) can result in severe neurological complications and even death among children under five years of age . In mainVarious vaccine candidates against EV71 virus, including inactivated vaccines , 11, livTM Core 700, CaptoTM Adhere resin and CaptoTM Butyl  columns, resulting in VLPs with ~31.52% yield and of > 95% purity [There are many VLPs of different viruses which have been produced and studied as candidate vaccines. Many VLP-based vaccines, such as influenza (Novavax), hepatitis B virus (Merck) and human papillomavirus (Merck) have been licensed. Given that VLPs resemble authentic virions in terms of their structural proteins and lack of a viral genome, they have been as promising candidates for vaccine development. Chung et al. , 19 have% purity . We also% purity . How the% purity , 24. Alt% purity , but theTM system  to co-express P1 and 3CD based on a Chinese endemic genotype C4 strain EV71, AH08/08 (GenBank no. HQ611148) to produce EV71 VLPs, as described previously [g for 30 min at 4°C, and the supernatant was obtained with a 0.45-μm cut-off filter  and then loaded onto chromatography columns for purification. A novel multistep chromatography process using CaptoTM Core 700, CaptoTM Adhere resin and CaptoTM Butyl  columns was used, resulting in EV71 VLPs with ~31.52% yield and of > 95% purity as previously described [The Sf9 insect cells derived from ATCC CRL-1711 were cultured in 2 L of Serum-Free Insect Cell Culture Medium (SFX) , which were infected with a recombinant baculovirus (Bac-P1-3CD) constructed using the Bac-to-Baceviously . The proescribed .4HCO3, alkylated with 50 mM indoleacetic acid (IAA) in 100 mM NH4HCO3 and divided into three parts. The three samples were digested for 12–16 h at 37°C with a solution of trypsin, chymotrypsin or endoproteinase Glu-C (protease-to-substrate ratio was 1:50). To identify the N-glycosylation sites of EV71 VLPs, a solution of PNGase-F (protease-to-substrate ratio was 1:20) was adopted to digest the purified VLPs samples at 37°C for 12–16 h. Thereafter, the molecular weights of the peptide fragments and their amino acid sequences were measured by tandem mass spectrometry (LC-MS/MS) by using Q Exactive . The HPLC system consisted of an Eksigent Nano2D LC  and an Acclaim® PepMap300 C18  . Electrospray MS/MS spectra were allocated to the EV71 VLPs primary sequence using Mascot 2.1.0  software, and an in-house protein sequence database was established. The mass tolerance on the parent and the fragment ions were 0.1 Da and 0.2 Da, respectively. The variable modifications allowed were carbamidomethyl (C), oxidation (M) and deamination (D and E). The N-glycosylation sites were determined by the quality drift (0.984 Da) of asparagine changed to aspartic acid [For the peptide mapping and N- and C-terminus sequencing by liquid chromatography-mass spectrometry, the purified EV71 VLPs samples were reduced with 10 mM dithiothretiol (DTT) in 100 mM NHtic acid .To identify the molecular weights of VP0, VP1 and VP3 capsid proteins of EV71 VLPs, the purified EV71 VLPs were mixed (1:1) with 5 mg/mL alpha-cyano-4-hydroxy-cinnamic acid (CHCA) containing 0.1% trifluoroacetic acid (TFA) and 50% acetonitrile. After mixing, 0.8 μL of the mixture was used for MS analysis, and then a MALDI-TOF/TOF mass spectrometer 4800 Proteomics Analyzer  was adopted. Data Explorer (TM) software (Applied Biosystems) was used to analyze the data.5 cells per well) onto 24-well plates 1 day prior to the experiment. On the second day, recombinant baculovirus Bac-P1-3CD diluted in SFX medium was added to each well at 1 multiplicity of infection (MOI), followed by incubation at 27°C for 24 h, 48 h or 72 h to observe the production of EV71 VLPs. Uninfected Sf9 cells were used as the blank control. Thereafter, the cells were prepared for immunostaining. In brief, 4% paraformaldehyde was used to fix cells at ambient temperature for 20 min, and then followed by treatment with permeabilization buffer (0.1%Triton X-100 in PBS) for 30 min. After cell fixation, the cells were blocked with PBS containing 5% bovine serum albumin (BSA) for 1 h and then incubated with the 1/50-diluted anti-VP1 rabbit polyclonal antibody for 1 h. Infected Sf9 cells incubated with the 1/50-diluted serum from rabbits immunized with PBS were used as the negative control to monitor non-specific staining. Next, the cells were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG  for 45 min. The PKH26 Red Fluorescent Cell Linker Kit  was used as the standard protocol to label the lipid bilayer of the cell membrane, followed by 4',6-diamidino-2-phenylindole (DAPI)  staining for 10 min. All incubation steps were carried out at 27°C, and the cells were washed thrice with PBS between steps. Finally, a Zeiss LSM 710 laser scanning confocal microscope  was used to test the stained samples at 400× magnification, and the Zeiss Zen Lite software was adopted to collect and analyze images.The Sf9 cells were seeded (5 × 103) were then cut and washed, and LR White Resin  was used to infiltrate the blocks for embedding at 4°C. The steps were: dehydration at 4°C in 50% ethanol (20 min); at 4°C in 70% ethanol (2 × 30 min) followed by a 1:2 mixture of 70% ethanol and LR White (60 min), LR White (60 min); then two changes of pure resin . After these processes, the blocks were first transferred into fresh LR White with LR White accelerator (2 μL/mL) capsules and then polymerized at 50°C for 24 h, which were trimmed to cut into 70 nm-thick sections using the LEICA EM UC7 ultramicrotome. Sections were mounted on formvar that was coated nickel grids (Gilder 200 mesh), and were then rehydrated by floating the grid on a droplet (20 μL) of PBS contained BSA  for 30 min at ambient temperature. A droplet of anti-VP1 rabbit polyclonal antibody, prepared in-house as previously reported [Pellets of EV71 VLP-producing cell cultures infected for 72 h were resuspended after centrifugation, and were then washed thrice with PBS before fixing for 2 h at room temperature in 4% paraformaldehyde. To remove the fixative agent, we washed the cell suspension thrice with PBS. The cells were resuspended in PBS rapidly and then centrifuged for 2 min at ambient temperature. Small blocks (<1 mmreported  and diluPurified EV71 VLPs were first treated with reducing SDS-PAGE sample buffer (pH 6.8) and run through a 13.5% separating gel. After the run, the electrophoretic profiles were revealed by silver staining with standard protocols. A Bio-Rad model 583 gel dryer  was used to dry the gels. Proteins (3 μg/well) were loaded for silver staining.After electrophoresis, the purified EV71 VLPs were then transferred onto nitrocellulose membranes (Bio-Rad) for Western blotting, and the membranes were incubated with an anti-VP1 rabbit polyclonal antibody, anti-VP2 mouse polyclonal antibody detecting VP0 protein or anti-VP3 mouse polyclonal antibody (prepared and supplied by our laboratory) for 90 min at ambient temperature. After incubating with the primary antibody, the membranes were incubated with the appropriate alkaline phosphatase (AP)-labeled secondary antibody  at ambient temperature for 45 min. The AP substrate solution of nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Sigma) was used to incubate at ambient temperature for 5 min, and distilled water was finally adopted to wash the membrane. The presence of VP0, VP1 and VP3 capsid proteins of EV71 VLPs in the purified sample was confirmed.2HP04 containing 100 mM NaCl (pH 7.4) before loading 50 μL of our final sample at 15 mL/h.For HPLC, as our previous study the HPLC TSK gel® G5000  column was applied to determine the purity of our EV71 VLPs samples , which wTo observe the morphology of VLPs, the EV71 VLPs in suspension were examined using an H-7650 (Hitachi) transmission electron microscope at 80 kV, the specific procedure was detailed as our previous study .The morphology of EV71 VLPs were studied by AFM. Following a published protocol , 0.01%  was adopted to determine the hydrodynamic diameter of EV71 VLPs. Measurements were carried out at 25°C using three 10 × 10-sec datasets. All data here are averages of three measurements of the same sample .For cryo-TEM imaging of EV71 VLPs, a 3.5 μL aliquot of EV71 VLPs (1.5 mg/mL) was applied to glow-discharged Quantifoil R2/2 , blotted for 2 s in a 100% humidity chamber under 0 blot force at 22°C and then plunged into liquid ethane in an FEI Vitrobot Mark IV vitrification robot. EV71 VLPs were imaged in an FEI 200-kV TECNAI SPIRIT transmission-electron microscope.A Beckman Coulter Proteome Lab XL-I analytical ultracentrifuge with a 4-hole An-60Ti rotor was used to determine the sedimentation coefficient distribution of EV71 VLPs (0.3 mg/mL) contained in 0.1 M PBS, and a continuous scan mode was adopted to measure the sedimentation at 15,000 rpm in 12-mm double-sector cells at 20°C (measured cell radius from 5.9 cm to 7.15 cm), the absorbance at 280 nm. The data in c(s) mode was analyzed by the program Sedfit to show a sedimentation coefficient distribution apparently, and the c(s) calculations were determined by the 3th to 40th scans of EV71 VLPs.® PepMap300 C18 column  was employed for HPLC. The amino acid sequences of VP0, VP1 and VP3 were gained by translation of the corresponding DNA sequences (176 (To verify sequences of the BEVS-produced EV71 VLPs capsid proteins, their amino acid sequence was determined by using Eksigent Nano2D LC and Q Exactive after protein hydrolysis of EV71 VLPs. The Acclaimequences . More thequences . Determices (176 .in situ immunogold staining of baculovirus-infected cells using anti-VP1 polyclonal rabbit antibodies specific to EV71 VLPs (In our study, anti-VP1 rabbit polyclonal antibody labeled by green fluorescence dye or gold particles were mainly detected in the cytoplasm of infected Sf9 cells, which indicated that EV71 VLPs were mainly expressed in cytoplasmic locations of the host cells. Thus, VLP formation occurred inside the Sf9 cells when they were infected with 1 MOI of the recombinant baculovirus Bac-P1-3CD. These findings were determined by immunofluorescence and V71 VLPs . CompareV71 VLPs . Hence, V71 VLPs , which mV71 VLPs . At thisTM system co-expressing P1 and 3CD derived from a Chinese endemic genotype C4 strain EV71 was generated as previously reported [et al. [In an attempt to produce and purify EV71 VLPs, a Bac-to-Bacreported . The recreported . Differe [et al. . These bA representative TEM image is provided in Cryo-TEM, which allows for direct imaging of fully hydrated biological specimens in a near-physiological environment, revealed that the EV71 VLPs were 28.74 nm in diameter , similarIn this work, evidence is presented that EV71 VLPs of the C4 strain produced from the BEVS and purified through our multiple chromatography process retained key molecular and structural features for the first time, compared with those reported for native empty virions  and othe176. This direct investigation into the glycosylation of BEVS-produced EV71 VLPs was important, and as their expression in eukaryotic cells resulting in post-translational modifications may affect their folding, localization, solubility and antigenicity [176 may need further study to verify whether the identified N-glycosylation site in EV71 VLPs at the VP1 residue N176 is essential. Using the site-directed mutagenesis, changing N176 into G176, we will test the effect of N-glycosylation site on the EV71 VLPs assembly used by TEM and AUC and HPLC, and on the humoral or cellular immune responses through immunization of BALB/c mice injected intraperitoneally.Analysis of mass and amino acid sequences suggested that the amino acid sequences of the EV71 VLPs produced by BEVS were consistent with the native empty capsid at the hydroxyl- and carboxy-terminal ends. Moreover, no mutations were observed in the peptide mapping of VP0, VP1 and VP3 subunits of the purified EV71 VLPs. Since insect cells infected with recombinant baculoviruses can usefully express recombinant proteins, particularly when high production levels and post-translational modifications of the protein are desired , a detergenicity . Proteingenicity , 41. Mamgenicity . Even usgenicity . Hyakumugenicity  have genRecombinant EV71 VLPs produced from yeast  and Sf9 The purified EV71 VLPs, with a diameter of approximately 38 nm as analyzed by DLS  and the 15 neutralization antibody titer and 100% immune protection against EV71 homologous virus attacks in suckling mice could be elicited [The culture and purification methods may also influence the antigenicity and immunogenicity. Interestingly, although a difference between the sedimentation coefficient of our EV71 VLPs and that previously reported , these Velicited , supportTaken together, this study located the distribution of EV71 VLPs expression at the cellular level, and our results demonstrated that EV71 VLPs produced from the BEVS and prepared through a multi-step purification process retained good characteristics as a vaccine antigen. Because of the well-characterized EV71 VLPs antigen tested in this study, the EV71 VLPs prepared by us for vaccine antigen is expected to lay a foundation for the vaccine against HFMD research and development.


The basic mechanism of the major neurodegenerative diseases, including neurogenic pain, needs to be agreed upon before rational treatments can be determined, but this knowledge is still in a state of flux. Most have agreed for decades that these disease states, both infectious and non-infectious, share arguments incriminating excitotoxicity induced by excessive extracellular cerebral glutamate. Excess cerebral levels of tumor necrosis factor (TNF) are also documented in the same group of disease states. However, no agreement exists on overarching mechanism for the harmful effects of excess TNF, nor, indeed how extracellular cerebral glutamate reaches toxic levels in these conditions. Here, we link the two, collecting and arguing the evidence that, across the range of neurodegenerative diseases, excessive TNF harms the central nervous system largely through causing extracellular glutamate to accumulate to levels high enough to inhibit synaptic activity or kill neurons and therefore their associated synapses as well. TNF can be predicted from the broader literature to cause this glutamate accumulation not only by increasing glutamate production by enhancing glutaminase, but in addition simultaneously reducing glutamate clearance by inhibiting re-uptake proteins. We also discuss the effects of a TNF receptor biological fusion protein (etanercept) and the indirect anti-TNF agents dithio-thalidomides, nilotinab, and cannabinoids on these neurological conditions. The therapeutic effects of 6-diazo-5-oxo-norleucine, ceptriaxone, and riluzole, agents unrelated to TNF but which either inhibit glutaminase or enhance re-uptake proteins, but do not do both, as would anti-TNF agents, are also discussed in this context. By pointing to excess extracellular glutamate as the target, these arguments greatly strengthen the case, put now for many years, to test appropriately delivered ant-TNF agents to treat neurodegenerative diseases in randomly controlled trials. Journal of Alzheimer’s Disease entitled “Time to Dismount”  eloquently again brings to the fore the long-held, widespread, and increasing unease among researchers [The amyloid theory of Alzheimer’s disease, and by extension other chronic neurodegenerative states, has dominated the field for decades. It has, however, in the face of the reality of numerous large clinical trials yielding no clinical improvement, lost momentum. A recent item on the Editor’s Blog of the webpage of the earchers –5. Likewearchers , 7. Receearchers , furtherWe have recently  reviewedWaning enthusiasm for the amyloid theory now allows many other approaches, including the last 10 years of animal studies, case reports, open trials, and off-label treatments of neurodegenerative diseases, based on neutralizing excessive levels of TNF within the brain, to receive more attention. Unaccountably, this neglected approach to neurodegenerative disease is sometimes still referred to as highly controversial . This reAs well as providing sufficient background to enable the bigger picture of TNF in brain disease pathogenesis to be understood, we focus here on the implications of newer data, largely neglected in the world of neurodegenerative disease, on how this cytokine evidently controls levels of extracellular glutamate in the synaptic cleft. In brief, glutamate is the chief physiological excitatory neurotransmitter, essential of course in memory and learning, and indeed is functionally involved in virtually all activities of the nervous system. Glutamate’s combination of functional importance and toxicity demands tight control over its release and re-uptake. Thus, as will be discussed, control by TNF of both of these functions gives treatments based on reducing excess cerebral levels of this cytokine a solid therapeutic foundation in neurodegenerative disease, in part because of its essential effects in driving excitotoxicity. In practice, we may usefully view TNF toxicity and glutamate toxicity as two perspectives on the one pathophysiological entity.TNF was recognized, and named, as an endogenous tumor killing agent , and 6 yAplysia kurodai, was observed to be reduced by human TNF [The extraordinarily broad relevance of TNF in biology can now be inferred by its strongly conserved state, traceable back through a remarkably ancient lineage including fish and insects, with the form generated by reef-building corals, and the TNF receptors on their cells, co-recognizing their human counterparts . Unsurpruman TNF , 27. Nexuman TNF . Neverthuman TNF –31 contiTwenty years ago, the involvement of unchecked chronic TNF generation, particularly within the brain, in the pathogenesis of stroke, traumatic brain injury (TBI), and AD began to be apparent –34. RefiThe rationale for treating chronic neurodegenerative states by reducing excess cerebral TNF extends far beyond the post-stroke syndrome, AD, and TBI noted above. Despite “belonging” to various disciplines, these cerebral states characterized by TNF excess clearly have much pathophysiology in common. They include Table  Parkinsol-glutamate, the most abundant extracellular amino acid in the brain, is, as reviewed over the decades [ decades –67, the  decades , as well decades , 70, as As reviewed , 71, shoAs reviewed in 2001 , glutamaIn the event, TNF became much more heavily incriminated in glutamate regulation than has been taken into consideration in the above models of excitotoxocity Fig. . Ten yeaIn passing, we note that glutamine deficiency is a long-recognized characteristic of chronic inflammatory stress and has made nutritionally motivated i.v. glutamine replacement therapy a routine, if formally untested, adjunct treatment in critical care wards . HoweverThe above data on TNF provide insights into the breadth of therapeutic relevance of the functional link between unbridled TNF production and glutamate neurotoxicity and how this adds immensely to the central argument of this review that TNF is a highly logical target in neurodegenerate disease. As but one example, the capacity of excess TNF to greatly increase glutamate output through activating glutaminase , 105 casImportantly, intra-amygdala infusion of TNF has been reported to elevate glutamate levels in this region of the brain . LikewisControl of glutamate by TNF might also explain why etanercept has often been reported to reverse a number of clinical manifestations of neurodegenerative disease surprisingly rapidly. It was shown 17 years ago  that turWhereas infliximab and adalimumab are essentially monoclonal antibodies directed at TNF itself, etanercept, the only anti-TNF biological drug yet tested in this context, is a fusion protein consisting of the p75 TNF receptor, joined to the constant end of the IgG1 antibody . An etanThe use of anti-TNF agents in neurodegenerative disease has its critics who largely base their concerns on whether the functional complexities of TNF science, such as the p55 and p75 TNF receptors and membrane versus soluble location of TNF, should be more fully elucidated beforehand , 135. ThAs other therapeutic molecules, the biological anti-TNF agent etanercept, though employed widely with great success systemically, is, as often noted, too large to cross the BBB in significant amounts unaided. Two BBB-crossing techniques now exist side by side in the literature, the earliest and simplest from a small group, the later technically complex, and not yet tried in patients. Here, we summarize their origins and rationale.Understanding the perispinal delivery of large molecular weight drugs into the central nervous system requires an appreciation of the cerebrospinal venous system. As recently reviewed in detail , this roAn alternative method of delivery of large molecules into the brain exists –147, butThirty years after being removed from the market in 1961 because of its disastrous effects on fetal development, thalidomide had begun to be explored to treat a number of intractable conditions in patients other than child-bearing age women. It was shown to selectively inhibit TNF production by stimulated human monocytes , and to Nilotinib, a tyrosine kinase inhibitor, is 30 times more potent than imatinib, which it is replacing for treating certain leukemias , 165. A The capacity of nilotinib (and indeed dasatinib ) to inhiHow might nilotinib reduce TNF levels? Tyrosine phosphorylation is central to TLR stimulation and subsequent activation of NF-kappaB  that genCannabis sativa have fascinated researchers for about two centuries. About 90 phytocannabinoids  have been identified, the two with the largest literatures being tetrahydrocannabinol (THC) and cannabidiol (CBD). The former is psychotropic and thus under a legal cloud, although a synthetic trans-9-delta isomer, termed dronabinol, is an example of forms of THC nowadays undergoing limited investigation [As reviewed , the thetigation . CBD, intigation , 185 forBoth endocannabinoids and CBD have been shown to be active in models for pain –190, AD Again, the list of model conditions investigated for therapeutic use of cannabinoids in the previous paragraph remarkably mirrors the list of previously discussed conditions associated with excessive cerebral extracellular levels of glutamate. Moreover, treatment with cannabinoids or altering the function of their cellular receptors , 216–221Plasmodium berghei ANKA [DON, a glutamine analogue, is studied largely with a view to reduce extracellular glutamate, and thus treat glutamate toxicity, through inhibiting glutaminase. Having been earlier shown  to possehei ANKA , and DONhei ANKA , 227. Ithei ANKA  and in bhei ANKA .These new data on DON and the relationship between TNF and glutamate excess through glutaminase enhancement put historic observations on malarial encephalopathy into clearer focus. As has been reviewed , malariaToxoplasma gondii-infected mice [T. gondii is a well-known TNF inducer. Being a pathogen, it possesses the PAMP activity discussed earlier.Ceftriaxone is a broad-spectrum beta-lactam antibiotic, largely reserved, in this context, for use against otherwise resistant bacteria. In contrast to cannabinoids and nilotinib, it has been shown not to reduce TNF release from LPS-treated human monocytes , implyinted mice . The autRiluzole (6-(trifluoromethoxy)benzothiazol-2-amine), a relatively toxic material nevertheless approved for treatment of ALS, has for some time been known to be a glutamate release inhibitor and thus affecting the glutamate functions discussed above. This has been reported to include enhancing levels of glutamate re-uptake transporters , includiAs discussed earlier, neurodegenerative diseases are characterized by excessive levels of extracellular cerebral glutamate that can be expected to have accumulated through its too rapid formation as well as its slowed re-uptake and conversion back to glutamine. The ideal therapy would be able to reverse both of these changes. So far as we are aware, TNF is the only endogenous mediator that, when in excess, as in the brain in these diseases, enhances cerebral glutaminase  and alsoSpecific anti-TNF biologicals are expensive, but can be very effective through neutralizing a precise target, in this case excessive cerebral TNF, known to be central to the disease in question. As discussed earlier, their large size need not be a problem. In contrast, pharmaceuticals such as dithio-thalidomides, nilotinib, cannabinoids, DON, ceftriaxone, and riluzole, although less expensive, may prove to be burdened with unknown targets that generate greater side effects than anti-TNF biologicals can. They have, however, a considerable advantage in neurodegenerative disease in that when administered orally or systemically, they can traverse the BBB and get to where they are needed , 231.We propose that the excess levels of TNF, and glutamate in the brain across a range of neurodegenerative diseases are crucially linked, high TNF causing extracellular glutamate to accumulate to levels high enough to inhibit synaptic activity and kill neurons by two synergistic mechanisms. As described, these are increasing glutamate production by enhancing glutaminase and simultaneously reducing glutamate clearance by inhibiting re-uptake proteins, thus causing it to accumulate in the synaptic cleft. The shared efficacy of specific anti-TNF biologicals and non-specific anti-TNF agents  on this superficially diverse range of conditions can thus be understood. The usefulness of DON, ceftriaxone, and riluzole, agents without apparent anti-TNF activity, but each possessing separate activities that counter one of these two influences of high TNF on glutamate accumulation, are similarly rationalized.


Xionghuang, in Chinese). It contains over 90% tetra-arsenic tetra-sulfide (As4S4). Currently, realgar has been confirmed the antitumor activities, both in vitro and in vivo, of realgar extracted using Acidithiobacillus ferrooxidans (A. ferrooxidans). Bioleaching, a new technology to greatly improve the use rate of arsenic extraction from realgar using bacteria, is a novel methodology that addressed a limitation of the traditional method for realgar preparation. The present systematic review reports on the research progress in realgar bioleaching and its antitumor mechanism as an anticancer agent. A total of 93 research articles that report on the biological activity of extracts from realgar using bacteria and its preparation were presented in this review. The realgar bioleaching solution (RBS) works by inducing apoptosis when it is used to treat tumor cells in vitro and in vivo. When it is used to treat animal model organisms in vivo, such as mice and Caenorhabditis elegans, tumor tissues grew more slowly, with mass necrosis. Meanwhile, the agent also showed obvious inhibition of tumor cell growth. Bioleaching technology greatly improves the utilization of realgar and is a novel methodology to improve the traditional method.Realgar is a naturally occurring arsenic sulfide (or  Niu Huang Jie Du Pian released into the blood system . Fr. FrA. fe in Eqs.  73]. Th. ThA. feModerate ferric ions were useful for efficient bioleaching. As an oxidant, ferric ions can promote the dissolution of realgar. These reactions can be characterized by Eqs. , 4), ,  49]. An. An49]. + includes metallic ions, such as As3+, K+, Na+, NH4+ or H3O+.XA. ferrooxidans. The reactions can be characterized by Eq. , the agent showed an obvious antitumor effect in vivo and in vitro and a significantly higher efficiency and safety than the raw realgar powder and other arsenic compounds . It has 180 cells). RBS remarkably induced cell apoptosis through inhibition of cell cycle pathways. This effect is expected to increase in a time- and concentration-dependent manner. Similarly, RBS has the exact same effect in Kunming mice.The arsenic concentration of RBS is approximately 15-fold 10.40–156.23 mg/l) higher than the sterile control [.23 mg/l Caenorhabditis elegans (C. elegans), the LC50 value of RBS was surpass the value of ATO which indicated that after bioleach the toxicity of RBS was less than other arsenic compounds such as ATO, raw realgar and the NPS. Meanwhile, as can be seen from the graph, there is a obvious increase in toxicity and bioavailability when processing raw realgar particles to the nano-size.RBS have a higher solubility and less toxicity when compare with raw realgar and ATO. Furthermore, the antitumor effect of it was enhanced, significantly Table . The mecIn order to prove the antitumor effect of the RBS in vivo the cancer cells was transplanted to the Kunming mice. Then, treat the bearing mice with RBS in difference concentration respectively. Two obvious curative effects were discovered in mice: the weight of tumor growth was less than that of the normal group, and the tumor inhibition rate (at 3.24 mg/kg) reach to 32.2%; additionally, RBS can extended mouse lifetimes, and the life extending rate (3.0 mg/kg) is 118% . More imCaenorhabditis elegans is a model organism that is used to measure the pharmacological activity of anticancer drugs. RBS can suppress the ras gene in C. elegans. Ras gene, one of the characteristic gene relate with cancer. The Ras/MAPK signaling pathway has associated with proliferation, growth, and other living activity of the germ line [C. elegans it will lead to the wild type become the multivulva (Muv) phenotypes [C. elegans absorbed RBS into their body the mutant phenotype was affected and the brood size decreased significantly. The research point out that RBS can inhibit the excessive expression of genes and was be most probably in a promising anti-tumor drug candidate. Meanwhile, RBS has less toxicity in C. elegans compared to ATO. The LD50 value of RBS is 81 μg/mL compare with the values of 55 μg/ml of ATO [erm line , 80. If enotypes . Therefol of ATO .C. elegans or cancer cells. There is a recognizable increase in the levels of ROS in cancer cells, and the level of intracellular ROS accumulation in the experimental group worms is two times (6.4 μg/ml) that of the control [The mechanism of apoptosis of RBS was verified when utilized in some tumor cells. RBS displayed significant activity against CML. RBS shown an obvious inhibiting effect in K562 cells (K562:CML cell line) in vitro cell culture experiment The research suggested that RBS might be a promising agent against CML of apoptosis and showed less toxicity and more effectiveness against K562/ADM cells compared with arsenic trioxide (ATO) and raw realgar . The con control . which s control . Further control .A more detailed description is that an arsenic ion in RBS was transported into intracellular by AQP9 (one of the aquaporin water channel family), When the arsenic ions transported into cytoplasm the expression of AQP9 was enhanced. That change increased the uptake of arsenic ions. Another kind of change is that the level of P-gp (a protein relate to the excretion of arsenic species) was suppressed which attenuate the metabolism of arsenic in cancer cells particularly. Those above variation of the cells clearly contributed to the increase of arsenic concentration in the cancer cell. And most of the arsenic was transported to the endoplasmic reticulum.The mechanism of RBS was inducing of apoptosis by increasing the concentration of ROS in the cancer cells. As stated previously, arsenic ions have been converted to the endoplasmic reticulum. The increased of arsenic could inhibit the cellular antioxidant defense system which led to abundant ROS accumulating. On one hand, the ROS accumulating can led the production of DNA damage. Once that happens, the gene of P53 and CADD45A will be activated which bring out the levels of protein expression increase in some certain apoptosis promoting proteins such as Bax, cyclin B1 and Cdc2, and the decrease in anti-apoptosis protein such as Bcl-2. On the other hand, ROS accumulating can make the effect of depolarization on the outer membrane of mitochondrial by the changes of Bax and Bcl-2. It will lead to the release of cytochrome c into the cytosol. For this reason, the level of pro-caspase-3 (Caspase-3): The important enzyme in the process of cell apoptosis) decreased and the cleaved-caspase-3 increased. Meanwhile, the obvious enhancement of the level caspase-3 was also observed in RBS treatment group. In short, the above two findings suggest that the apoptosis of the cancer cell by RBS via controlling the activation of P53 and other related proteins . The posNiuhuang Jiedu Wan, Jufang Zhibao San, Fufang Qingdai Pian, Angong NiuHuang San, Wansheng Huafeng Dan, among others, containing realgar [Compared with traditional Chinese patent medicine, the novel medicine containing RBS has great advantages in application. First, the pharmacological activity was greatly improved and side effects, such as toxicity and gastrointestinal tract irritation, were significantly reduced, . Chinese realgar –92. Beca realgar . Replace realgar . Moreove realgar . The nov realgar , 82. TheA. ferrooxidans for bioleaching. A. ferrooxidans was cultured in medium at pH 1.5 and 35 °C, with 20% v/v inoculum, a ferrous concentration of 1.0 g/l and a 1.5% w/v pulp density. Under these conditions, the efficiency of bioleaching was highest and the function of bacteria was maximal. Furthermore, RBS showed an obvious antitumor effect in vivo and in vitro through induction of apoptosis. More importantly, RBS had a higher efficiency and safety than raw realgar powder and other arsenic compounds.The solubility of realgar was increased significantly when using


Extracellular vesicles (EVs) are membrane-contained vesicles shed from cells. EVs contain proteins, lipids, and nucleotides, all of which play important roles in intercellular communication. The release of EVs is known to increase during neuroinflammation. Glutaminase, a mitochondrial enzyme that converts glutamine to glutamate, has been implicated in the biogenesis of EVs. We have previously demonstrated that TNF-α promotes glutaminase expression in neurons. However, the expression and the functionality of glutaminase in astrocytes during neuroinflammation remain unknown. We posit that TNF-α can promote the release of EVs in astrocytes through upregulation of glutaminase expression.N-acetyl-l-cysteine, or genetic knockout of glutaminase, suggesting that glutaminase plays an important role in astrocyte EV release during neuroinflammation.Release of EVs, which was demonstrated by electron microscopy, nanoparticle tracking analysis (NTA), and Western Blot, increased in mouse astrocytes when treated with TNF-α. Furthermore, TNF-α treatment significantly upregulated protein levels of glutaminase and increased the production of glutamate, suggesting that glutaminase activity is increased after TNF-α treatment. Interestingly, pretreatment with a glutaminase inhibitor blocked TNF-α-mediated generation of reactive oxygen species in astrocytes, which indicates that glutaminase activity contributes to stress in astrocytes during neuroinflammation. TNF-α-mediated increased release of EVs can be blocked by either the glutaminase inhibitor, antioxidant These findings suggest that glutaminase is an important metabolic factor controlling EV release from astrocytes during neuroinflammation.The online version of this article (doi:10.1186/s12974-017-0853-2) contains supplementary material, which is available to authorized users. In the central nervous system (CNS), abnormal protein aggregation, autoimmunity, and infection can lead to neurodegenerative conditions, such as amyotrophic lateral sclerosis (ALS) , AlzheimGls and Gls2, presumably derived from a common ancestral gene by duplication and divergent evolution [Glutamate metabolism is one of the major metabolic pathways in the CNS. Glutaminase , an enzyme localized in the inner membrane of the mitochondria, is a rate-limiting enzyme in glutamate metabolism that catalyzes the conversion of glutamine to glutamate and ammonia . Glutamivolution . Furthervolution –14.Extracellular vesicles (EVs) are membrane-contained vesicles that include exosomes, microvesicles, and apoptotic bodies . EVs canN-acetyl-l-cysteine, or using astrocytes that had genetic knockout of glutaminase (Gls−/−) reduced TNF-α-mediated EV release, suggesting that glutaminase is required for EV release in astrocytes during neuroinflammation. Understanding the mechanism of EV release in astrocytes during neuroinflammation is important in identifying novel therapeutic targets in the relevant neurological diseases.To determine the mechanism of EV release during neuroinflammation, we used TNF-α to stimulate primary mouse astrocytes. TNF-α induced the increased release of EVs and elevated protein levels of GLS in mouse astrocytes. Furthermore, TNF-α induced the generation of reactive oxygen species (ROS) in astrocytes through GLS. Treatment with either a glutaminase inhibitor, an antioxidant N-Acetyl-l-cysteine , 6-Diazo-5-oxo-l-norleucine , BPTES (SML0601), and GW4869 (D1692) were obtained from Sigma-Aldrich.C57BL/6J mice were housed and maintained in the Comparative Medicine Facility of the Tongji University School of Medicine . All procedures were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee at the Tongji University School of Medicine. Postnatal (P1 to P2) brain tissues were used for mouse astrocyte cultures. Gls knock in mouse embryonic stem (ES) cells were obtained from the Knockout Mouse Project (KOMP) Repository . Recombinant mouse TNF-α and IL-1β were obtained from R&D Systems. v/v) and penicillin/streptomycin (1% v/v). Cultures were maintained in a humidified chamber . After 7 to 10 days, the astrocytes were harvested by trypsinization.Cortices of C57BL/6J mice were dissected and mechanically dissociated using forceps to remove the membranes and large blood vessels. Brain tissues were digested by Trypsin-EDTA (Life Technologies) and then plated on cell culture flasks in Dulbecco’s modified Eagle medium Nutrient Mixture F-12 (DMEM/F-12). Culture medium was supplemented with FBS .The method for the isolation of extracellular vesicles has been described previously . EVs werExtracellular vesicles were characterized at 25 °C using Nano ZS90 . Eighty microliter samples were loaded into a microcuvette  for measurement.The size and number of extracellular vesicles were assessed with NanoSight NS300 system . Astrocytes were cultured in 6-cm culture dishes. At 24 h after medium change, EVs were isolated from normalized volumes of serum-free culture supernatants through differential centrifugation and resuspended with 150 μl PBS. The supernatant was diluted at 1:100 in PBS, and 1 ml solution was used for NanoSight analysis.Mouse astrocytes were grown on a glass coverslip, fixed with 2.5% glutaraldehyde, and washed three times with PBS. The cells were then dehydrated in a series of increasing ethanol concentrations and transferred for critical drying. After coating with platinum/palladium using a sputter coater, the sample was imaged with a scanning electron microscope .EVs were negatively stained and then spread on the copper grids. The droplets of EVs were removed with filter paper and air-dried at room temperature. Images were obtained using transmission electron microscopy .Cells or EV pellets were lysed in M-PER mammalian protein extraction reagent (Thermo Scientific). Proteins from lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoretic transfer to polyvinyldifluoridene (PVDF) membranes , proteins were treated with purified primary antibodies for glutaminase , GFAP , β-actin , flotillin-2 , and ALG-2 interacting protein (Alix)  overnight at 4 °C followed by a horseradish peroxidase-linked secondary anti-rabbit or anti-mouse antibody . Antigen–antibody complexes were visualized by Pierce ECL Western Blotting Substrate. Mitochondrial protein was isolated by the Mitochondria Isolation Kit for Cultured Cells . ATP5A  was used as a mitochondrial marker to indicate that there is no mitochondria contamination in the cytosol.Mouse astrocytes cultured on cover glasses were fixed with 4% PFA, rinsed with PBS, and then blocked by 2% BSA in PBS. Cells were incubated overnight at 4 °C with primary antibodies anti-GFAP . Cover glasses were washed and incubated for 1 h at room temperature with secondary antibodies including anti-mouse IgG . Nuclear DNA was stained with DAPI. Cover glasses were mounted on glass slides with mounting buffer (Sigma-Aldrich). Morphological changes were visualized by a Zeiss 710 confocal laser scanning microscope.2 and then were washed with PBS. The ROS were determined using conventional fluorescence microscopy (ZEISS).ROS measurement was assayed by dichloro-dihydro-fluorescein diacetate (DCFH-DA). Mouse astrocytes were incubated in 10 μM DCFH-DA  for 30 min at 37 °C, 5% COIntracellular glutamate detection was performed with the Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit from Invitrogen following manufacturer’s procedure. High performance liquid chromatography (HPLC) analysis for extracellular glutamate was performed as previously described .Briefly, mouse astrocytes were treated with TNF-α for 24 h, the cell viability was assayed by cell viability assay kit . The luminescent signal recording from the reaction was as the standard of cell viability.p < 0.05, p < 0.01, and p < 0.001 in comparison to control, respectively. #, ##, and ### denote p < 0.05, p < 0.01, and p < 0.001 in comparison to TNF-α-treated groups, respectively.Data were evaluated statistically by the analysis of variance (ANOVA), followed by Tukey’s test for multiple comparisons. Data were shown as mean ± SD. *, **, and *** denote Mouse astrocytes were established from the cortices of neonatal mice as previously described  mice were used. Since Gls−/− mice died shortly after birth , Gls−/− Taken together, our data unveiled a relationship between glutaminase and EV release in mouse astrocytes. TNF-α treatment upregulated protein levels of GLS and increased the production of ROS. GLS and ROS could influence each other, and both of them could promote EV release.First, TNF-α treatment significantly promoted the release of EVs in mouse astrocytes. Second, TNF-α upregulated the protein level of glutaminase and pretreatment with a glutaminase inhibitor blocked TNF-α-mediated generation of ROS in astrocytes. Last, the TNF-α-mediated increased release of EVs can be inhibited by either the glutaminase inhibitor, antioxidant NAC, or genetic knockout of glutaminase. These observations suggest that GLS may contribute to EV release in mouse astrocytes.In this study, our results present three important new findings regarding EV release. EVs are associated with the message delivery of the nervous system and may involve in the pathogenesis of many neuroinflammatory disorders, both infectious and neurodegenerative . HoweverGlutaminase is one of the key enzymes in cell metabolism. We have previously studied the effects of neuron, macrophage, and microglia GLS in brain injury, infection, and inflammation , 29, 43.Astrocytes use gliotransmitters to modulate neuronal function and plasticity. Recently, others discovered a fundamentally different form of long-term potentiation (LTP) that is induced by glial cell activation and mediated by diffusible, extracellular messengers, including D-serine and tumor necrosisfactor (TNF), and were spread widely in nociceptive pathways . AstrocyIn summary, we have identified glutaminase as an important player in mediating EV over-release during inflammatory cytokines stimulation. EVs in inflammatory stress may be associated with the occurrence and development of the disease and cellular immune regulation. Uncovering the important role of glutaminase in inflammation-induced EV release, GLS may provide a potentially pathological mechanism in neuroinflammation, and a possible therapeutic target of inflammatory brain diseases.In summary, our studies further explored the question about the cellular mechanisms of EV release, implicating an important role of glutaminase. These new findings support the notable role of glutaminase and suggest a potential regulatory mechanism in which glutaminase may represent a promising therapeutic target that mediates CNS inflammation.Additional file 1: Figure S1.Dynamic light scattering measurements demonstrate that treatment of mouse astrocytes with TNF-α does not affect the distribution of EVs. a Immunofluorescent staining for GFAP (red) in primary mouse astrocytes. Scale bars all indicated 50 μm. b A viability assay for astrocytes treated with TNF-α after 24 h. c Western Blot for calreticulin in astrocytes and EVs, calreticulin was a marker of endoplasmic reticulum, actin was a loading control. d EVs were isolated from serum-free culture of control and TNF-α-treated group after 24 h, and the size of EVs were determined by dynamic light scattering (DLS). The results were shown by intensity percent. (PDF 1048 kb)Additional file 2: Figure S2.TNF-α induces GLS release from the mitochondria in mouse astrocytes. a After 72 h stimulation, mitochondria from mouse astrocytes were isolated, and then subjected to Western blotting analysis using anti-glutaminase and anti-ATP5A antibodies. ATP5A was a mitochondria marker and actin was a loading control. Gls−/− mouse astrocytes were treated with TNF-α for 48 h and then stained with DCFH-DA. Cells were washed with serum-free culture medium, and then subjected to fluorescence microscope (b) and fluorescent microplate reader (c). (PDF 769 kb)


Even as we advance the frontiers of physics knowledge, our understanding of how this knowledge evolves remains at the descriptive levels of Popper and Kuhn. Using the American Physical Society (APS) publications data sets, we ask in this paper how new knowledge is built upon old knowledge. We do so by constructing year-to-year bibliographic coupling networks, and identify in them validated communities that represent different research fields. We then visualize their evolutionary relationships in the form of alluvial diagrams, and show how they remain intact through APS journal splits. Quantitatively, we see that most fields undergo weak Popperian mixing, and it is rare for a field to remain isolated/undergo strong mixing. The sizes of fields obey a simple linear growth with recombination. We can also reliably predict the merging between two fields, but not for the considerably more complex splitting. Finally, we report a case study of two fields that underwent repeated merging and splitting around 1995, and how these Kuhnian events are correlated with breakthroughs on Bose-Einstein condensation (BEC), quantum teleportation, and slow light. This impact showed up quantitatively in the citations of the BEC field as a larger proportion of references from during and shortly after these events. According to Karl Popper, science progresses through repeated hypothesis testing . HypotheMany historians of science have noted the strongly reductionistic flavor of scientific research in the last couple of centuries . Startinw common references, we draw an edge with weight w between them  , proposethem see . The BCNTo determine the statistical significance of our empirical BCNs , we built, build its BCN, extracting the community structure with sizes {s1, s2, …, sn}, and then randomly assign papers in year t into n pseudo-communities of the same sizes, to remove any potential size effects. For a community of size s, we then identify the largest subset of papers sharing the same PACS number. This PACS number can represent the subfield of the community to a certain extent, and the fraction of papers in the largest subset reflect the homogeneity of the community. The results show that the communities extracted are papers really focused on closely related topics, so we refer to these validated units of knowledge as topical clusters (TCs).After building the BCN, we applied the Louvain method based on the maximization of modularity, to extract the community structure . In the t become t + 1. The papers published in different years are distinct, but they do overlap in their references. Therefore we use this fact to define a forward intimacy indexbackward intimacy indext and t + 1 are N is the number of times element occurs in list, and L(list) is the length of list. Both forward and backward indices take on values between 0 and 1. The larger the intimacy index, the clearer the inheritance relationship between two TCs. In this definition, we assume each citation instance in t will be uniformly distributed over all instances of the same citation in t + 1, while each citation in t + 1 receives equal contributions from all instances of the same citation in t. In general, this index is asymmetric, i.e. To study how knowledge evolves, we investigate how TCs According to the above mentioned methodology we visualize the sequence of TCs and their intimacy indices, the evolution of physics research they represent in the form of alluvial diagrams. Before that, we first discuss the TC validation results. As shown in n papers can appear with its observed frequency, within random collections of n papers. The largest subset of papers sharing the same PACS number in a random collection of s papers is typically small. Dividing the sizes of the largest subsets in the empirical communities and in the random collections, we find ratios are larger than 1 for most cases. That is to say, for most communities, this is highly unlikely, so we conclude that the groupings of papers extracted are meaningful. , quantum information (QI), and Bose-Einstein condensation (BEC). These three fields experienced breakthroughs in the 1990s. In et al.  and Kett  is the relative size fraction of subsubcommunity j inside subcommunity i. The more fragmented a community is, i.e., more and smaller subcommunities, the closer F is to 0. Quantile regression between F and Mf gives very similar results as B and Mf, i.e., for the decile of most strongly splitting TCs, increasing the standardized F by one standard deviation will decrease Mf by about 0.06, whereas for the decile of the least strongly splitting TCs, there is no obvious trend as β close to 0, as shown in We also define a Finally, we want to know the impacts of such merging and splitting events. We first check for an increase in the number of publications after such events, but found an insignificant difference in paper numbers in strongly and weakly mixing TCs see . We suspFocusing on the highly productive chain of knowledge processes that led to experimental realizations of BEC, quantum teleportation and slow light, we checked the citation profiles between 1995 and 1998. While the 1995 BEC+QI+QO TC cited a slightly lower proportion of 1995 papers than the APS 0-year average, the 1996 BEC+QI+QO, the 1997 BEC TC, the 1998 BEC TCs all cited significantly more 0-year papers. The full effect of this BEC breakthrough can be seen in the large proportions of 1996 papers cited by the 1997 and 1998 TCs and the proportion of 1997 papers cited by the 1998 TC see . Indeed,In this paper we have studied the knowledge evolution in physics restricted to the APS citation network. We built yearly bibliographic coupling networks, identified TCs and calculated the intimacy indices between TCs in successive years. Based on these results, we visualized the long-term knowledge evolution in physics research in the form of alluvial diagrams. From these alluvial diagrams, we see not only the split of PR into PRA, PRB, PRC and PRD in 1970, and the split of PRA into PRA and PRE in 1993, but also complex interactions between QO, QI and BEC. Overall, we find that the sizes of TCs are governed by a simple linear combination law. We find that merging events are highly correlated with inter-TC connections, making it possible to statistically predict merging events, while splitting events are more complex, as they are weakly correlated with the sizes or internal structures of the TCs. Finally, we showed using the QO, QI, and BEC case study that a strong merging/splitting event will significantly alter the field’s reference structure. In contrast with the well-known Popperian/Kuhnian qualitative and descriptive picture of science, which relate natural phenomena and their explanations with the practices of scientists, our approach leverage on the large and relatively complete data set on scientific publications, to look at changes at a whole-of-science (whole-of-physics in this paper) level, to quantify the types of changes  and their impacts on scientific research.We should emphasize that our results should still be interpreted cautiously because of a number of limitations. One obvious limitation is that our citation network is restricted to APS journals, which are prestigious journals and likely reflect the frontiers of physics research, but are incomplete in two ways. First, many important physics papers are published outside of the APS journals, like Nature, Science and Applied Physics Letters. These papers are clearly important components of physics research. The second limitation is that even for the APS papers, we only have citations pointing back to APS papers. This means that the BCN between APS papers is not complete. For example, if two APS papers cite the same Nature paper, but no APS paper in common, this information will not be captured in the data set. The distortion due to the network being incomplete is hard to estimate, because we are dealing not only with missing nodes, but also with missing edges. There is also a third limitation, not due to the choice of the APS data set. This has to do with our choice to only consider pair-wise relations on merging prediction for simplicity. This is appropriate as a first step, but may not accurately reflect the many multiple-party merging events that we also see in our study.While we believe we have made the correct first step towards a quantitative science of knowledge evolution, many open questions remain. The most crucial one is on the microscopic mechanisms governing merging and splitting. Can we build a model to simulate such evolution? How will this evolution picture change if we use more complete data set, like the Web of Science? It is well known that citation conventions in different disciplines are very different. What is therefore the evolution picture in other disciplines, like biology or social science? We seek to address these questions and more in future investigations.S1 File(PDF)Click here for additional data file.


Previous studies have shown that cyclooxygenase-2, a key enzyme that converts arachidonic acid to prostaglandins, is involved in anxiety and cognitive processes, but few studies have investigated the effects of chronic administration of cyclooxygenase-2 inhibitors on anxiety, learning and memory under normal physiological conditions. The aim of the study was to investigate the effects of chronic administration of parecoxib, a cyclooxygenase-2 inhibitor, on anxiety behavior and memory performance under normal physiological conditions and to explore the possible neural mechanism underlying parecoxib-mediated effects.Adult male ICR mice were randomly divided into four groups: the control group and three parecoxib groups. Mice received normal saline or parecoxib  intraperitoneal injection once a day for 21 days, respectively. Elevated plus-maze, novel object recognition and Y maze tests were conducted on day 23, 24 and 26, respectively. Four additional groups that received same drug treatment were used to measure synaptophysin protein levels by western blot and prostaglandin E2 (PGE2) levels by ELISA in the amygdala and hippocampus on day 26.Chronic parecoxib exerted an anxiolytic-like effect in the plus-maze test test, and enhanced memory performance in the novel object recognition and Y maze tests. Western blot analysis showed that chronic parecoxib down-regulated synaptophysin levels in the amygdala and up-regulated synaptophysin levels in the hippocampus. ELISA assay showed that chronic parecoxib inhibited PGE2 in the hippocampus but not amygdala.Chronic parecoxib exerts anxiolytic-like and memory enhancing effects, which might be mediated through differential modulation of synaptophysin and PGE2 in the amygdala and hippocampus. Cyclooxygenase is a key enzyme that converts arachidonic acid to prostaglandins. Cyclooxygenase comes in two isoforms: cyclooxygenase-1 and cyclooxygenase-2. In the brain, cyclooxygenase-2 is expressed in neurons in both physiological and pathological processes. It is enriched in brain regions that are critically involved in anxiety behavior and cognitive function . Within Recent studies have suggested that cyclooxygenase-2 is involved in anxiety behavior. Mice with traumatic brain injury show increased anxiety-like behavior and cyclooxygenase-2 expression . Stress A considerable body of evidence indicates that cyclooxygenase-2 plays an important role in learning and memory. Under normal physiological conditions, cyclooxygenase-2 inhibitors impair memory acquisition and/or retention in the water maze task in rats and in the one-trial passive avoidance task in chicks , 14. UndSynaptophysin is a membrane protein in synaptic vesicles, and is used as a specific marker of synaptic vesicles/synaptic terminals . This prWe investigated the effects of chronic administration of parecoxib on anxiety behavior in the elevated plus-maze test, and on memory performance in the novel object recognition and Y maze tests under normal physiological conditions in mice. In addition, synaptophysin protein and PGE2 levels in the amygdala and hippocampus was measured, to explore the possible underlying mechanisms.National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the University of South China Animal Care and Use Committee approved experimental protocols 2010–0006).We used naive adult male ICR mice, 15 weeks old and weighing 25–35 g, obtained from the Hunan SJA Laboratory Animal Co.,Ltd. , for this experiment. The animals were housed in groups of 4–5 per cage in a temperature- and humidity-controlled environment, with ad libitum access to food and water. They were maintained on a 12 h light/dark schedule, and lights were switched on at 7 A.M. The animals were handled  for 1 week, after being housed, to habituate them to the experimenter. Experiments were conducted according to the n = 10) and three parecoxib groups (n = 9 per group). Parecoxib sodium  was injected intraperitoneally into the mice, once a day for 21 days. The doses were chosen according to a pharmacological profile study of parecoxib [The mice were randomly divided into four groups: the control group (arecoxib . The conn = 4 per group) and prostaglandin E2 (PGE2) (n = 5 per group) levels in the amygdala and hippocampus. The control and parecoxib groups received saline or parecoxib  injection once a day for 21 days as described above. To avoid the potential effect of behavioral tests on synaptophysin protein expression and prostaglandin E2, the four groups were not submitted to behavioral tests. The mice were anesthetized and sacrificed by decapitation 5 days after the cessation of drug administration. The time point corresponded to the end of the behavioral tests. The amygdala and hippocampus were quickly removed from the skull and then extracted. The tissues were stored at −80 °C until use.Four additional groups were used to measure synaptophysin protein (The elevated plus-maze test is a proven model used to assess the level of anxiety in rodents. Details of the apparatus and the procedure were described in our previous study . BrieflyDetails of the apparatus and the procedure were described in our previous study . HoweverThe apparatus consisted of a Y-maze with three arms (30 cm × 8 cm), surrounded by black Plexiglass walls (15 cm high). The central platform of triangular shape measured 8 × 8 × 8 cm. The Y-maze test was conducted as previously described . BrieflyTissues were homogenized in RIPA Lysis Buffer . They were centrifuged at 12000 rpm/min for 6 minutes. We collected the supernatant and kept it at −70 °C. Protein content was further determined with the BCA assay kit . Sample proteins were separated by sodium dodecyl sulfate polyacrylamide gel electropheresis, and then transferred to polyvinylidene fluoride membranes . The membranes were blocked for 60 min in Tris-buffered saline containing 0.05% Tween-20 and 5% bovine serum albumin. The membranes were then incubated with mouse anti-synaptophysin antibody  or with mouse anti-β-actin antibody  and shaken for 1 hour at 37 °C. The membranes were then washed and incubated with secondary antibody (Goat Anti-Rabbit IgG/HRP) and shaken for 45 min at 37 °C. The membranes were washed and the specific proteins were then visualized using an enhanced chemiluminescence detection kit . The intensity of protein bands of synaptophysin and β-actin were quantified by densitometry.As previously described , PGE2 let-test or one-way analysis of variance (ANOVA) (Sigma Stat 3.2). Post-hoc comparisons were performed with the Tukey HSD method. In addition, one-sample t-test was used to compare the discrimination index of the control group with chance performance (discrimination index = 0%). The data were represented as mean ± SEM. The significance level was set at P < 0.05.Statistical analyses were performed using unpaired F = 0.184, P > 0.05), indicating that chronic parecoxib administration does not affect locomotor activity. For the percentage of the open arms entries  = 8.454, P < 0.001). Post-hoc comparisons showed that when compared with the control group, mice treated with parecoxib at 5 and 10 mg/kg showed higher levels of open arms entry percentages . For the percentage of time spent in the open arms  = 6.278, P < 0.01). Post hoc comparisons showed that when compared with the control group, mice treated with parecoxib at 5 and 10 mg/kg showed higher levels of percentage of time spent in the open arms . These results suggest that chronic administration of parecoxib exerts an anxiolytic-like effect in the elevated plus-maze test.The effects of chronic administration of parecoxib in the elevated plus-maze test are shown in Fig. ies Fig. , a one-wrms Fig. , a one-w = 0.181, P > 0.05) . A one-way ANOVA revealed a significant difference in the discrimination index between the four groups  = 4.358, P < 0.05) . There was no significant difference in the discrimination index between the control mice and those treated with parecoxib at 2.5 or 5 mg/kg (both P > 0.05). These results suggest that chronic administration of parecoxib enhances memory performance in the novel object recognition test.A one-way ANOVA revealed no significant difference in the total time spent exploring both objects between the four groups in the 5 minutes sampling session (F05) Fig. . In the 05) Fig. . Post-ho = 0.111, P > 0.05)  = 4.054, P < 0.05) . There was no significant difference in the alternation ratio between the control mice and the mice treated with parecoxib, at doses of 2.5 and 5 mg/kg . These results suggest that chronic administration of parecoxib enhances memory performance in the Y-maze test.There was no significant main effect of groups in the number of total entries (F05) Fig. , indicat05) Fig. . Post-ho = 12.162, P < 0.001) . In the hippocampus, a one-way ANOVA revealed a significant difference in the synaptophysin protein levels between the four groups  = 4.445, P < 0.05) .The effects of chronic administration of parecoxib on synaptophysin protein expression in the amygdala and hippocampus are shown in Fig. 01) Fig. . Post-ho05) Fig. . Post-ho = 0.845, P > 0.05)  = 9.149, P < 0.001) .The effects of chronic administration of parecoxib on PGE2 in the amygdala and hippocampus are shown in Fig. 05) Fig. . In the 01) Fig. . Post-hoIn this study, we first investigated the effects of chronic administration of cyclooxygenase-2 inhibitor parecoxib on anxiety behavior in the elevated plus-maze test, and on memory performance in the novel object recognition and Y maze tests in mice. The results indicate that chronic administration of parecoxib exerts anxiolytic-like and memory enhancing effects under normal physiological conditions. It could be argued that the non-cognitive components, such as motor stimulant effects or anxiolytic-like activity, could be confounding factors in the evaluation of memory performance of the animals. Indeed, our current results found that chronic parecoxib administration did not affect locomotor activity as indicated by the number of the closed arms entries in the elevated plus-maze test and the number of total entries in the Y-maze test. Furthermore, the novel object recognition and Y-maze tasks require little training of animals and do not induce high levels of stress and arousal . Thus, tOur finding is in accordance with previous observations that cyclooxygenase-2 inhibition reduces anxiety-like behavior –12. The The specific mechanism underlying chronic parecoxib-induced bi-directional change of synaptophysin in the amygdala and hippocampus is unclear. COX-2 and the PGE2 subtype 2 receptor are colocalized with synaptophysin in presynaptic terminals . CyclooxPrevious studies have suggested that cyclooxygenase-2 is involved in learning and memory. In some of the investigations, administration of cyclooxygenase-2 inhibitors caused impairment in memory performance in rats , 14. OurPrevious researches have suggested that synaptophysin is implicated in learning and memory. For example, heat stress-induced memory impairment in the novel object recognition and Y-maze tasks is associated with a decreased level of synaptophysin expression in the hippocampus . SitagliIn conclusion, chronic administration of parecoxib exerts anxiolytic-like and memory enhancing effects under normal physiological conditions in mice. These effects might be mediated through differential modulation of synaptophysin expression in the amygdala and hippocampus. Our finding extends the researches on the effects of cyclooxygenase-2 inhibitors on anxiety, learning and memory.


ALCANZA assessed the anti-CD30 antibody-drug conjugate brentuximab vedotin gen CD30 . In ALCACurrently available systemic therapies for CTCL offer limited efficacy benefits, with low response rates and durations of response . For advn = 64 in each arm; ORR4 56.3% vs 12.5%; p < 0.0001). The proportion of patients achieving an objective response of any duration was also higher with brentuximab vedotin vs physician’s choice , as was the complete response rate . Additionally, a significant progression-free survival benefit was shown with brentuximab vedotin over physician’s choice  0.27 ; p < 0.0001), and time to next therapy was 14.2 vs 6.1 months; HR 0.335; p < 0.001 [Objective response rates (ORRs) with currently available systemic CTCL therapies range from 30-40% . ALCANZAORR4 was high with brentuximab vedotin across key ALCANZA subgroups, including CTCL subtypes (pcALCL and MF) and in skin-only or extracutaneous disease. Alongside MF and pcALCL, brentuximab vedotin has previously demonstrated activity in patients with Sézary syndrome and lymphomatoid papulosis , 7, who In ALCANZA, CD30 positivity was defined as ≥10% CD30-positive malignant cells or lymphoid infiltrate in ≥1 biopsy samples. There was no correlation between CD30 expression and ORR4, with sustained responses occurring across the range of CD30 expression levels . Since bvs physician’s choice [Advanced-stage CTCL is associated with reduced patient quality of life (QoL). Symptoms including pain, pruritus, and fatigue place a significant physical burden on patients, and the chronic and morbid nature of the disease is detrimental to psychological and emotional wellbeing. Brentuximab vedotin-treated patients in ALCANZA experienced meaningful reduction in patient-reported skin symptom burden, using the Skindex-29 measure, showing significant reduction in symptoms with brentuximab vedotin s choice .Adverse events (AEs) are also associated with impaired QoL, and AE profiles for individual agents may vary between diseases. However, AEs reported in ALCANZA were consistent with the established tolerable and manageable safety profile of brentuximab vedotin . While tThe results of the ALCANZA study provide compelling evidence that brentuximab vedotin is now an important treatment option for patients with R/R CD30-positive pcALCL and MF. Indeed, in November 2017, the Food and Drug Administration approved brentuximab vedotin for use in this setting in the USA, and European Medicines Agency recommendation for approval was received in December 2017. Moreover, looking forward, brentuximab vedotin may offer promise in other settings, including in patients with lesions exhibiting low CD30 expression, in other CD30-positive CTCL subtypes, or as retreatment in CTCL, providing a potential post-relapse option. Different dosing schedules are also of interest; dose reduction is a key tactic in AE management, and comparative studies could assess the impact of altered dosing schedules on safety and efficacy. Brentuximab vedotin may offer potential in combination with other therapies; combination regimens could provide another step forward in identifying treatments that further improve and prolong responses.