Mice bearing 5–10-day-old intracranial GL-261 gliomas were intratumorally administered Ad-RTS-mIL-12 in which IL-12 protein expression is tightly controlled by the activator ligand, veledimex. Local tumor viral vector levels concomitant with veledimex levels, IL-12-mRNA expression, local and systemic cytokine expression, tumor and systemic flow cytometry and overall survival were studied. Ad-RTS-mIL-12+veledimex elicited a dose-related increase in tumor IL-12 mRNA and IL-12 protein and discontinuation of veledimex resulted in a return to baseline levels. These changes correlated with local immune and antitumor responses. Veledimex crossed the blood–brain barrier in both orthotopic GL-261 mice and cynomolgus monkeys. We have demonstrated that this therapy induced localized controlled production of IL-12 which correlates with an increase in tumor-infiltrating lymphocytes (TILs) leading to the desired biologic response of tumor growth inhibition and regression. At day 85 (study termination), 65% of the animals that received veledimex at 10 or 30 mg/m2/day were alive and tumor free. In contrast, the median survival for the other groups were: vehicle 23 days, bevacizumab 20 days, temozolomide 33 days and anti-PD-1 37 days. These findings suggest that the controlled intratumoral production of IL-12 induces local immune cell infiltration and improved survival in glioma, thereby demonstrating that this novel regulated immunotherapeutic approach may be an effective form of therapy for glioma.The purpose of this study was to determine if localized delivery of IL-12 encoded by a replication-incompetent adenoviral vector engineered to express IL-12 via a RheoSwitch Therapeutic System Glioblastoma (GBM) is the most common and aggressive primary brain tumor. These tumors are characterized by invasiveness coupled with neovascularization with a median survival of ~15 months from diagnosis through treatment with current standard of care . PatientTraditionally, it was believed that the central nervous system was an immuno-privileged compartment. However, recent data have shown the presence of lymphatic drainage, increased permeability in the presence of tumor as well as antigen-presenting capability of microglial cells . The GBMCancer immunotherapy attempts to harness the specificity and cytotoxic activity of the immune system to control growth and destroy tumor cells. Numerous strategies are in progress to reverse these immunosuppressive signals, including the use of immune checkpoint inhibitors which have been shown to partially reverse the immunosuppressive signals resulting in a reduction in tumor mass in some subjects –9. In adIL-12, a heterodimeric protein, plays a pivotal role in linking the innate and adaptive immune systems , 14. IL-Studies with recombinant IL-12 protein have been performed in multiple murine tumor models using systemically administered IL-12. Results of these studies clearly demonstrated a reduction in tumor growth rate coupled with no appreciable toxicity .Based on these results, phase 1 studies were performed where recombinant IL-12 was administered systemically to human subjects. However, in the expanded phase 2 study, severe toxicity was observed and the study halted , therebySeveral groups demonstrated that in vitro transduction of tumor cells with IL-12 genes could be therapeutically useful and avoid the severe toxicities observed with systemic administration , 20. Dir® (RTS®) provides a gene expression control switch platform that confers tightly regulated, inducible gene that has been validated in clinical trials involving IL-12 [® gene switch consists of multiple inter-dependent functional components: (1) two transcription factors, (2) an inducible promoter and (3) a small molecule activator ligand. One of the transcription factors serves as a co-activation partner, and is a fusion between a transcription activation domain and a nuclear factor domain. The second transcription factor serves as a ligand-inducible transcription factor and is a DNA binding domain fused to a nuclear factor ligand binding domain. The RheoSwitch activator ligand (veledimex) is a synthetic analog of the insect molting hormone ecdysone [® [The RheoSwitch Therapeutic Systemng IL-12 , 24. An ecdysone , 26. Botysone [® . We haveysone [® .In this study, we explore the mechanism of action of a direct intratumoral injection of Ad-RTS-mIL-12 plus orally administered veledimex and correlate it with antitumor activity and induced systemic immunity in an orthotopic syngeneic mouse GBM model. The successful preclinical studies led to ongoing studies of Ad-RTS-hIL-12+veledimex in cancer subjects with relapsed and refractory GBM (NCT01397708).2. Female C57BL/6 mice, age 6–7 weeks or 20–23 weeks for the control arm of the rechallenge study, were obtained from Harlan Laboratories  and Charles River Laboratories . All animal care and experimental procedures used in this study were performed in accordance with the protocol approved by the Institutional Animal Care and Use Committee guidelines.A murine glioma tumor cell line, GL-261, was purchased from American Type Culture Collection . Cells were maintained in complete RPMI containing 10% heat-inactivated fetal bovine serum . Cells were grown and maintained at 37 °C in a humidified atmosphere with 5% CO2, 0.02% PS-80, 100 µM EDTA and 0.5% EtOH, pH 7.4 [Adenoviral shuttle vectors were generated as previously described by Komita and colleagues  containi, pH 7.4 . All pre® and RiboGreen® assay kits from Life Technologies . RNA quality was also assessed using the Agilent RNA Nano kit . Absolute quantification was performed on DNA samples to determine gene copy numbers. Standard curves were generated using murine IL-12 shuttle plasmid . Each reaction involved 100 ng of template DNA to determine the vector copy number and samples were run in triplicate. Real-time quantitative polymerase chain reaction (PCR) assays were performed using an ABI 7300 and/or 9300 HT system .Mouse genomic DNA and RNA was isolated from snap-frozen tumors using the DNeasy and RNeasy nucleic acid isolation kits from Qiagen . Isolated RNA was also further treated with RNase-free DNAse (Qiagen). Quantification of isolated DNA and RNA was performed on a Nanodrop spectrophotometer , and further quantified using Quant-IT PicoGreenFor relative gene expression analysis, RNA was converted to complementary DNA (cDNA) using the qScript™ cDNA SuperMix , per manufacturer’s protocol. Equivalent amounts of cDNA were used per reaction and run in triplicate. Isolated RNA was analyzed by quantitative reverse transcription PCR  for mIL-12 and a house-keeping gene panel (mACTB). The relative expression levels of mIL-12 was based on ∆∆CT method. Assays were run using an ABI 7300 and/or 9300 HT system .g for 5 min and used for cytokine analysis. The mIL-12p70 concentrations in the sera and tumor lysates were measured by ELISA using a Quantikine mIL-12 Immuno-assay kit . Total protein concentration in each tumor lysate was determined using the bicinchoninic acid (BCA) method  to calculate the pg cytokine levels /mg of tumor or /mL of sera.Murine IL-12-p70 (mIL-12-p70) levels were measured by enzyme-linked immunosorbent assay (ELISA) in sera and in tumor cell lysates. Frozen tumors were lysed with SDS Free Cell Culture Lysis Buffer  and then pulverized with a tissue homogenizer, QIAGEN  Tissue Lyser Bead Mill, followed by 3× freeze–thaw cycles. The supernatant was removed after centrifugation at >10,000 × Brain tumors or whole blood (50 µL) samples were stained per Flow Contract Site Laboratory standard operating procedures. Briefly, samples were incubated for 30–35 min in the dark at room temperature and then washed twice with 1 mL 1× Permeabilization Buffer. The samples were resuspended in 125 µL of 1× calcium- and magnesium-free Dulbecco's phosphate-buffered saline for acquisition on the flow cytometer.One negative sample (no antibody) was used for gating purposes. Cell populations were determined by electronic gating based on forward versus side scatter. The flow cytometer collected 20,000 CD45+ events in each tube. The CD45+ population was further characterized for T cells (CD3+/CD4+ and CD3+/CD8+), macrophages (CD11b+/Ly6G-/Ly6C-F4/80+), B cells (CD3-CD19+), NK cells (CD49b), T-cell exhaustion (LAG3+) and regulatory T cells (CD4+CD25+FoxP3+). Flow cytometric data acquisition was performed using the Facscanto ii™ flow cytometer (BD Biosciences). Data were acquired using BD FACSDiva™ software (version 8.0.1).Brain tumor, plasma and cerebrospinal fluid (CSF) samples were analyzed for veledimex using liquid chromatography/tandem mass spectrometry method. Brain tumor samples were homogenized in homogenization solution (10:90/v:v/acetonitrile:0.1% Tween 80 in water) with ratio of 1 g to 9 mL and proteins precipitated. Veledimex concentrations were calculated with a Quadratic 1/x concentration weighting linear regression over concentration ranges of 0.0500 to 50 ng/mL using deuterated (D3) veledimex as an internal standard.5 cells/mouse. Implant coordinates were 1 mm anterior and 2 mm lateral to the bregma, at a depth of 3 mm. Buprenorphine  was administered to the mice approximately 30 min prior to surgery. After 5 days, mice were randomized into one of the treatment groups. For those mice administered Ad-RTS-mIL-12, the vector was administered through the same bur hole at the coordinates above in a constant volume of 3–5 μL. Veledimex was administered via oral gavage once daily for 14 consecutive days.At 5 days prior to treatment initiation, female C57BL/6 (C57BL/6NHsd) mice 6–7 weeks old  were inoculated intracranially with 3 µL of GL-261 murine glioblastoma tumor cells at 1 × 10P < 0.05. Pharmacokinetics and tissue levels of veledimex was determined using non-compartmental analysis module of the pharmacokinetic software Phoenix 64 WinNonlin.All values are expressed as the mean ± standard error of the mean. Statistical analysis was performed using a one-way analysis of variance with Dunnets post hoc test to compare differences between the groups versus control. Increased survival fractions were studied using Kaplan–Meier survival plot followed with a log-rank and Gehan–Wilcoxon tests to assess the significance of the differences. All statistical analyses were performed using GraphPrism 5 . Differences between groups were considered significant when 2. Terminal blood and CSF and brain samples were collected at 1, 2, 4, 6, 24 and 48 h post dose. Following a single oral dose of 225 mg/m2 veledimex, Cmax and AUC0-t in plasma were 4153 ng/mL and 4057 ng h/mL while the CSF Cmax and AUC0-t were 14 ng/mL and 466 ng h/mL. For veledimex detected in the brain, Cmax and AUC0-t were 1794 ng/mL and 25325 ng h/mL. The veledimex Cmax and AUC0-t ratio between CSF and plasma was 0.34%.Veledimex was administered to separate groups of C57BL/6 mice at a single dose via oral gavage (PO) at 225 mg/mmax, and AUC0-t in plasma and CSF were 327 ± 142 and 2.07 ± 0.91 ng/mL and 5887 ± 2203 and 42.5 ± 17.4 ng h/mL, respectively. The mean plasma Tmax was observed at ~4 h post dose with elimination t1/2 25.0 ± 6.1, respectively. There was ~1% of veledimex brain uptake, Cmax, and AUC0-t ratios between CSF and plasma were 0.6 ± 0.2 and 0.7 ± 0.2.A single oral dose of veledimex at 120 mg was administered to six cynomolgus monkeys (3/gender) and plasma and CSF levels were assessed through 48 h post treatment. Since no gender-related differences were observed, the data from male and female animals were pooled. After a single oral dose of 120 mg veledimex, CIn summary, these data show that orally administered veledimex crosses the blood–brain barrier in both mice and cynomolgus monkeys at sufficient levels to warrant the assessment of Ad-RTS-IL-12+veledimex in glioma.8 to 5 × 109 vp intracranial at the coordinates stated above with veledimex administered gavage at a fixed dose of 100 mg/m2/day PO, and brain veledimex, viral copy number and the ability to turn on the switch to elicit local production of IL-12 mRNA and IL-12 protein expressions were assessed. The results of this study demonstrated that increasing doses of the vector in the presence of a fixed dose of activator ligand, veledimex, elicited a dose-related increase in tumor viral particles, IL-12 mRNA (activating the switch) and localized production of IL-12   sustained and prolonged survival with the median survival of greater than 85 days with ~66% of the mice surviving to terminal killing at day 85. At terminal killing, most Ad-RTS-mIL-12-treated animals were tumor free. Ad-RTS-mIL-12+veledimex 30 mg/m2/day elicited a moderate reduction in body weight during the veledimex dosing period which rapidly rebounded on discontinuance of veledimex  15 mg/m2 q4dx5. The anti-PD-1 and temozolomide slightly prolonged median survival to 37 and 33 days, respectively with 24 and 14% of the animals surviving to terminal killing with all mice having tumor present at terminal killing. Bevacizumab failed to prolong survival when compared to vehicle with a median survival of 20 days and 4% survival to terminal killing , we observed sustained increase in tumor cytotoxic T cells (CD3+CD8+) concomitant with sustained reductions in T regulatory cells  concomitant with sustained reductions in Tregs (CD4+CD25+FoxP3+) on day 7 . In the tumor at those doses which markedly prolonged survival (Ad-RTS-mIL-12+veledimex 10 and 30 mg/m10 vp)+veledimex (450 mg/m2/day PO) were rechallenged with 1 × 105 GL-261 cells at the site of original implantation. In addition, 12 age-matched (20–23 weeks) C57BL/6 mice were administered intracranial 1 × 105 GL-261 cells and survival monitored for an additional 73 days. The results of this study clearly show that Ad-RTS-mIL-12+veledimex demonstrated a significant increase in survival with ~90% of the animals surviving until the end of post rechallenge monitoring period vs. 50% for naive age-matched control group (P < 0.05)  promoter of transgene expression which can be controlled by a small molecule ligand. RTS-inducible transgene expression is “off” in the absence of veledimex, whereas transgene expression is turned “on” by the administration of veledimex. In the GL-261 orthotopic model of glioma, we demonstrated a dose-related increase in tumor IL-12 and downstream IFN-γ, demonstrating that the IL-12 produced was biologically active.In the present study, we assessed an adenoviral vector that expresses IL-12, i.e., Ad-RTS-mIL-12 or Ad-RTS-hIL-12 under the control of a conditional (regulated) promoter, administered together with an orally bioavailable small molecule activator ligand, veledimex. The RTS2. The increase persisted for at least 14 days after the last dose of veledimex. Concomitant with the increase in cytotoxic T cells there was a marked decrease in Tregs, thus demonstrating that Ad-RTS-mIL-12+veledimex could stimulate and restore local immune function in a dose-related manner. Numerous investigators have also demonstrated that tumor-infiltrating T cells and more importantly the ratio of cytotoxic T cells/Tregs provide a favorable prognostic marker to predict the success of immunotherapies in breast [Successful immunotherapy involves overcoming the immunosuppressive tumor environment as demonstrated by low levels of cytotoxic T cells and increased Tregs. We have shown that Ad-RTS-mIL-12+veledimex elicited a dose-related increase in tumor IL-12 which in turn elicited a dose-related increase in CD3+CD8+cytotoxic T cells concomitant with a reduction in FOXP3+ regulatory T cells. On day 14, cytotoxic T cells were increased by ~ 6-fold over vehicle and at the efficacious doses of veledimex 10 and 30 mg/mn breast , ovariann breast , melanomn breast  and glion breast –45. In t5 GL-261 tumor cells 100 days after Ad-RTS-IL-12+veledimex. We observed ~90% of the animals surviving until the end of the 73-day post-rechallenge monitoring period vs. 50% for the naive control group, thus demonstrating the induction of local immune response induced by vector intratumoral administration and oral veledimex.The local increase in IL-12 increase coupled with the local restoration of the immune system resulted in a concomitant increase in long-term survival without significant adverse events. At day 85 (study termination), over 95% of the Ad-RTS-IL-12+veledimex-treated animals were tumor free. In contrast, bevacizumab, temozolomide and anti-PD-1 therapy only slightly prolonged survival. Similar survival benefit was observed by others –46. To aIn summary, our results demonstrate that the localized delivery of IL-12 encoded by the replication-incompetent adenoviral vector Ad-RTS-IL-12, and controlled by the oral activator veledimex is an effective gene and immunotherapeutic strategy in preclinical studies. We have demonstrated that this therapy induced localized controlled production of IL-12 which correlates with an increase in tumor-infiltrating lymphocytes leading to the desired biologic response of tumor growth inhibition and regression. These results demonstrate the need to study Ad-RTS-IL-12+veledimex in the patients with glioma. Indeed, clinical trials based on these data have been initiated in the treatment of glioma (NCT02026271) via direct intratumoral injection of adenoviral particles carrying a gene switch and the human IL-12-p70 transgene and oral administration of veledimex to produce hIL-12. The preliminary results of this study are encouraging . Thus, t


MUC1 (mucin 1), a membrane‐tethered mucin glycoprotein, is highly expressed on the surface of respiratory epithelial cells and plays a key role in anti‐inflammatory and antiapoptotic responses against infections. However, little is known about the link between MUC1 and necroptosis in asthma. This study aimed to investigate the effects of MUC1 on TNF‐α‐induced necroptosis in human bronchial epithelial (16HBE) cells and the underlying molecular mechanism. Negative control and MUC1‐siRNA cells were treated with TNF‐α in the presence or absence of necrostatin‐1 (Nec‐1). Necroptosis was investigated using flow cytometry analyses, and the protein expression levels of MUC1, receptor‐interacting protein kinase‐1 (RIPK1), RIPK3, and phosphorylated RIPK1 were detected by western blot analysis. In addition, the interactions between RIPK and MUC1 were analyzed by coimmunoprecipitation. The results demonstrated that TNF‐α could induce necroptosis of 16HBE cells, and MUC1 expression was increased upon treatment with TNF‐α. The coimmunoprecipitation outcomes showed that MUC1 interacted with RIPK1 but not with RIPK3 in 16HBE cells, and the interaction was augmented by TNF‐α. Furthermore, MUC1 downregulation obviously increased the TNF‐α‐induced necroptosis of 16HBE cells and enhanced the expression of p‐RIPK1‐Ser166 and RIPK3, whereas these phenomena were partially attenuated by Nec‐1. These results may provide a new insight into the mechanism of severe asthma‐related necroptosis and lay a foundation for the future development of new anti‐inflammatory drugs for asthma. It consists of two noncovalently associated polypeptide subunits, an N‐terminal extracellular subunit and a C‐terminal subunit containing its cytoplasmic tail (MUC1‐CT). MUC1‐CT can shed from the cell membrane and together with some of its binding partners, kinases and adapter proteins, induce signal transduction events  cells in vitro. Furthermore, we aimed to elucidate the underlying molecular mechanisms, specifically, whether this effect was completed through the RIPK1/RIPK3 pathway.22.12 atmosphere at 37℃. The study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.The 16HBE cell line was purchased from Wuhan Boster Biotech Co. Ltd. (People’s Republic of China), and cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in a 5% CO2.2TNF‐α ‐induced cellular death in 16HBE cells was investigated using the Annexin V/PI Apoptosis Detection Kit . Following incubation of cells with TNF‐α (300 ng/ml), they were washed in phosphate buffered saline and resuspended in 500 μl of binding buffer containing 5 μl of annexin V‐FITC and 5 μl of PI. Afterwards, cells were incubated for 15 min at room temperature in the dark. Samples were analyzed by flow cytometry using a Becton Dickinson LSR flow cytometer. The data were analyzed with Cell Quest software. Annexin V(+)/PI(+) cells in upper right quadrant were considered as necrotic.2.3MUC1 siRNA and nontargeting negative control siRNA were synthesized by Viewsolid Biotech . The target sequences were as follows: MUC1‐siRNA sense 5′‐GUU CAG UGC CCA GCU CUA CTT‐3′, and MUC1‐siRNA antisense 5′‐GUA GAG CUG GGC ACU GAA CTT‐3′. SiRNAs were transfected into 16HBE cells using Lipofectamine 2000 according to the manufacturer’s protocol . Following incubation for 48 hr, MUC1 protein levels were determined by western blot analysis.2.4Extracts of 16HBE cells were prepared with the lysis buffer, and the protein concentration in homogenates was measured by the BCA Protein Assay Kit . Western blots were performed following standard procedures. Briefly, equal protein aliquots were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gel, and then the proteins were transferred to polyvinylidene fluoride membranes, which were incubated with primary antibodies against RIPK1 , p‐RIPK1 , RIPK3 , and MUC1‐CT . The intensity of individual bands was quantified using ImageJ . For coimmunoprecipitation assays, equal protein aliquots of cell lysates were incubated overnight at 4°C with immunoprecipitating antibodies against RIPK1 , RIPK3 , MUC1‐CT , or with isotype‐matched nonimmune IgG. Protein A/G agarose beads  were added. The beads were washed five times at 4°C with lysis buffer and eluted by boiling in SDS‐PAGE loading buffer. Immunoprecipitated proteins were separated by SDS‐PAGE and subjected to immunoblot analysis for the protein of interest.2.5SD. A two‐tailed Student t‐test was used for comparison between two groups and one‐way analysis of variance for more than two groups. A value of p < 0.05 was considered as an indicator of a statistically significant difference.Statistical analysis was performed using GraphPad Prism V6.0 , and the data are presented as the mean ± 33.1First, we determined whether TNF‐α could initiate necroptosis in 16HBE cells after treatment with TNF‐α (300 ng/ml). Our findings indicated that TNF‐α induced 16HBE cell necroptosis compared with control group (Figure 3.2The RIPK1 and RIPK3 signaling pathways have been strongly involved in necroptosis. To understand the effect of MUC1 on the activation of these two pathways, the expression levels of RIPK1 and RIPK3 were examined. Western blot analysis revealed that MUC1‐siRNA cells showed a significant increase in RIPK3 following TNF‐α stimulation compared with negative control group Figure b. Howeve3.3The previous results demonstrated that MUC1 downregulation increased the expression of RIPK3 but not RIPK1 and that MUC1 could bind RIPK1. Therefore, we focused on whether MUC1 downregulation alters the phosphorylation of RIPK1 expression. First, we used Nec‐1 to treat 16HBE cells, and the results proved that Nec‐1 efficiently reduced TNF‐α‐induced necroptosis in 16HBE cells (Figure 3.4To further verify whether Nec‐1 inhibits 16HBE cells necroptosis caused by MUC1 downregulation in this study, we used the specific RIPK1 inhibitor Nec‐1 to treat 16HBE cells. The results indicated that Nec‐1 significantly protected TNF‐α‐induced necroptosis in 16HBE cells regardless of the MUC1 presence. However, the MUC1‐siRNA + TNF‐α + Nec‐1 group still showed higher cell necroptosis in comparison with the NC + TNF‐α + Nec‐1 group Figure a, which 4In this study, we provided the first evidence that TNF‐α could induce 16HBE cells necroptosis accompanied by the upregulation of MUC1. Furthermore, we demonstrated that MUC1 downregulation contributed to cell necroptosis through a mechanism that involved the RIPK1/RIPK3 signaling and it could be partially blocked by Nec‐1.Asthma is characterized by airway inflammation, remodeling and hyperresponsiveness (Saglani & Lloyd, To elucidate the mechanisms responsible for the promotion of TNF‐α‐induced 16HBE cell necroptosis by MUC1 downregulation, we examined the RIPK1/RIPK3 signaling because its role is widely accepted in the regulation of necroptosis Newton, . RIPK3 hIn recent years, many studies have confirmed that TNF‐α induces cell necroptosis partly via TNF‐α‐mediated phosphorylation and ubiquitination of RIPK1. Phosphorylated RIPK1 is thought to have a crucial role in activating RIPK3 (Cho et al., 5In conclusion, we have demonstrated that MUC1 downregulation aggravates TNF‐α‐induced necroptosis in 16HBE cells by mediating the RIPK1/RIPK3 pathway, and the effect could be partially blocked by Nec‐1. This may provide a new insight into the effects of severe asthma on necroptosis and lay a foundation for the future development of new anti‐inflammatory drugs for asthma.The authors declare that there are no conflicts of interest.


BH, as the consultant gynaecologist, consented patients and conducted the hysterectomies. AA, as the consultant histopathologist, reviewed and released cervical tissue under ethical approval to FM's study. CB was responsible for donor recruitment, consenting and clinical administration of the study. JF and RW collected tissue, JF, RW and FM prepared and cryopreserved it. JF undertook the viability experiments described and JM performed the tissue sectioning and H&E staining. JF, RW and FM wrote the manuscript; all authors approved the final version.JF and FM were responsible for designing the research project. FM secured the grant funding and national and local ethical approval for the project. Mr Bill Hunter was mistakenly omitted from the original version of the manuscript; this corrigendum rectifies this error.The authors would like to apologize for any inconvenience caused.https://doi.org/10.1016/j.cryobiol.2017.06.004.DOI of original article:


P=1.00, B: P=0.764), and the results of the Egger’s test showed no evidence of publication bias . Seven studies assessed the relationship between TNF-α-308 gene polymorphisms and risk of bone-joint and spinal tuberculosis risk. The heterogeneity (2I) of GG vs. AA or AG was 0% and there was no heterogeneity (χ2 = 0.06 and P=0.944) in a fixed-effects model. There was also a lack of association between TNF-α-308 polymorphism and bone-joint and spinal tuberculosis risk under the recessive model. The remaining models of the TNF-α-308 genotype and further studies of TNF-α-238 did not show a noteworthy association. Overall, there was no significant association between TNF-α-308, TNF-α-238 gene polymorphisms and bone-joint and spinal tuberculosis risk. Our study suggests that tumor necrosis factor α (TNF-α) gene polymorphisms may not contribute to bone-joint and spinal tuberculosis based on the current evidence.The aim of the present study was to investigate the association of TNF-α-308 and TNF-α-238 gene polymorphisms with the risk of bone-joint and spinal tuberculosis (TB) by meta-analysis. By searching PubMed, Web of Science, Wanfang databases, CNKI, Medline, and Cochrane Library, the published articles about studies of the association of the TNF-α-308, TNF-α-238 gene polymorphisms with risk of bone-joint and spinal tuberculosis were collected by two reviewers. Begg’s and Egger’s tests were performed to assess publication bias. Stata 12.0 software was used for data analysis. The symmetry of the funnel plot indicated no significant publication bias in the Begg’s test (A:  Mycobacterium tuberculosis (Mtb) is an infectious disease that severely endangers human health in the world. Twenty-two countries in the world experience high morbidity due to tuberculosis, while China has the second largest incidence of tuberculosis [Tuberculosis (TB) caused by rculosis . Excludirculosis . Tumor nrculosis . The phyrculosis .TNF-α is a potent pleiotropic proinflammatory and immunoregulatory cytokine, which contributes to the initiation, up-regulation, and perpetuation of the inflammatory response. TNF-α is produced by many cell types including neutrophils, fibroblasts, NK cells, T and B cells, and macrophages infiltrating tissue as the part of host defense against brucellosis infection ,6. IncreStudies have shown that TNF-α can induce osteoclast differentiation, activation and apoptosis directly or indirectly by binding to the corresponding receptor of osteoclast precursor cells . Its proBy searching PubMed, Web of Science, Wanfang databases, CNKI, Medline and Cochrane Library databases for articles published prior to January 2018, there were seven studies identified by two reviewers finally. The search strategy included using the keywords, such as ‘bone-joint & spinal tuberculosis’, ‘TNF-α’, ‘gene’, ‘polymorphisms’. To identify titles and abstracts of relevant literature, the reference that may have been missed during search or indexing were checked manually. All papers included in the meta-analysis met the following criteria: (i) investigating the association between TNF-α gene polymorphism and bone and joint tuberculosis, (ii) case-controlled studies and the controls had no malignant disease, (iii) the number of studies of genotypes must be completely provided in both experimental and control groups directly or indirectly. Exclusion criteria: (i) repeated studies; (ii) the control group did not meet the Hardy–Weinberg equilibrium (HWE) ; (iii) iData were extracted by two independent reviewers. Extraction of literature, including: first author, year of publication, country of study, method of gene detection, number of various genotypes. In the process of extraction, if there is a dispute, it is resolved through negotiation. Any disagreements on data extraction were resolved through discussion with other researchers.The quality of all research was assessed by the Newcastle–Ottawa Scale (NOS). The quality assessment for each research included the following aspects: (i) Queue selection; (ii) Comparability; (iii) Result measurement. According to the three aspects, the number of stars was divided. The lowest was 0 stars, the highest 10 stars, 0–3 stars were level C, the 4–6 stars were level B, and the 7–10 stars were level A . If therI2 test. . If the I2 was lower than 50%, the pooled OR estimate of the study was calculated by a fixed-effects model. Otherwise, the random-effects model was used. (5) P<0.05 was considered statistically significant.Stata 12.0 software was used for meta-analysis as following: (1) Test of HWE. (2) Funnel plot analysis and Begg’s test, Egger’s test for publication bias. (3) Five different odds ratio (ORs) were computed: (i) G vs. A ; (ii) GG vs. AA (homozygous model); (iii) GG vs. AA or AG (recessive model); (iv) GG or GA vs. AA (dominant model); and (v) GA vs. GG or AA (overdominance model). (4) Heterogeneity between studies was evaluated by chi-square-based Q test and According to the above retrieval methods, a total of 125 relevant studies were selected. After skimming the titles, abstracts and reviewing the full text, 118 studies were excluded due to lack of detailed data or lack of data on target polymorphisms. Finally, seven case–control studies involving 1600 subjects were selected for the meta-analysis . The maiI2 value was 0%, indicating no heterogeneity. Meta-analysis results were as follows: χ2 = 0.06 and P=0.944 in a fixed-effects model, indicating there was no association between TNF-α-308 polymorphism and bone-joint and spinal tuberculosis under the recessive model. Neither the remaining models of the TNF-α-308 genotype nor further studies in TNF-α-238 revealed a noteworthy association  indicated obvious heterogeneity in I2 test and its pooled OR estimate of the study was calculated by the random-effects model. The same was true for the recessive model (GG vs. AA + AG) of TNF-α-238 in the I2 test . The other I2 test  of genotype indicated no obvious heterogeneity, and the fixed-effects model was used. One study with small number of cases was eliminated for sensitivity analysis, and the other six studies were analyzed again. The results showed that lack of cases still had no effect on the results of meta-analysis in the six studies.Based on the results of heterogeneity test, the recessive model (GG vs. AA or AG) of TNF-α-308 , and the results of Egger’s test suggest no evidence of publication bias .Begg’s test and Egger’s test were performed to evaluate the publication bias A,B. SymmOur study showed that there was a lack of association between TNF-α-308 polymorphism and bone-joint and spinal tuberculosis risk under the recessive model. The remaining models of the TNF-α-308 genotype and further studies of TNF-α-238 did not show a noteworthy association. There was no significant association between TNF-α-308, TNF-α-238 gene polymorphisms and bone-joint and spinal tuberculosis risk.Bone-joint tuberculosis is a common infectious disease in the world, which is common in children and adolescents. However, with the increase in life expectancy, the probability of bone-joint tuberculosis in the elderly has also been greatly increased . Bone-joThe susceptibility to TB depends upon different factors and the risk of developing diseases after infection with Mtb ranges from 5 to 10%. This suggests that besides the mycobacterial itself, the host genetic factors may determine the differences in host susceptibility to TB . Among tThere are four steps between TNF-α and anti-tuberculosis immunity : (i) TNFTNF-α is encoded by the TNF-α gene, and its expression is mainly focused on the promoter region of the TNF-α gene . The sinIn the present study, four randomized controlled trial (RCT) showed that bone-joint tuberculosis was associated with TNF-α-308 and TNF-α-238 genetic polymorphisms, and allele ‘A’ may play a protective role. One study showed that bone-joint tuberculosis was associated with TNF-α-308 genetic polymorphisms, and allele ‘A’ played a protective role but no obvious genetic polymorphisms with TNF-α-238. Only two studies showed that bone-joint tuberculosis was not associated with genetic polymorphisms of TNF-α -308 or 238. The main contribution of this meta-analysis is to systematically evaluate the TNF-α gene polymorphism in patients with bone-joint tuberculosis, and to draw a comprehensive conclusion. Our analysis suggested that there was no correlation between TNF-α-308 or TNF-α-238 gene polymorphisms and bone-joint tuberculosis. This conclusion is similar to the research pulmonary tuberculosis by Sharma et al.  and VejbHowever, the results of this analysis are subject to large samples, multicenter collaboration and further randomized controlled trials to validate. Our study does not imply that all polymorphisms in the TNF-α locus are completely unrelated to the susceptibility of bone and joint tuberculosis. Gene–environment interactions might also affect the results.There were also some limitations in our study. Our research focuses on the Chinese population. Non-Chinese studies were not excluded from this review, and according to our inclusion/exclusion criteria, only Chinese populations were finally included. In the future, we should analyze the correlation between bone and joint tuberculosis and TNF-α gene polymorphism in various groups. Therefore, more scholars are expected to explore more genes related to tuberculosis susceptibility, promote the development of tuberculosis gene therapy, and benefit more patients with tuberculosis. Further study on subgroup analysis should be performed based on ethnicity, countries, regions, type of control source and so on. Moreover, the study may be influenced by false-positives, gene–environment interactions and longitudinal studies were needed to infer the causation.


Staphylococcus aureus-mediated lung damage. However, depletion of JNK protein using siRNA sensitized macrophages to necroptosis that was triggered by LPS or poly I:C but still inhibited TNF-induced necroptosis. Mechanistic studies revealed that RIPK1 recruited JNK to the necrosome complex and their kinase activity was required for necrosome formation and the phosphorylation of MLKL in TNF- and TLRs-induced necroptosis. Loss of JNK protein consistently suppressed the phosphorylation of MLKL and necrosome formation in TNF-triggered necroptosis, but differentially promoted the phosphorylation of MLKL and necrosome formation in poly I:C-triggered necroptosis by promoting the oligomeration of TRIF. In conclusion, our findings define a differential role for JNK in regulating TNF- and TLRs-mediated necroptosis by their kinase or scaffolding activities.Tumor necrosis factor (TNF) and Toll-like receptor (TLR)3/TLR4 activation trigger necroptotic cell death through downstream signaling complex containing receptor-interacting protein kinase 1 (RIPK1), RIPK3, and pseudokinase mixed lineage kinase-domain-like (MLKL). However, the regulation of necroptotic signaling pathway is far less investigated. Here we showed that c-Jun N-terminal kinases (JNK1 and JNK2) displayed kinase-dependent and -independent functions in regulating TNF- and TLRs-mediated necroptosis. We found that RIPK1 and RIPK3 promoted cell-death-independent JNK activation in macrophages, which contributed to pro-inflammatory cytokines production. Meanwhile, blocking the kinase activity of JNK dramatically reduced TNF and TLRs-induced necroptotic cell death. Consistently, inhibition of JNK activity protected mice from TNF-induced death and  Various stimuli can trigger necroptosis, including activation of specific death receptors, interferon receptors, or TLRs6. Upon the stimulation of TNF, RIPK1 and RIPK3 form a protein complex called necrosome through their RIP homotypic interaction motif (RHIM), which leads to the phosphorylation and oligomerization of RIPK39. Activated RIPK3 subsequently phosphorylates the downstream pseudokinase MLKL11. Ultimately, phosphorylated MLKL undergoes oligomerization and translocates to the cell membrane to execute necroptosis13. Excessive and uncontrolled necroptosis lead to embryonic development failure. Also, under many pathophysiology settings, including viral or bacterial infection, pancreatitis, multiple sclerosis, neurodegeneration, and systemic inflammatory response syndrome, the significance of necroptosis has been revealed17.Necroptosis is a type of programmed cell death, independent of caspases activity, and characterized by membrane swell and rupture, which is mediated by RIPK1 and RIPK318. Several apoptosis-related proteins, including caspase 8, FADD, and cFLIP, inhibit the activation of necroptosis. Deficiency of these genes causes embryonic lethality in mice which is dependent on RIPK3 and MLKL-mediated necroptosis21. Other reports showed that several kinases, including IKKα, IKKβ, and MK2 directly phosphorylated RIPK1 and restricted the autophosphorylation of RIPK1, which inhibited the activation of TNF-induced apoptosis and necroptosis25. Recently, it has been reported that ppm1b dephosphorylated RIPK3 and CHIP mediated the ubiquitination and lysosome-dependent degradation of RIPK327. Besides the negative regulation of necroptotic signaling pathway, some genes positively regulated necroptotic signaling pathway. Cyld can deubiquitylate RIPK1 and facilitate TNF-induced necroptosis, which can be cleaved by caspase 829. Additionally, necrosome recruits molecular chaperone HSP90 and CDC37 to facilitate necroptotic signaling transduction30.Necroptotic signaling pathway has been reported to be tightly regulated, especially in the embryonic development31. JNK1 and JNK2 are ubiquitously expressed, while JNK3 mainly exists in neuronal and heart tissues32. JNKs have been reported to be involved in the regulation of multiple cellular processes, from apoptosis to gene expression33. JNKs show a critical role in death receptors induced by extrinsic and mitochondrial intrinsic apoptotic pathways. Other studies also have showed that JNK function as a double-edged sword in the regulation of apoptosis and had pro- or antiapoptotic functions, dependent on different cell types and death stimulus32. Although JNK has been reported to regulate apoptosis, the functional roles of JNK in necroptosis have not been well characterized. Here we report that JNK differentially regulate TNF- and TLRs-mediated necroptosis by their kinase and scaffolding activities.c-Jun N-terminal kinases or JNKs are a subfamily of the mitogen-activated protein kinase superfamily, which contains three isoforms  with multiple splice variantsThe following compounds were used: Necrostatin-1 , SP600125 , SB203580 , PD98059 , zVAD , Smac , butylated hydroxyanisole (BHA) , and protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) . LPS and poly I:C were purchased from InvivoGen. Mouse recombinant TNF was purchased from Ebioscience. Propidium iodide (PI) was from Sigma.MEF cells, L929 cells, and Raw 264.7 cells were cultured in complete Dulbecco's modified Eagle's medium supplemented with 10% FBS, 100U/ml penicillin, and 100μg/ml streptomycin.Bone marrow-derived macrophages (BMDMs) were prepared from bone marrow collected from the femurs and tibias of wild-type mice. Cells were depleted red cells with ammonium chloride and were differentiated over 6 days in the presence of conditional media . Fresh media were changed every 2 days. After 6 days, adherent cells were collected and reseeded for further experiments.34. Briefly, wild-type mice or TNF-deficient mice were injected intraperitoneally (i.p.) with 2ml of 4% thioglycolate medium (FTG from BD Biosciences). After 3 days, cells were collected by peritoneal cavity lavage with sterile PBS and seeded in the plates in DMEM complete media. After 2 h, the supernatant cells were discarded and the adherent cells were regarded as peritoneal macrophages.Peritoneal macrophages (PMs) were isolated and cultured as described previouslytm1Gkl/J) were from Model Animal Research Center of Nanjing University. All mice were maintained in specific pathogen-free conditions at the Institute of Health Sciences. Six to ten weeks old and gender-matched mice were used in all experiments. All animal experiments were performed according to the guidelines for the care and use of laboratory animals and are approved by the institutional biomedical research ethics committee of the Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences).Wild-type C57BL/6 mice were purchased from Slac Laboratory Animal Center, Chinese Academy of Sciences. TNF-deficient mice . Cells were transiently transfected with scrambled siRNA (negative control), RIPK3 siRNA (5′-gcucucgucuucaacaacu-3′), MLKL siRNA (5′-gaaccugcccgaugacauu-3′), JNK oligo , JNK-oligo-2 siRNA , and RIPK1 siRNA (5′-gcauuguccuuugggcaauau-3′). siRNAs were transfected using Lipofectamine RNAiMAX (Life Technologies), according to the manufacturer’s protocol. The cells were incubated with the transfectin mixture for 3 days, and then treated with indicated ligands and inhibitors. Knock down efficiency was determined by detecting protein levels or mRNA levels.Peritoneal macrophages were seeded in 12-well plates and then treated as indicated. RNA of peritoneal macrophages was prepared using the TRIzol reagent according to the manufacturer’s protocol. cDNA was produced by retro-transcribing RNA using PrimeScript RT reagent kit (Takara). Real-time PCR was performed using SYBR Premix ExTaq kit (Takara) on ABI PRISM 7900 Sequence Detection System (Applied Biosystems), all according to the manufacturer’s instructions. Detected gene expression was normalized to the housekeeping gene (Rpl13a). The real-time PCR primers were shown in Supplementary Table Cell death was determined by detecting the level of released cytoplasmic enzyme lactate dehydrogenase (LDH) or propidium iodide (PI)-positive cells through FACS. For the LDH release assay, cells were seeded in 96-well plates. Before stimulation, media was changed with OPTI MEM. Cells were treated as indicated and the supernatant was collected to detect the released LDH using the CytoTox96 LDH release kit (Promega), according to the manufacturer’s protocol. Total LDH was produced by adding lysis buffer to liberate all cytoplasmic LDH. Cell viability was determined with the percentage of total LDH. For PI staining, peritoneal macrophages were seeded in 12-well plates and then treated as indicated. The cells were collected with EDTA-trypsin and washed in PBS. Peritoneal macrophages were stained with 2.5μg/ml PI in sterile PBS for 20min. After that, the cells were centrifuged and washed in sterile PBS. PI-positive cells were analyzed using a FACScan flow cytometer (BD Biosciences).35. Briefly, the cells were treated as indicated and washed with cold PBS. The cells were harvested with lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.0% Triton X-100, 1 mM EDTA, and 10% glycerol containing protease inhibitors). Cell extracts were left on ice for 30 min and centrifuged at 13,000 r.p.m for 30 min. Cell lysates were incubated with 2μg of anti-RIPK1 antibody or 2μg of normal mouse IgG antibody (Santa Cruz) overnight at 4°C and then incubated with 15μl of protein A beads for an additional 3h at 4°C. The beads were washed with lysis buffer three times and then boiled in 20μl of 1×SDS loading buffer for 10 min. Samples were loaded on SDS-PAGE gels followed by electroblotting onto PVDF membranes (Millipore). To determine protein levels, immunoblot was performed using the following antibodies: anti-RIPK3 (2283) from Pro Sci; anti-pMLKL (ab196436) from Abcam; anti-RIPK1 (610459) from BD Biosciences; anti-pP38 (9211S), anti-JNK (9252), anti-pERK1/2 (4370), anti-pRIPK1 (S166) (31122S), anti-pJNK (9251) from Cell Signaling, anti-Tim23 (11123-1-AP) from Proteintech, anti-GAPDH (sc-32233) from Santa Cruz, and anti-TRIF (657102) from Biolegend.Immunoprecipitation and immunoblot analysis were performed as described previouslyPeritoneal macrophages were seeded in 12-well plates and treated as indicated. Cells were washed with cold PBS and harvested with 2×DTT-free sample buffer . Protein samples were loaded on SDS-PAGE gels followed by electroblotting onto PVDF membranes (Millipore). Immunoblot analysis was performed with RIPK3 and p-MLKL antibody.Staphylococcus aureus were sacrificed. Liver, ileum, and cecum from mice treated with TNF and lung from mice infected with Staphylococcus aureus were fixed in 4% paraformaldehyde for at least 2 days. Fixed samples were embedded into paraffin and sliced into 5-μm sections. Five-micrometer sections were stained with H&E, according to the standard procedures described previously34. The images were captured with a Leica FDM2500 microscope.Mice treated with TNF or infected with Eight to ten weeks old C57BL/6 female mice were used for experiments. Mouse recombinant TNF, DMSO, and SP600125 were diluted in endotoxin-free PBS. Mice were injected i.p. with DMSO or SP600125 for 30 min. And then mice were injected intravenously (i.v.) with 15μg of TNF. Mortality of mice was monitored after TNF injection. Plasma samples and tissue samples of ileum, liver, and cecum were collected at indicated times after injection.Staphylococcus aureus USA300 was from ATCC. Eight to ten weeks old C57BL/6 female mice were injected intraperitoneally with DMSO or SP600125 for 1h. And then mice were intranasally infected with 107 colony-forming units (CFU)/mouse Staphylococcus aureus. Twenty-four hours later, lungs from infected mice were removed for histology and measured with CFU. Homogenized lungs were diluted with sterile PBS and plated on Luria Bertani (LB) agar plates. CFU were counted after 12h on LB agar plates at 37°C.t test was used to compare differences between two groups. Survival curves were presented using Kaplan–Meier method and significance was calculated by log-rank (Mantel–Cox) test. Statistical significance was defined as P < 0.05. *P <  0.05, **P <0.01, and ***P < 0.001.Prism software (GraphPad Software) was used to perform statistical analysis and graph development. Two-tailed Student’s 37. Therefore, we wonder whether other MAPKs (P38 and JNK) also participate in the RIPK1- and RIPK3-dependent pro-inflammatory signaling pathways. We also found that, compared to the treatment of LPS or poly I:C alone, LPS or poly I:C in the presence of caspases pan-inhibitor zVAD significantly increased the pro-inflammatory cytokines  production in peritoneal macrophages  and a specific short- interfering RNA (siRNA) targeting RIPK3. We found that Nec-1 treatment abolished P38 and JNK activation induced by LPS, poly I:C, or TNF plus zVAD in peritoneal macrophages  to pretreat peritoneal macrophages and then induced necroptosis with TNF or TLRs. As expected, P38 inhibition sensitized macrophages to necroptosis induced by TNF or TLRs , as well as pRIPK3 in peritoneal macrophages stimulated by TNF and zVAD Fig. . Similar39. To exclude the off-target effect of the JNK inhibitor, we treated the knockdown macrophages with JNK inhibitor SP600125, and found that SP600125 showed no effect on both TNF- and TLR-induced necroptosis in these cells  to interfere the expression of the ubiquitously expressed JNK1 and JNK2. Loss of JNK1 significantly suppressed the cell death of peritoneal macrophages in TNF-induced necroptosis, while JNK2 absence had only a weak suppressive effect in TNF-induced necroptosis Fig. . Howeverlls Fig. , which iI:C Fig. . TherefoNext, we investigated the necroptotic signaling pathway in the absence of JNK protein by siRNA interference. Our results showed that JNK proteins, especially JNK1 proteins, were required for TNF-induced phosphorylation of MLKL in peritoneal macrophages Fig.  and Raw 42. Then we investigated whether JNK regulated TNF- and TLRs-induced necroptosis through ROS production. We used protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) to deplete the mitochondria of peritoneal macrophages43. The CCCP-treated macrophages had almost no mitochondria compared with untreated cells , which contributes to TNF-induced necrosis41.Therefore, we suspected that JNKs were likely to function similarly in a RIPK1-dependent way to regulate necroptosis. We found that, as loss of JNK, RIPK1 depletion by siRNA showed a similar distinguishable phenotype in TNF- and poly I:C- induced necroptosis oup Fig. . Consistoup Fig. . In addioup Fig. , althougoup Fig. . RecentlLKL Fig. . HoweverStaphylococcus aureus-mediated lung damage in mice. Other study showed a lethal phenotype of JNK1 and JNK2 double knockout mice46, which indicate that the JNK scaffold function within their proteins may serve as a brake for their kinase activities to prevent the uncontrolled necroptosis and tissue damage. Thus, our study determines a dual role for JNK as necroptosis regulators in vitro and in vivo.The knowledge on programmed necrotic cell death has advanced rapidly in recent years. However, the regulatory mechanisms of TNF- and TLRs-induced necroptosis remain unclear. Our study has revealed that JNK differentially regulate TNF- and TLRs-induced necroptosis in macrophages. We discovered that both JNK activities and their scaffold functions were required for TNF-induced necroptosis, while JNK showed kinase-dependent and independent roles in TLRs-induced necroptosis Fig. . We foun36. The previous paper has reported that LPS plus zVAD induced more ERK activation in BMDMs36. However, we found that in peritoneal macrophages, LPS plus zVAD trigger comparable ERK activation and even lower ERK activation at 6h  via its scaffold activity and not its kinase activity48. Therefore, we speculated that JNK could promote selective autophagy of TRIF to inhibit TLRs-induced necroptosis via scaffold activity.TRIF has been reported to mediate TLRs-induced necroptosis by recruiting RIPK1 and RIPK3 via its RHIM domain, which is dispensable for TNF-mediated necroptosisSupplementary Information


Piper sarmentosum is an herb that has shown stimulation on endothelial NO production by increasing both expression and activity of eNOS. Thus, this study determined whether the positive effect of P. sarmentosum on NO production is related to its modulation on the DDAH–ADMA pathway in cultured human umbilical vein endothelial cells (HUVEC) exposed to tumor necrosis factor-α (TNF-α). HUVEC were divided into four groups: control, treatment with 250 µg/ml of aqueous extract of P. sarmentosum leaves (AEPS), treatment with 30 ng/ml of TNF-α, and concomitant treatment with AEPS and TNF-α for 24 h. After treatments, HUVEC were collected to measure DDAH1 messenger RNA (mRNA) expression using quantitative real-time polymerase chain reaction. DDAH1 protein level was measured using enzyme-linked immunosorbent assay (ELISA), and DDAH enzyme activity was measured using colorimetric assay. ADMA concentration was measured using ELISA, and NO level was measured using Griess assay. Compared to control, TNF-α-treated HUVEC showed reduction in DDAH1 mRNA expression (P < 0.05), DDAH1 protein level (P < 0.01), and DDAH activity (P < 0.05). Treatment with AEPS successfully increased DDAH1 mRNA expression (P < 0.05), DDAH1 protein level (P < 0.01), and DDAH activity (P < 0.05) in TNF-α-treated HUVEC. Treatment with TNF-α caused an increase in ADMA level (P < 0.01) and a decrease in endothelial NO production (P < 0.001). Whereas treatment with AEPS was able to reduce ADMA level (P < 0.01) and restore NO (P < 0.001) in TNF-α-treated HUVEC. The results suggested that AEPS promotes endothelial NO production by stimulating DDAH activity and thus reducing ADMA level in TNF-α-treated HUVEC.Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide synthase (eNOS). ADMA is degraded by dimethylarginine dimethylaminohydrolase (DDAH). Elevated levels of ADMA lead to reduction in nitric oxide (NO) production, which is linked to endothelial dysfunction and atherosclerosis.  Atherosclerotic cardiovascular disease is the leading cause of mortality worldwide . The earEndothelium-derived NO is synthesized by endothelial nitric oxide synthase (eNOS). NO acts as an endogenous anti-atherosclerotic molecule by inducing vasodilation  and inhivia the kidneys, while most ADMA is degraded by dimethylarginine dimethylaminohydrolase (DDAH) enzyme to dimethylamine and l-citrulline  leaves is rich in flavonoids and possesses numerous pharmacological properties such as anti-inflammatory, antioxidant, antibacterial, and anti-osteoporosis activities  (specimen voucher number: UKMB40240). AEPS was prepared according to the previous method  in a reflux extractor machine. The extract was boiled at 80°C for 3 h, and the resulting solution was filtered and frozen at −80°C. Finally, the extract was freeze-dried and stored at 4°C. Liquid chromatography (LC)–mass spectrometry (MS) Orbitrap full-scan analysis was conducted to identify the compounds present in AEPS. LC–ultraviolet (UV) analysis was performed using the Accela™ ultrahigh-performance LC (UHPLC) system  equipped a with quarternary pump, a build degasser, a photodiode array (PDA) detector, and an autosampler. The column used for the chromatographic separation was a Luna Kinetex Reversed Phase C18 column . The conditions consisted of a gradient elution using acetonitrile as mobile phase A and 0.1% formic acid as phase B at a flow rate of 200 µl/min over 40 min. The following gradient was applied: 0–20 min, 5% A; 20–30 min, 35% A; 30–35 min, 100% A; 35–35.01 min, 5% A, returning to 5% A in 5 min, with 10 µl of sample injected. The compounds eluted and separated were further analyzed with a linear trap quadrupole) LTQ Orbitrap mass spectrometer  operating in a negative ion mode. The operating parameters were as follows: source accelerating voltage, 4.0 kV; capillary temperature, 350°C; sheath gas flow, 40 arb; auxiliary gas, 20 arb. Mass spectra were acquired with a scan range of 300–1,000 m/z. All data were processed with the Xcalibur™ 2.1 software . LC–MS Orbitrap full-scan analysis identified a total of 19 compounds in AEPS (t) and mass spectra, as provided by electrospray ionization (ESI)–MS . Informed consent was obtained from all volunteers prior to delivery. HUVEC were isolated using collagenase infusion as described previously . The celnditions . HUVEC w4 HUVEC per well were seeded in a 96-well plate and allowed to attach for 24 h. The cells were then exposed to 10 to 40 ng/ml of TNF-α for 24 h. MTT assay was used to measure the viability of HUVEC while Griess assay was used to measure NO level in the culture medium. MTT assay was done according to a previous method  for 24 h. AEPS at these concentrations was used as it had shown potent antioxidant effects in a previous study . Subsequ5 cell density were seeded onto six-well tissue culture plates and allowed to grow until it reached 80% confluency. The cells were then divided into four groups: control, treatment with 250 µg/ml of AEPS, treatment with 30 ng/ml of TNF-α, and concomitant treatment with 250 µg/ml of AEPS and 30 ng/ml of TNF-α for 24 h. The dose of TNF-α (30 ng/ml) was chosen as it was the first dose that significantly reduced HUVEC viability and NO production. As for AEPS, 250 µg/ml of AEPS was used as it significantly increased HUVEC viability and NO production when exposed to 30 ng/ml of TNF-α.HUVEC at 1 × 10® III First-Strand Synthesis SuperMix for qRT-PCR kit . Master mixes containing SYBR® Select Master Mix , cDNA samples, RNase- and DNase-free distilled water, and forward and reverse primers were prepared and pipetted into the PCR plate. Specific forward and reverse primers were used as follows: DDAH1 (GenBank accession no. BC_033680), forward: 5′-ggacaaatcaacgaggtgct-3′, reverse: 5′-tagcggtggtcactcatctg-3′; and GAPDH (GenBank accession no. NM_002046), forward: 5′-tccctgagctgaacgggaag-3′, reverse: 5′-ggaggagtgggtgtcgctgt-3′. The reaction was conducted at initial denaturing at 95°C for 3 min; then involved 40 cycles of 61°C for 30 s, 95°C for 1 min, 55°C for 1 min, 70 cycles of 60°C for 10 s; and terminated by a cooling step at 4°C. Each experiment was performed in duplicate. The specificity of the PCRs was verified by analysis of melting curves and 1.5% agarose gel electrophoresis. The threshold cycle (CT) value was determined, and the relative mRNA expression of DDAH was analyzed as follows:Extraction of RNA was done using TRI reagent  as described previously . FollowiDDAH1 protein level in HUVEC was measured using a human DDAH1 ELISA kit  according to the manufacturer’s instructions. Briefly, 100 µl of HUVEC lysates was added into the plate and incubated for 2 h at 37°C. After incubation, liquid from each well was aspirated, and biotin-conjugated antibody specific to DDAH 1 was added. After washing, avidin conjugated to horseradish peroxidase (HRP) was added followed by 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. Optical density (OD) of the wells was measured at 450 nm using a spectrophotometer.The activity of DDAH was determined based on a previous method . The priADMA concentration in HUVEC was determined using a human ADMA ELISA kit  according to the manufacturer’s instructions. HUVEC lysate and biotin-labeled ADMA were added into their respective wells. The mixture was mixed and incubated for an hour at 37°C. After incubation, liquid was aspirated and washed. Then, avidin conjugated to HRP was added to each well and incubated for 30 min at 37°C. The processes of aspiration and washing were repeated. After the last wash, TMB substrate solution and sulfuric acid were added. OD of each well was measured at 450 nm using a spectrophotometer.post hoc Tukey test. The data were presented as mean ± standard error for the mean (SEM). Differences were considered significant at P < 0.05.Data were analyzed using SPSS version 21.0 software. The Kolmogorov–Smirnov test was used to measure the normality of the data. Then, the data were analyzed using one-way ANOVA and P < 0.05) and NO level (P < 0.05) (P < 0.05) (P < 0.05) (P < 0.05). However, there was no significant difference in the NO level between 250 and 300 μg/ml of AEPS. Based on the effects of AEPS on cell viability and NO production, 250 μg/ml of AEPS was selected as the dose for subsequent experiments. HUVEC exposed to 250 μg/ml of AEPS showed a normal cobblestone appearance and had a similar density as the control group (Exposure to varied concentrations of AEPS up to 300 μg/ml did not cause noticeable toxicity to HUVEC whose viability was 80% and above (DDAH1 mRNA expression for the AEPS group compared to the control group (P = 0.061) (DDAH1 mRNA expression by 0.52-fold compared to control (P = 0.011). Treatment of TNF-α-induced HUVEC with AEPS increased DDAH1 mRNA expression by 0.47-fold compared to the TNF-α group (P = 0.033).There was no significant difference in = 0.061) Figure 3P = 0.001) (P = 0.004). Treatment of TNF-α-induced HUVEC with AEPS increased DDAH1 protein level compared to the TNF-α group (P = 0.002).AEPS increased DDAH1 protein level compared to control (= 0.001) Figure 4P = 0.023) (P = 0.016). Treatment of TNF-α-induced HUVEC with AEPS increased DDAH activity compared to the TNF-α group (P = 0.039).AEPS induced DDAH activity compared with control (= 0.023) Figure 5P = 0.076) (P = 0.0008). Treatment of TNF-α-induced HUVEC with AEPS successfully reduced ADMA concentration compared to the TNF-α group (P = 0.001).There was no significant difference in ADMA level between the AEPS and control groups (= 0.076) Figure 6P = 0.0001) (P = 0.0007). On the other hand, treatment of TNF-α-induced HUVEC with AEPS promoted NO production compared to the TNF-α group (P = 0.0001).There was a significant increase in NO level produced by HUVEC treated with AEPS compared to the control group ( 0.0001) Figure 7DDAH1 mRNA expression compared to control. This is in accordance with a previous study whereby mRNA expression of DDAH1 is decreased in HUVEC treated with TNF-α or oxidized low-density lipoprotein (ox-LDL)  activation and cellular adhesion molecule expression in HUVEC treated with TNF-α  . Inducti(ox-LDL) . AEPS wath TNF-α . Anti-TNth TNF-α . Therefoin vitro study reported that HUVEC exposed to TNF-α for 48 h showed eight times increase in ADMA level  butanoic acid had reduced DDAH activity and increased ADMA formation   . Even inhe cells . In thisAntioxidants are known to enhance NO by scavenging reactive oxygen species (ROS), hence protecting NO against oxidative destruction by ROS . As AEPSLC–MS Orbitrap full-scan analysis of AEPS leaves used in this study showed the presence of flavonoids such as naringenin, quercetin, and vitexin. Naringenin had been shown to stimulate eNOS and NO in endothelial cells exposed to high glucose . QuercetHowever, to date, there is no study done on the effect of vitexin on the DDAH–ADMA–NO pathway yet. Nevertheless, vitexin was proven to promote cell viability, suppressed apoptosis and reduced oxidative stress in HUVEC treated with ox-LDL . BesidesP. sarmentosum contains other active compounds such as rutin, vitamin E, carotenes, tannins, and xanthophyll . Informed written consent was obtained from all participants.AU and CH conceptualized the study and designed the experiment. US and JL performed the experiments and analyzed the data. AU, CH, AA, JL, and US analyzed and interpreted the data. AU, CH, AA, JL, and US wrote the manuscript and performed critical analysis and the necessary revisions of the work. All authors read and approved the final manuscript.This study was supported by research grants from the Ministry of Higher Education, Malaysia (Grant No.: FRGS/1/2014/SKK03/UKM/02/1), and Universiti Kebangsaan Malaysia (Grant No: GGPM-2013-029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.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.


Tumor necrosis factor alpha (TNF-α) is a potent cytokine involved in systemic inflammation and immune modulation. Signaling responses that involve TNF-α are context dependent and capable of stimulating pathways promoting both cell death and survival. TNF-α treatment has been investigated as part of a combined therapy for acute myeloid leukemia due to its modifying effects on all-trans retinoic acid (ATRA) mediated differentiation into granulocytes. To investigate the interaction between cellular differentiation and TNF-α, we performed RNA-sequencing on two forms of the human HL-60/S4 promyelocytic leukemia cell line treated with TNF-α. The ATRA-differentiated granulocytic form of HL-60/S4 cells had an enhanced transcriptional response to TNF-α treatment compared to the undifferentiated promyelocytes. The observed TNF-α responses included differential expression of cell cycle gene sets, which were generally upregulated in TNF-α treated promyelocytes, and downregulated in TNF-α treated granulocytes. This is consistent with TNF-α induced cell cycle repression in granulocytes and cell cycle progression in promyelocytes. Moreover, we found evidence that TNF-α treatment of granulocytes shifts the transcriptome toward that of a macrophage. We conclude that TNF-α treatment promotes a divergent transcriptional program in promyelocytes and granulocytes. TNF-α promotes cell cycle associated gene expression in promyelocytes. In contrast, TNF-α stimulated granulocytes have reduced cell cycle gene expression, and a macrophage-like transcriptional program. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytotoxic cytokine  which acIL-6  supplemented with 10% fetal bovine serum (Moregate Biotech), 1% penicillin/streptomycin (ThermoFisher), 1% GlutaMAX (ThermoFisher) and 2mM CaCl2.HL-60/S4 cells (available from ATCC #CCL-3306) were cultured at 37°, 5% CO6 cells/mL; or differentiated = 5x105 cells/mL) in fresh media (with 1µM ATRA for differentiated cells). Nuclear morphology and cell surface receptor changes during differentiation are available from Jacobson et al. 2018  dissolved in ethanol (Sigma Aldrich) for four days as previously described (6 per flask) were incubated at 37° for 2 hr before addition of 16ng/mL TNF-α or vehicle in triplicate per condition. After 2 hr treatment, cells in each flask were lysed with TRIzol LS (Life Technologies), phase separated with chloroform, and RNA extracted with the Qiagen RNeasy mini kit . A summary of the conditions in this study, and the public data from Welch 2017 .The full experiment was repeated and samples analyzed with RT-qPCR of Total RNA libraries were prepared by Annoroad Gene Technology Co., Ltd.  using ribosomal RNA depletion with RiboZero Magnetic Gold Kit (Human/Mouse/Rat) and sequenced on an Illumina Hi-seq X 150PE (150 base pair paired end reads).http://www.ncbi.nlm.nih.gov/bioproject/303179). These conditions are: HL-60/S4 (promyelocytes), HL-60/S4 differentiated with ATRA (granulocytes), and HL-60/S4 differentiated with TPA (macrophage-like).mRNA-seq data of HL-60/S4 cells in three conditions in quadruplet  and differentially expressed genes  < 0.05) identified in DEseq2  v2.28.0 Mycoplasma hominis ATCC 23114 (NC_013511.1), M. hyorhinis MCLD (NC_017519.1), Mycoplasma fermentans M64 (NC_014921.1) and Acholeplasma laidlawii PG-8A (NC_010163.1). The fasta genome sequences and genome annotation files were downloaded from NCBI Genome, and genome indices were created with STAR 2.6.0c. Due to their small genome size, the parameter genomeSAindexNbases was adjusted for each genome to the recommended log2(GenomeLength)/2 – 1. Zero reads from any of the 12 libraries aligned to any of the four mycoplasma genomes, confirming that our cultures were free from mycoplasma contamination.RNA-seq data were also used to check for mycoplasma contamination, as performed in . All libGAPDH and COX4I1). Log2 fold changes, standard errors, and confidence intervals were calculated in RStudio . RNA was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies). Isolated RNA was DNase I treated (Life Technologies). Single-stranded cDNA was synthesized from 1 µg of RNA using a High capacity cDNA RT kit (Thermofisher # 4368814), according to the manufacturer’s protocol. Real-time quantitative PCR analysis was carried out using predesigned PrimeTime Mini qPCR assays  on a Lightcycler 480 (Roche). mRNA levels were normalized using reference genes  were analyzed by RT-qPCR qPCR in a set of independent samples treated as above i.e., ± 1 µM ATRA and ± 16ng/mL TNF-α (Supp table 2). Consistent with RNA-seq analysis, RT-qPCR demonstrated that TNF-α stimulated promyelocytes and granulocytes have increased levels of TNF and VCAM1. CDK1 increases expression in promyelocytes and decreases expression in granulocytes following TNF-α treatment.In order to investigate transcriptional responses to TNF-α, the promyelocytic cell line HL-60/S4 was differentiated into a granulocytic form by treatment with ATRA for 96 hr, as described previously . The TNF-α treatment resulted in more genes being upregulated than downregulated in both the promyelocytic and granulocytic forms of the HL-60/S4 cells .The promyelocytic and granulocytic forms of HL-60/S4 cells both exhibited strong transcriptional responses to TNF-α treatment (Supp table 1). In promyelocytes, 1,312 genes were significantly increased and 980 significantly decreased . There was a small subset of differentially expressed genes that exhibited changes in opposite directions in promyelocytes and granulocytes .Genes that were significantly differentially expressed after TNF-α treatment in both the promyelocytic and granulocytic forms of HL-60/S4 cells exhibited strongly correlated log2 fold changes  hallmark gene sets  is a method that allows analysis of an entire differential expression dataset without using arbitrary significance thresholds . We found a strong upregulation response to TNF-α, in both the promyelocytic and granulocytic forms of the HL-60/S4 cells, that was characterized by immune and cell death pathways . HoweverAs observed in the differentially expressed genes, GSEA showed a strong conserved response with notable divergences between TNF-α stimulated promyelocytes and granulocytes.i.e., the genes were significantly differentially expressed in the same direction in both promyelocytic and granulocytic forms of the HL-60/S4 cells. The next two groups represent cell type-specific responses – i.e., genes that were only differentially expressed in either promyelocytes or granulocytes in response to TNF-α treatment. The final group is comprised of the 157 genes that were significantly upregulated in the first cell type and significantly downregulated in the other cell type, or vice versa.In order to further investigate the similarities and differences in the TNF-α response, we grouped the list of analyzed genes based on the direction and significance of the expression change in each cell type. Genes that were differentially expressed following TNF-α treatment were divided into four groups according to the nature of the correlation between the promyelocytic and granulocytic responses: 1) conserved; 2) promyelocyte-specific; 3) granulocyte-specific; and 3) opposite responses (Supp table 1). The first group represents the conserved response, Having identified groups of genes that change in a conserved, cell-type specific, and opposite manner, we performed functional enrichment analyses to further investigate the responses to TNF-α in promyelocytes and granulocytes.Genes that exhibited significant changes in the same direction in response to TNF-α treatment in both promyelocytic and granulocytic forms of the HL-60/S4 cells were more frequently upregulated than downregulated (∼3:1). Notably, this trend was not maintained for cell type-specific gene expression changes . There were nine enriched MSigDB hallmark gene sets . The topIn summary, using three different annotation databases and four different analysis approaches, we have shown that TNF-α treatment of both promyelocytic and granulocytic forms of HL-60/S4 cells induces a transcription profile associated with inflammatory responses and NFKB signaling.The cell type-specific promyelocytic DE genes had no significant enrichments of MSigDB gene sets, KEGG pathways, or GO terms. Cell type-specific granulocytic DE genes were enriched for MSigDB sets related to cell cycle and energetics . The set of genes that was significantly expressed in opposite directions was also enriched for three terms, including mitotic chromosome condensation, within the GO database (Supp table 11). There was no enrichment for pathways within KEGG.Genes that were significantly differentially expressed in opposite directions in promyelocytes and granulocytes were enriched for G2M checkpoint and E2F targets within the MSigDB database  and macrophages (with TPA). Principal component analysis of variance stabilized transcripts confirmed clustering by differentiation status (Supp figure 1C).TNF expression was upregulated during differentiation into either granulocytic or macrophage forms of HL-60/S4 cells. The magnitude of TNF transcript upregulation was different in granulocytes (log2 fold change = 1.37) and macrophages (log2 fold change = 4.08) (Supp table 1). Differentiation also induced changes in the expression levels of the TNFRs. Consistent with previous observations (TNFRSF1A gene expression was increased (log2 fold change = 0.87) after ATRA treatment, but there was no significant change in expression following TPA treatment. By contrast, TNFRSF1B gene expression was increased in both conditions . This observed increase in mRNA expression of TNF and both TNF-α receptors may be one explanation for why granulocytes have an enhanced transcriptional response to TNF-α, compared to promyelocytes.Consistent with previous reports  were downregulated, while genes associated with inflammatory terms  were upregulated in both differentiation experiments . The first group consists of genes that significantly change expression levels in the same direction after differentiation into either the granulocytic from with ATRA, or the macrophage form with TPA. The second and third groups contain genes that are differentially expressed only after differentiation into granulocytes or macrophages, but not both. The fourth group represents genes that are differentially expressed after differentiation into granulocytes and macrophages, but are upregulated in granulocytes and downregulated in macrophages, or vice versa. We analyzed these groups of genes for enrichment of MSigDB gene sets, KEGG pathways, and GO terms. GSEA identified the response that is conserved during differentiation into granulocytes or macrophages as being characterized by pro-inflammatory cytokine associated gene sets , and cell cycle associated gene sets   conserved; 2) granulocyte-specific; 3) macrophage-specific; and 4) opposite, spindle) . This fii.e., carbon metabolism and protein processing in endoplasmic reticulum), and the TNF-α signaling pathway (Supp table 17). Enriched GO terms included cell division, and terms related to RNA and protein regulation .Genes that changed expression after differentiation into granulocytes, but not macrophages, were not enriched for MSigDB gene sets or GO terms, but they were enriched for three KEGG pathways related to protein processing . In contrast, genes that changed expression after differentiation into macrophages, but not granulocytes, were enriched for several MSigDB gene sets related to cell cycle  increased expression after macrophage differentiation and granulocytes treated with TNF-α, but decreased expression after granulocytic differentiation, which indicates a shared change in transcriptional programming. CD83, which is a marker of transdifferentiation of neutrophils into a dendritic-type cell, increased in both TNF-α treated cell types  and TNFR2 (TNFRSF1B), but soluble TNF-α can only activate TNFR1  and concurrently higher levels of both TNFRSF1A and TNFRSF1B gene expression (There are several factors that could explain the different responses of promyelocytes and granulocytes to TNF-α. First, granulocytes have increased levels of endogenous TNF-α production. Not only does this increase the total TNF-α the cells are exposed to, but endogenously produced TNF-α is membrane-bound prior to processing (e.g., TNFR-associated factors (The TNFR1 and TNFR2 receptors have uniquely stimulated pathways (A reduction in the expression of cell cycle genes at the population level indicates a change in the proportion of cells at different stages of the cell cycle. Due to the massive transcriptional changes that occur during the progression through cell cycle , this maA large-scale TNF-α response experiment treating many different cell types at various stages of differentiation would allow a network analysis of the transcriptional responses and annotation-free pathway discovery (Despite being one of the most highly studied genes in the human genome , the comTNF expression, and increased levels of TNF-α receptors. Transcriptional changes indicate that TNF-α treatment represses cell cycle progression in granulocytes, but has the opposite effect on promyelocytes. This effect may be sensitive to the sum total of the exogenous and endogenous TNF-α levels. Finally, comparisons of transcriptional changes during differentiation and TNF-α treatment suggest that TNF-α treatment of granulocytes pushes them toward a macrophage transcriptional program. The context specific effects of TNF-α are likely mirrored in other models of innate immune signaling, and may contribute to the disparities seen between in vitro and in vivo studies of innate immune signaling.HL-60/S4 cells show a conserved set of core responses to TNF-α treatment irrespective of the differentiation state of the cell. This is expected, since functional annotations represent canonical pathways and responses. However, granulocytes are more responsive to TNF-α, possibly due to priming from increased endogenous


In addition, PPRV VLPs induced a strong Th1 class response in mice, as indicated by a high IgG2a to IgG1 ratio. Goat studies demonstrated that PPRV VLPs can induce the production of antibodies specific for F and H proteins and can also stimulate the production of virus neutralizing antibodies to the same magnitude as the PPRV Nigeria 75/1 vaccine. Higher amounts of IFN-γ in VLP-immunized animal serum suggested that VLPs also elicited a cellular immune response in goats. These results demonstrated that VLPs elicit a potent immune response against PPRV infection in small ruminants, making PPRV VLPs a potential candidate for PPRV vaccine development.Peste des petits ruminants is a highly contagious acute or subacute disease of small ruminants caused by the peste des petits ruminants virus (PPRV), and it is responsible for significant economic losses in animal husbandry. Vaccination represents the most effective means of controlling this disease, with virus-like particle (VLP) vaccines offering promising vaccine candidates. In this study, a PPRV VLP-based vaccine was developed using a baculovirus expression system, allowing for the simultaneous expression of the PPRV matrix (M), hemagglutinin (H), fusion (F) and nucleocapsid (N) proteins in insect cells. Immunization of mice and goats with PPRV VLPs elicited a robust neutralization response and a potent cellular immune response. Mouse studies demonstrated that VLPs induced a more robust IFN-γ response in CD4 Morbillivirus, family Paramyxoviridae [Peste des petits ruminants virus (PPRV) is the causative agent of peste des petits ruminants (PPR), a highly contagious disease that affects small ruminants and results in extremely high rates of mortality and morbidity. PPR is currently endemic in most of Africa, South Asia, the Middle East, and China . PPRV isoviridae , and hasoviridae ,4,5. Matoviridae . PPRV cooviridae ,8. Amongoviridae . In addioviridae ,11,12. Boviridae ,13,14. Toviridae ,16.PPR is classified as a notifiable disease by the World Organization for Animal Health . AccordiVaccination is the most effective method for preventing recurrence of PPRV infection. Attenuated PPRV Nigeria 75/1 and Sungri 96 are among the most widely used vaccines worldwide and are considered to be completely attenuated, safe, and potent. These two vaccines also offer lifelong protection to the immunized animals . HoweverAlthough previous studies have been successful in producing PPRV VLPs using sequences without codon optimization, the yield of VLPs is often low (3–4.5 mg/L) , thus inSpodoptera frµgiperda Sf9 (ATCC® CRL-1711TM) insect ovary cells in suspension culture were grown in Sf-900TM II serum-free medium  at 27 °C on a temperate orbital shaker at 200 rpm. Vero (ATCC® CCL-81TM) cells were grown in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum  at 37 °C with 5% CO2 for PPRV propagation and titration. Nigeria 75/1, the vaccine strain of PPRV, was purchased from Tecon, Xinjiang, China.The open reading frames (ORFs) for the PPRV (China/Tibet/geg/07-30) M, F, H and N genes were obtained from NCBI (Gen-Bank Access NO.FJ90530401). Codons in the four ORFs were optimized based on Sf9 cells to eliminate rare codons .Ecoli DH10Bac  competent cells, after which recombinant bacmids were identified by PCR using the two primer pairs M13F/P10R and M13R/P10F and purified as previously described [The synthetic genes M, F, H and N were each sub-cloned into the Puc57-Simple plasmid  to produce Puc57-Simple-M, Puc57-Simple-F, Puc57-Simple-H, and Puc57-Simple-N, respectively. All the primer sequences are shown in escribed .®II reagent  following the manufacturer’s protocol. In brief, 3 μg of bacmid DNA was diluted in 100 μL unsupplemented Grace’s Insect Medium  and vortexed briefly. Diluted DNA (100 μL) and 100 μL diluted Cellfectin®II reagent were combined and vortexed briefly, followed by incubation at room temperature for 15–30 minutes. DNA–lipid mixture was added onto the Sf9 cells and the cells were incubated at 27 °C for 3 h. Insect medium containing 10% FBS with supplements was used to replace the old medium. The first generation of virus (P1) was harvested after 4 days incubation at 27 °C. In order to obtain high-titer baculovirus, cells were continuously passaged for several generations.Recombinant baculoviruses (rBVs), named rpFBD-2M, rpFBD-2F, rpFBD-2H and rpFBD-2N expressing M, F, H and N proteins, respectively, were rescued using the CellfectionSf9 cells were infected with four rBVs at a multiplicity of infection (MOI) of 1. After 48 hours, Sf9 cells were fixed in 80% acetone for 30 min at room temperature, after which, they were incubated with one of the following primary antibodies: anti-M (1:100 dilution), anti-F (1:1000 dilution), anti-H (1:100 dilution), or anti-N (1:1000 dilution). A FITC-labeled goat anti-mouse antibody  was used as a secondary antibody to detect the expression of PPRV M and F proteins, and a FITC-labeled mouse anti-goat  was used as a secondary antibody to detect the expression of PPRV H and N proteins. Cells were incubated with primary and then secondary antibodies at room temperature for 30 min each and rinsed three times with PBS  between steps. The plate was observed using a ZEISS Axio Vert.A1 Inverted Microscope and FITC filter . Uninfected Sf9 cells and cells infected with wild-type baculovirus were used as negative and positive controls, respectively. 7/mL. Cells were cultured in suspension at 27 °C and 120 rpm. At 120 hours post-infection, supernatant was harvested and centrifuged at 5000 rpm for 20 min to remove cells and debris, after which, clarified supernatant was centrifuged at 30,000 rpm for 90 min at 4 °C to pellet the VLPs. VLPs were subsequently purified through a 20%–40%–60% (w/v) discontinuous sucrose gradient prepared in PBS, with centrifugation at 35,000 rpm for 90 min at 4 °C. Bands between 40% and 60% sucrose, corresponding to purified VLPs, were harvested, and the VLP concentration was determined to be 8–10 mg/L by BCA kit .To generate PPRV VLPs, Sf9 cells were infected with rBVs at an MOI of 5 in a volume of 300 mL with a cell density of 1–2×10Samples collected from Sf9 cells infected with rBVs were treated with 5×SDS Loading Buffer for 10 min at 100 °C and separated using 12% SDS-PAGE gels. For Western blot analysis, samples separated through SDS-PAGE were transferred onto polyvinylidene fluoride (PVDF) membranes , after which, the membranes were blocked for 1 hour in 5% milk in Tris-buffered saline with Tween 20 (TBST). Two different antibodies were used to detect the presence of M, F, H and N: mouse polyclonal antibody for M protein and sheep polyclonal antibody for N, F, and H protein. HRP-labeled goat anti-mouse IgG  and HRP-labeled rabbit anti-sheep IgG  were used as secondary antibodies. The membranes were washed in TBST and visualized using Western Blue stabilized substrate  to detect alkaline phosphatase.A copper–rhodium (Cu–Rh) grid with a mesh size of 200 was incubated with VLPs at room temperature for 60 min and then incubated with a 200-fold dilution of sheep anti-PPRV sera produced in-house at 37 °C for 60 min. The copper mesh was then incubated with a 50-fold dilution of 10 nm gold-conjugated goat anti-mouse IgG  at room temperature for 60 min. VLPs were stained with 1% phosphotungstic acid, and the excess dye solution was removed after 3 min. Samples were observed under a transmission electron microscope.All live animal work was performed in accordance with guidelines from the Animal Welfare and Ethics Committee of the Changchun Veterinary Research Institute (Permit No. SCXK-2012-017). The environment and housing facilities satisfied the National Standards of Laboratory Animal Requirements (GB 14925-2001) of China.3 TCID50 PPRV Nigeria 75/1 diluted in 100 μL DMEM. Mice in the control group were given 100 μL of PBS alone. All groups received a second and third immunization two and four weeks following the primary immunization. The booster immunizations were identical to the original immunization, with the exception of the VLP group, in which Freund’s incomplete adjuvant was used instead of Freund’s complete adjuvant. Blood samples were collected by retro-orbital plexus puncture at 2, 4, and 6 weeks post-vaccination. Serum was separated and stored at −80 °C for analysis of antibody and cytokine levels.BALB/c mice  were purchased from Changchun Animal Breeding Center for Medical Research . Animals were randomized into three groups of 10 and immunized either subcutaneously (s.c.) with 50 μg VLPs in 50 μL PBS mixed with an equal volume of Freund’s complete adjuvant , or immunized s.c. with 10TM Alum adjuvant . The third group was inoculated with 105 TCID50 PPRV Nigeria 75/1 in 1 mL DMEM as a positive control. The fourth group was inoculated subcutaneously with 1 mL PBS or alum adjuvant as a negative control. All goats received a second and third identical vaccination 3 and 6 weeks after the first. Blood was collected through the jugular vein at different time-points post-immunization using 10 mL vacuum blood collection tubes; serum was separated and stored at −80 °C for antibody titration.Outbred goats  were purchased from the Changchun Institute of Biological Products Co., Ltd, China and randomized into five groups of five animals each. The first and second groups were vaccinated s.c. in the gastrocnemius muscle with 150 or 300 μg/goat of PPRV VLPs, respectively, mixed with an equivalent volume of ImjectMouse sera obtained at 2 weeks post-immunization was analyzed for PPRV-specific IgM, while sera obtained at 6 weeks post-immunization was analyzed for PPRV-specific IgA, IgG, IgG1, and IgG2a. Antibody titers were measured using an indirect ELISA, as described elsewhere . The coa3 TCID50/mL) in a 96-well cell culture plate. Virus-only control wells and uninfected-cell control wells were also included. After incubation at 37 °C for 1 h, Vero cell suspension was added (50 mL/well). The cytopathic effect (CPE) was observed after 8 days incubation. VNA titer was expressed as the highest dilution at which the CPE was inhibited by at least 50%, according to previously described methods [PPRV-specific virus neutralization antibody (VNA) titers were analyzed in serum samples collected from immunized mice and goats using a micro-neutralization assay. Serially diluted  inactivated serum was mixed with an equal volume of PPRV . IFN-γ- or IL-4-secreting cells were quantified using ELISpot kits , according to the manufacturer’s instructions. Spot-forming cells (SFCs) were counted on an automated ELISpot reader  ,30.7 cells/mL) were harvested two weeks after the second vaccination. Cells were stimulated with inactivated PPRV (10 μg/mL) containing monensin  for inhibiting protein transport. At 6 h post-stimulation, anti-CD8 and anti-CD4 monoclonal antibodies (BD Biosciences) were used to quantify surface expression of CD8 and CD4. After permeabilization with Cytofix/Cytoperm (BD Biosciences), cells were stained with anti-IFN-γ and anti-IL-4 monoclonal antibodies (BD Biosciences). All stained cells were detected using a FACSAria TM CELL Sorter (BD Biosciences), as previously described [Mouse splenocytes  bacteria (Takara) transformed with a T7 promotor plasmid that expresses the open reading frame of F, H, or N protein fused to a His-tag. Purification of the His-tagged protein from the inclusion bodies was done using HisPurTM Ni-NTA Spin Columns , as per the manufacturer’s protocol. Purified F, H, and N proteins (0.5 µg) were used to coat 96-well, flat-bottomed plates overnight at 4 °C. The following steps were performed according to a previous study . The totTo determine cytokine responses, Goat Interleukin 4 (IL-4), Goat Interleukin 10 (IL-10) and Goat Interferon γ (IFN-γ) ELISA Kits were purchased from Cusabio. The reagent, samples and standards were prepared according to the manufacturer’s protocol.Figures were generated using GraphPad Prism 8.0 software. Differences between means were evaluated using the one-way ANOVA or two-way ANOVA and were deemed significant at P values of 0.05 or less.To generate PPRV VLPs, we first constructed a series of bacmids, each expressing two copies of the codon-optimized genes for M, H, F, or N from two separate promoters (p10 and pH) in order to enhance protein expression a. From t3 TCID50 of the commercial live-attenuated vaccine PPRV Nigeria 75/1 were used as positive controls, while mice immunized with PBS served as negative controls. Inactive sera collected at different time-points after immunization were serially diluted and incubated with live PPRV Nigeria 75/1. The virus was then added to Vero cells, and the development of CPE was monitored. As shown in To assess the immunogenicity of VLPs, mice were vaccinated with 50 µg VLP and boosted two weeks later, after which, neutralizing antibody titers were determined using a micro-neutralization assay. Mice immunized with 10To further investigate the humoral response to PPRV VLPs, antigen-specific total IgG and antibody subtypes were confirmed by ELISA . The totAfter confirming that VLPs successfully induced a broad antibody response in mice, we next sought to evaluate the induction of a cell-mediated immune response. The antigen-specific IFN-γ and IL-4 activities in splenocytes were evaluated by ELISpot assays a. Spot-f+ and CD8+ splenocytes were labeled for IFN-γ or IL-4. VLPs elicited a robust IFN-γ-secreting CD4+ T cell response in contrast to PPRV Nigeria 75/1, which elicited a significantly greater IL-4-secreting T cell response  than PBS alone; however, PPRV Nigeria 75/1 induced significantly higher levels of CD19+CD40+ cells than VLPs (+CD80+ and CD11c+CD86+ cells (DCs) in the lymph nodes of VLP-immunized animals were significantly higher than those immunized with PPRV Nigeria 75/1 and the PBS control group (To investigate whether VLPs can induce the production of B cells and dendritic cells (DC), cells from inguinal lymph nodes were analyzed using flow cytometry. Mice immunized with VLPs or PPRV Nigeria 75/1 induced a greater production of CD19han VLPs a. Converol group b,c. Compol group d,e. Take5 TCID50 Nigeria 75/1, while control animals were immunized with either PBS or the alum adjuvant alone  and PPRV Nigeria 75/1. The data showed that the neutralizing antibody response in PPRV Nigeria 75/1 is higher than that induced by VLPs in goats , although the differences were not statistically significant. Goats immunized with 300 µg of VLPs induced higher levels of neutralizing antibodies—starting from the third week after the first immunization—compared to goats immunized with 150 µg of VLPs, but the differences were not significant until the twelfth week after immunization, and no neutralization activity was detected in pre-immune serum or in serum from animals immunized with either PBS or adjuvant  in the inguinal lymph nodes than immunization with PPRV Nigeria 75/1. Our results also demonstrate that PPRV VLPs stimulate greater expression of MHC I and MHC II molecules on the surface of DCs. Whether VLPs induce immunity through similar mechanisms in goats requires further research, especially considering the differences in mouse and goat immune systems, including differences in Toll-like receptors. High-level expression of cytokines and co-stimulatory molecules likely elicited enhanced cellular immune and humoral responses compared with PBS-immunized mice. Furthermore, VLPs derived from rabbit hemorrhagic disease virus and hepatitis B virus have been found to be efficiently processed by DCs for cross-presentation of an MHC class I-restricted epitope, a process that induced strong protective cytotoxic T lymphocyte responses [DCs are the most potent antigen-presenting cells (APCs) in the body, and they play a crucial role in promoting the proliferation and differentiation of T cells. A previous study demonstrated that papillomavirus VLPs bind to DCs and stimulate their maturation, including the upregulation of MHC class I and II, CD80, CD86, and CD40 costimulatory molecules and cytokine production ,47. Simiesponses ,49. Our A previous study showed that PPRV VLPs could be readily assembled by the co-expression of M and N proteins in Sf9 cells infected with rBVs; however, these VLPs did not induce neutralizing antibodies in mice or goats . AlthougOur results indicate that PPRV VLPs can produce neutralizing antibodies although the titers are a little lower compared to the attenuated vaccine PPRV Nigeria 75/1. PPRV VLPs also induced a higher level of activation of cell-mediated immune responses. Our results demonstrate that the PPRV VLP-vaccine containing all four proteins is highly immunogenic. In conclusion, we demonstrate that PPRV VLPs can be successfully produced and that the resulting vaccine has the ability to induce cellular and humoral immune responses in mice, as well as neutralizing antibodies and enhanced IFN-γ secretion in goats. These findings suggest that PPRV VLPs containing all four viral proteins  may be a promising vaccine candidate to facilitate the control and eradication of PPR. Whether the VLP vaccine induces a long-term humoral response, like the attenuated vaccines, remains to be investigated.