The gastroduodenal artery (GDA) is frequently embolized in cases of upper GI bleed that has failed endoscopic therapy. Additionally, it may be done for GDA pseudoaneurysms or as an adjunctive procedure prior to Yttrim-90 (Y90) treatment of hepatic tumors. This clinical review will summarize anatomy and embryology of the GDA, indications, outcomes and complications of GDA embolization. Upper gastrointestinal (GI) bleeding is a common and potentially deadly medical condition with an estimated mortality of 10% . The gasThe GDA, like other mesenteric arteries, originate from the embryological descending aorta, which is in turn developed from the primitive ventral segmental arteries . Most ofThe GDA arises from the common hepatic artery. Lateral to the GDA origin, the common hepatic artery becomes the proper hepatic artery. The variable origins of the GDA include the superior mesenteric artery, the left hepatic artery, and the right hepatic artery . It courGastrointestinal bleeding is a significant public health issue, with an annual estimated direct cost to the health care system of five billion dollars . PatientUpper GI bleed can be variceal or non-variceal. The former is a sequela of portal hypertension with treatment options including endoscopic banding, and/or sclerotheraphy, transjugular intrahepatic portalsystemic shunt (TIPS) placement +/− embolization, and/or balloon occluded retrograde transvenous obliteration/variants (BRTO) ,10. The Helicobacter pylori (H. pylori) infection. With the advent of the improved detection and treatment of H. pylori, non-steroidal anti-inflammatory drugs (NSAIDs) have become the most common cause . Ot. Ot26]. Historically, psuedoaneursyms were treated surgically, through ligation and/or resection. Transarterial embolization has now become the preferred treatment method in GDA pseudoaneurysms, given superior success rate and improved mortality, when compared to surgery . The tecEndovascular embolization can be utilized in the cases of hemobilia. The causes of hemobilia could include trauma (often iatrogenic), vasculitis, hepatobiliary tumors, and vascular malformations. Hemobilia presents with melena in 90% of cases, hematemesis in 60% of cases, biliary colic in 70% of cases, and obstructive jaundice in 60% of cases . AngiogrThe coil embolization of GDA, prior to Y-90 treatment, is sometimes performed in order to prevent non-target embolization. Selective internal radiation therapy (SIRT) with Yttrium-90 (Y-90) is performed in order to provide oncologic control of liver tumors, including primary liver cancer, (hepatocellular carcinoma [HCC]) and for liver dominant metastases, such as in unresectable colorectal hepatic metastases. Commercially available resin or glass microspheres  contain yttrium, a β-emitting radioisotope, which leads to local irradiation . Dependi® catheter , as they may obviate the need for the coil embolization of the GDA all together [Several studies showed that no difference was seen in the incidence of gastrointestinal complications in patients with prophylactic embolization of the gastroduodenal artery, when compared to the patients without embolization ,42. A fetogether .The main complication after transarterial embolization is ischemia. Although the upper gastrointestinal tract has a rich collateral blood supply, ischemic complications can still occur in 7 to 16% of cases ,47. PrevUpper GI bleeding is a potentially life threatening condition with the overall mortality rates for non-variceal bleeding approaching 10% . Pseudoa


The article entitled “Effect of Tumor Necrosis Factor-Alpha (TNF-α) on Erythropoietin- and Erythropoietin Receptor-Induced Erythroid Progenitor Cell Proliferation in β-Thalassemia/Hemoglobin E Patients”, written by Tanyong et al.  and publSplenectomy can increase the release of TNF-α and cell apoptosis in experimental and clinical studies in different diseases ,3,4.Increased serum TNF-α was reported in E/b-Thal patients, particularly after splenectomy ,4. In siBanyatsuppasin et al. suggested the role of the spleen in controlling mononuclear phagocytic activity in E/b-Thal patients . TNF-α p


Inhalation of diesel exhaust particles (DEP) induces an inflammatory reaction in the lung. However, the underlying mechanisms remain to be elucidated. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that operates by binding to tumor necrosis factor receptor 1 (TNFR1) and tumor necrosis factor receptor 2 (TNFR2). The role of TNF-α signaling and the importance of either TNFR1 or TNFR2 in the DEP-induced inflammatory response has not yet been elucidated. TNF-α knockout (KO), TNFR1 KO, TNFR2 KO, TNFR1/TNFR2 double KO (TNFR-DKO) and wild type (WT) mice were intratracheally exposed to saline or DEP. Pro-inflammatory cells and cytokines were assessed in the bronchoalveolar lavage fluid (BALF). Exposure to DEP induced a dose-dependent inflammation in the BALF in WT mice. In addition, levels of TNF-α and its soluble receptors were increased upon exposure to DEP. The DEP-induced inflammation in the BALF was decreased in TNF-α KO, TNFR-DKO and TNFR2 KO mice. In contrast, the inflammatory response in the BALF of DEP-exposed TNFR1 KO mice was largely comparable with WT controls. In conclusion, these data provide evidence for a regulatory role of TNF-α in DEP-induced pulmonary inflammation and identify TNFR2 as the most important receptor in mediating these inflammatory effects. In controlled human exposure studies, inhalation of diesel exhaust or DEP was associated with increased airway resistance and airway inflammation in healthy individuals; as demonstrated by increased neutrophils, lymphocytes and mast cells and elevated IL-8, IL-6 and myeloperoxidase levels in bronchoalveolar lavage fluid (BALF), bronchial biopsies and sputum6. Also in murine studies, we have previously demonstrated that exposure to DEP induces an inflammatory response in the lung characterized by the accumulation of neutrophils, monocytes, dendritic cells and T-cells8. Moreover, DEP exposure increased pro-inflammatory cytokine and chemokine expression in BALF and lung tissue9. However, the precise molecular and cellular events underlying the DEP-induced pulmonary inflammation remain unclear. Understanding these mechanisms could provide new tools to protect and treat at-risk individuals from the adverse effects of particulate air pollutants.In the last decades, several epidemiological studies have shown an association between inhalation of particulate matter pollutants and adverse pulmonary health effects including increased risk for respiratory tract infections, asthma, chronic bronchitis, chronic obstructive pulmonary disease and lung cancer. Particulate matter emissions from diesel engines  are a major contributor to the ambient air pollution problem10.Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine and is an important mediator of inflammation. It is generated as a type II transmembrane protein that can be proteolytically processed by TNF-converting enzyme (TACE or ADAM17) into a soluble form. Both the transmembrane and soluble protein mediate their biological effects by binding to TNF receptor 1  and TNF receptor 2 , which differ in structure, expression patterns and signaling pathways that they induce13, others described no or rather a decreased TNF-α release from macrophages in response to DEP14. Interestingly, increased TNF-α levels in plasma were found in healthy individuals that were exposed to diesel exhaust15. In animal studies, acute and chronic exposure to DEP was reported to be associated with elevated TNF-α levels in lung tissue19, however single or multiple DEP exposures up to 4 days in TNF-α knockout (KO) mice showed no role for TNF-α in the acute DEP-induced neutrophilic response20. The involvement of TNF-signaling in other DEP-induced inflammatory cell responses has not yet been addressed thus far.The contribution of TNF-α/TNFR signaling to the DEP-induced lung inflammation remains poorly understood and controversial. Whereas several studies reported that in vitro stimulation with particulate matter and DEP resulted in increased TNF-α secretion from lung epithelial cells, alveolar macrophages and peripheral blood mononuclear cells from human or miceIn this manuscript, we examined the role of TNF-α/TNFR signaling in DEP-induced lung inflammation. For that, we evaluated TNF-α knockout (KO) mice as well as TNFR1 KO, TNFR2 KO and TNFR1/TNFR2 double KO (TNFR-DKO) mice in an established murine model of DEP-induced pulmonary inflammation.+ T-cells and CD8+ T-cells in a dose-dependent manner  mice were exposed to saline and increasing doses of DEP  and analyzed at day 9. Exposure to DEP increased the number of total BALF cells, including neutrophils, monocytes, dendritic cells, CD4ner Fig. . When coner Fig. . Since t21. To examine this in our murine model, lung function was measured in WT mice that were exposed to saline and 100 µg DEP by assessing AHR to carbachol. As shown in Fig. Exposure to DEP has been associated with increased airway hyperresponsiveness (AHR)in vivo exposure to DEP induces a TNF-α response in the lungs. Exposure to 100 µg DEP was associated with significantly elevated TNF-α levels in BALF in comparison with saline-exposed mice  versus non-hematopoietic (CD45−) cells from lung tissue of saline and DEP-exposed WT mice, and examined the mRNA expression. We found that TNF-α mRNA was preferentially expressed in the CD45+ hematopoietic lung cell population, while limited TNF-α mRNA expression was seen in the CD45− lung cell population , CC chemokine ligand 2 (CCL2), and CC chemokine ligand 20 (CCL20) in BALF Fig. . DEP-exp+ T-cells and CD8+ T-cells in comparison with WT controls , can bind the TNF receptors10. Since exposure to particulate matter can increase the mRNA expression of TNF-β in lung tissue30, it would be worthwhile to investigate how TNF-β signaling relates to pollutant-mediated TNF receptor signaling.TNF-α exerts its biological functions via interaction with its receptors: TNFR1 and TNFR2. We demonstrate that TNFR2 is the most important receptor in eliciting the DEP-induced lung inflammation, as revealed by decreased inflammatory cell numbers and cytokine expression in DEP-exposed TNFR2 KO mice when compared with DEP-treated WT controls. In contrast, the DEP-induced accumulation of inflammatory cells was similar in TNFR1 KO and WT mice. Levels of DEP-induced CXCL1, CCL2 and CCL20 were however also slightly reduced in absence of TNFR1, thus a minor role for the TNFR1 axis cannot be ruled out. Similar to our current findings, TNFR2 was identified as the most active receptor in cigarette smoke-induced pulmonary inflammation and emphysema2. In line with literature10, we demonstrated that TNF-α and TNFR2 were dominantly expressed by lung hematopoietic cells. Given that TNFR2 is preferentially activated by membrane-bound TNF-α10, one can speculate that TNF/TNFR2 signalling via hematopoietic cell-cell contacts contributes to the DEP-induced cellular inflammation and cytokines. In contrast, TNFR1 is activated by both membrane-bound and sTNF-α, and is reported to be expressed more uniformly10, although we showed the highest expression on CD45− cells. The mild reduction of chemoattractants in DEP-exposed TNFR1 KO mice could therefore suggest a role for TNFR1 in epithelial produced CXCL1, CCL2 and CCL20.Inhaled DEP targets multiple cell types including hematopoietic cells as well as structural cells such as the epithelium10. Although exposure to DEP is associated with increased activation of these pathways2, the specific contribution of DEP-induced TNF-α herein is unclear. Moreover, in an attempt to explain the more crucial role for TNFR2 in our model, it would be interesting to compare the activation of TNFR downstream signaling pathways between WT, TNFR1 and TNFR2 KO mice that were exposed to DEP.TNF-α binding on its receptors activates a complex network of signaling pathways including NF-κB and mitogen activated protein kinases (MAPK)et al. previously showed no difference in DEP-induced neutrophils in the BALF of TNF-α KO and WT controls20, suggesting that TNF-α signaling is not implicated in the DEP-induced neutrophilic inflammation. Although we obtained inconsistent results for DEP-induced neutrophils in preliminary experiments31, our current data support the statement of Saber et al. that TNF-a signaling is not crucial in the DEP-induced neutrophilic response. Indeed, neutrophils are recruited very early after exposure to DEP or particulate matter , whereas TNF-α levels increased relatively late (i.e. 24 hours) after exposure to DEP17. Also upon exposure to ozone, TNF-α signaling only contributed to neutrophilic inflammation induced upon subacute, but not acute ozone exposure26. A possible candidate that is important in the (early) DEP-induced neutrophil inflammation could be IL-1β. In this regard, we previously showed that blocking IL-1β or the IL-1R during a single dose of DEP was associated with a strong attenuation of the DEP-induced neutrophilic inflammation9.Concerning the DEP-induced neutrophils, Saber 35. A limitation of our work is that we could not investigate the contribution of TNF-α signaling to DEP-induced AHR, due to the absence of AHR in our model in WT mice. In experimental DEP exposure models, some authors have reported increased AHR36, whereas others failed to find AHR in response to DEP38. This discrepancy between studies could be due to the (duration of) experimental protocol, the mouse strain that was used, and the method of assessing pulmonary function (i.e. invasive lung function measurements versus noninvasive whole-body plethysmography). On exposure to ozone, airway hyperreactivity to methacholine and acetylcholine was diminished in TNFR1 and TNFR2 KO mice when compared with WT controls39.In controlled chamber studies, exposure to diesel exhaust was shown to increase airway resistance and hyperresponsiveness in healthy persons or individuals with asthma40. In this regard, human and animal studies have reported increased TNF-α levels in plasma and cardiac tissue in response to diesel exhaust and particulate matter inhalation42. Furthermore, blocking TNF-α with a monoclonal antibody attenuated the cardiac dysfunction that was induced by exposure to particulate matter41. This suggests that TNF-α signaling can contribute to various adverse health effects that are attributed to particulate matter inhalation.Besides causing inflammatory responses in the lung, exposure to DEP and ambient particulate matter also associates with adverse cardiovascular events. Indeed, pro-inflammatory cytokines that are produced on exposure to particulate matter can elicit systemic inflammation that increase the risk for cardiovascular diseases43, clinical improvement is suggested in subpopulations of patients with corticosteroid-refractory asthma and high TNF-α expression45. At least in mice, TNF-α neutralization restored the therapeutic effects of corticosteroids in glucocorticoid-resisted asthma46. Of interest, corticosteroid-refractory asthma has been associated with exposure to particulate air pollutants such as DEP47.The clinical use of TNF-α inhibitors in the treatment of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease is controversial and has been reported to be associated with potential adverse side effects. Whereas TNF-α antagonism in a general population of moderate-to-severe asthmatics failed to improve asthma outcomeIn conclusion, we demonstrated a role for TNF-α signaling in DEP-induced lung inflammation. In addition, we showed that TNFR2 is the most important receptor in pulmonary inflammation upon DEP exposure, although a contribution for TNFR1 cannot completely be ruled out. Our data provide insights into the mechanisms by which exposure DEP induces inflammatory responses in the lung. This could be of interest in the search for new tools to protect and treat at-risk individuals from the adverse effects of particulate air pollutants.in vivo manipulations were approved by the Animal Ethical Committee of the Faculty of Medicine and Health Sciences of Ghent University and were performed in accordance with institutional guidelines for animal care.Female C57BL/6 J WT mice were obtained from The Jackson Laboratory. TNF-α, TNFR1 and TNFR2 KO breeding pairs were purchased from The Jackson Laboratory and bred in the animal breeding facility of Ghent University Hospital. TNFR-DKO breeding pairs were obtained from the French National Center for Scientific Research and bred in the animal facility of the Faculty of Medicine and Health Sciences of Ghent University. Animals aged 6–8 weeks were included in the experiments. All 9, DEP (SRM 2975) were obtained from the U.S. National Institute for Standards and Technology. DEP were suspended in sterile saline containing 0.05% Tween 80 to a concentration of 2 mg/ml, 1 mg/ml, 0.5 mg/ml or 0.25 mg/ml (i.e. 100 µg (5 mg/kg), 50 µg (2.5 mg/kg), 25 µg (1.25 mg/kg) and 12.5 µg (0.625 mg/kg) dose respectively) and instilled on day 1, 4 and 7. The control group received sterile saline containing 0.05% Tween 80. Prior to instillation, mice were anesthetized with an intraperitoneal ketamine/xylazine injection (70 mg/kg ketamine (Ketamine 1000 CEVA); 7 mg/kg xylazine (Rompun 2%)). Anesthetized mice were held vertically and 50 µl saline or DEP solution was pipetted just above their vocal cords. On day 9, the animals were sacrificed by a lethal dose of intraperitoneal pentobarbital.As described previously2+ or Mg2+ supplemented with 1% BSA (for cytokine and chemokine measurements) and 6 × 500 μl HBSS without Ca2+ or Mg2+ supplemented with 0.6 mM sodium EDTA. The lavage fractions were pooled. Total cell counts were performed using a Bürcker chamber. Differential cell counts were performed on cytospin preparations after May-Grünwald-Giemsa staining. The remaining cells were used for flow cytometry.A tracheal cannula was inserted and BALF was recovered by instillation of 3 × 300 μl HBSS without Ca2+ or Mg2+ containing 5 mM EDTA and 1% BSA. To minimize nonspecific bindings, cells were pre-incubated with anti-CD16/CD32 (2.4G2). Cells were labeled with fluorochrome-conjugated monoclonal antibodies against the following cell markers: CD11c (HL3), MHCII (2G9), CD11b (M1/70), Ly6C (AL-21), Ly6G (1A8), CD3 (145-2C11), CD4 (GK1.5) and CD8 (53-6.7). 7-Aminoactinomycin D was used for dead cell exclusion. Data acquisition was performed on a FACSCalibur flow cytometer running CellQuest software. FlowJo software was used for data analysis. Following gating strategy was used: neutrophils were CD11c−, CD11b+, Ly6C+, Ly6G+ cells; monocytes were CD11c-, CD11b+, Ly6C+, Ly6G− cells; dendritic cells were CD11c+, low autofluorescent, MHCII+ cells; CD4+ T-cells were CD3+, CD4+, CD8− cells; CD8+ T-cells were CD3+, CD8+, CD4− cells.All staining procedures were performed in PBS without CaTNF-α, sTNFR1, sTNFR2, CXCL1, CCL2, and CCL20 protein levels in BALF were measured using commercially available ELISA kits (R&D Systems) following the manufacturer’s instructions.2. RBCs were lysed using ammonium chloride buffer. Hematopoietic (CD45+) cells and nonhematopoietic (CD45−) lung cells were sorted using an OctoMACS separator and CD45 MicroBeads . The sorted populations showed >95% purity (data not shown). Next, RNA extraction was performed (miRNeasy Mini Kit), and cDNA (Transcriptor First Strand cDNA synthesis kit) was obtained. Expression of TNF-α, TNFR1 and TNFR2 mRNA, relative to hypoxanthine guanine phosphoribosyl transferase (HPRT) was analyzed using TaqMan gene expression assays. qRT-PCR was performed using a LightCycler 96 system. Data was processed using the standard curve method.To remove the intravascular pool of cells, lung circulation was rinsed with saline, supplemented with EDTA. Lung tissue were minced and incubated in digestion medium  for 45 min at 37 °C and 5% COAirway resistance was measured in anaesthetized tracheostomized mice using the Flexivent system. Neuromuscular blockade was induced using intravenously injected pancuronium bromide (1 mg/kg). Mice were challenged with increasing doses of carbachol . The resistance (R) of the whole respiratory system  was measured.U), according to standard statistical criteria. Reported values were expressed as mean ± SEM. P-values under 0.05 were considered as significant.Statistical analysis was performed with SPSS, version 22.0. Groups were compared using nonparametric tests (Kruskal-Wallis and Mann-Whitney


Cancers have killed millions of people in human history and are still posing a serious health problem worldwide. Therefore, there is an urgent need for developing preventive and therapeutic cancer vaccines. Among various cancer vaccine development platforms, virus-like particles (VLPs) offer several advantages. VLPs are multimeric nanostructures with morphology resembling that of native viruses and are mainly composed of surface structural proteins of viruses but are devoid of viral genetic materials rendering them neither infective nor replicative. In addition, they can be engineered to display multiple, highly ordered heterologous epitopes or peptides in order to optimize the antigenicity and immunogenicity of the displayed entities. Like native viruses, specific epitopes displayed on VLPs can be taken up, processed, and presented by antigen-presenting cells to elicit potent specific humoral and cell-mediated immune responses. Several studies also indicated that VLPs could overcome the immunosuppressive state of the tumor microenvironment and break self-tolerance to elicit strong cytotoxic lymphocyte activity, which is crucial for both virus clearance and destruction of cancerous cells. Collectively, these unique characteristics of VLPs make them optimal cancer vaccine candidates. This review discusses current progress in the development of VLP-based cancer vaccines and some potential drawbacks of VLPs in cancer vaccine development. Extracellular vesicles with close resembling to viral particles are also discussed and compared with VLPs as a platform in cancer vaccine developments. Streptococcus pyogenes and Serratia marcescens, which became known as Coley’s Toxin in 1891  is one of the primary factors that impede the development of cancer vaccines . SeveralSuccessful vaccination relies heavily on the immunogenicity and presentation efficiency of the tumor antigen. Various entities have been employed to function as tumor antigen delivery platforms. In this regard, virus-like particles (VLPs) offer several advantages over other substances. VLPs are multimeric nanostructures morphologically resembling authentic viral particles. They are mainly composed of viral structural proteins with inherent self-assembly properties but are devoid of viral genetic materials . Thus, V+ cytotoxic T lymphocytes (CTLs) and CD4+ T helper cells by activated DCs elicits potent antitumor immune responses and breaks self-tolerance  VLPs were employed to display an HBV antigenic determinant known as the “a” determinant  in yeasts or mammalian expression systems . In an apatients . Recentlerminant , an immuerminant . Immunizat study .in vitro studies also demonstrated the protective effects of the vaccine  . In the  vaccine . SurprisHCV VLPs are commonly synthesized by expressing viral glycoproteins E1 and E2. The efficacy of HCV VLP vaccine was extensively tested in different animal models, including mouse and baboon, and yielded satisfactory results . RecentlNon-virus induced HCC cannot be treated with preventive vaccine, but a VLP-based antitumor vaccine has been previously described by Recently, chimeric HBc VLPs bearing the HBV x protein (HBx) were produced by Nicotiana benthamiana and L2 was found to be exposed on the surface of L1/L2 chimeric VLPs. Immunization studies demonstrated that L1/L2 chimeric VLPs induced both L1- and L2-specific humoral immune responses and conferred cross-protection upon immunized mice against HPV-16 and HPV-52 . According to the American Cancer Society, the survival rate of CC patients varies between 15% and 93%, depending on cancer stage. It was reported that nearly 100% of women with CC were found to be HPV positive, of which, HPV-16 and HPV-18 accounted for approximately 70% of all CC cases . To dated HPV-52 . Similard HPV-52 . Remarkasubtypes .To date, all commercially available CC vaccines are prophylactic HPV vaccines. The lack of a therapeutic vaccine makes it impossible to cure pre-existing HPV-associated CC. Nevertheless, an attempt to develop a therapeutic vaccine against HPV was undertaken by inserting the non-structural E7 protein of HPV into infectious bursal disease virus VLPs (VLP E7) . The E7 To improve CTL activity, an MHC class II restricted T-cell epitope was inserted into VLPs. In particular, Pancreatic cancer is a highly aggressive cancer, which is currently ranked as one of the top five causes of cancer-related mortality . The aveTrop2 is one of the tumor-associated antigens (TAAs), which is overexpressed in many epithelial carcinomas. Overexpression of this protein is often associated with tumorigenesis, metastasis, decreased overall survival rate, and tumor grade . LimitedIntriguingly, a recombinant simian/human immunodeficiency virus (SHIV) also has been recruited as backbone for the production of VLP-based pancreatic cancer vaccine. The first SHIV VLPs were produced by a baculovirus expression system using the protein transfer technique . FollowiIn addition to its association with pancreatic cancer, MSLN is also commonly expressed in mesothelioma, ovarian cancer, lung cancer, acute myeloid leukemia, and uterine serous carcinoma . As a coin vitro DC activation  conferred partial protection from prostate tumor challenge in mice and the maximum protection was achieved only when PSA-MPy VLPs were loaded onto DCs in the presence of CpG as an adjuvant . Both CT B cells . Loading1−683Py VLPs  and drops to 22% at stage IV or metastatic stage . The mos3Py VLPs . Her2 ha3Py VLPs . In vivo vaccine . However vaccine . Humoralinto DCs . Polyomainto DCs .Furthermore, enveloped VLPs also have been used to display Her2. in vivo, it is expected that it will be a suitable vaccine candidate to elicit strong cellular immunity 257−264 epitope and H-2Kb-restricted CTL epitope of melanoma differentiation antigen tyrosinase-related protein 2 (TRP2) into VP1 of murine polyomavirus VLPs (PLP), generating chimeric PLP (VP1-OVA252−270 PLP) and chimeric murine polyomavirus like pentamers (VP1-TRP2180−192PP), respectively. In vivo studies of VP1-OVA252−270 PLP in mice showed that notable protection against OVA-expressing melanoma cells was conferred by the induction of strong CTL activity. Although VP1-TRP2180−192PP failed to assemble into VLPs, surprisingly it partially protected immunized animals from lethal melanoma challenge  VLPs have been recruited as tumor antigen delivery system because this virus does not infect humans . RHDV VLA VLP-based vaccine against lung metastases has been established by incorporating the TAA isoform 2 of the tight junction molecule claudin-18 (CLDN18.2) into HBc VLPs . Immunizin vitro  infected individuals . In factin vitro . In addiin vitro . EBV VLPin vitro . CTL immin vitro . Howeverin vitro .Extracellular vesicles (EVs) refer to microvesicles and exosomes released by cells into their extracellular space . For decDespite promising results presented by VLPs, there are two major drawbacks of using VLPs as a platform in cancer vaccine development. One of the major concerns is the pre-existing immunity of an immunized individual against the VLPs which function as nanocarriers for epitopes or therapeutic peptides. This phenomenon is known as carrier induced epitopic suppression (CIES), characterized by suppression of antibody responses directed against the antigen conjugated to an immunogenic carrier due to the pre-existing immunity against the carrier . HoweverAnother impediment in VLP-based vaccine development is the cost of production. A vaccine candidate is unlikely to be competitive in the market if its manufacturing process is not scalable due to cost ineffectiveness even if it yields a promising pre-clinical outcome. For instance, current HPV VLP-based vaccine costs approximately US$360 for the full regimen in the United State, a price which is not widely affordable in many developing countries where the CC is actually most prevalence . ProductCancers that emerge following infectious diseases are notorious killers worldwide. Vaccination against such cancers is speculated to be the most effective approach in cancer prevention and treatment. VLPs are promising candidates in cancer vaccine development due to their characteristics such as stability and capability of self-assembly. These prerequisite features are required for packaging and delivery of bioactive materials, such as tumor antigens, antibody fragments, immunodominant regions, short peptides, DNA, or RNA. Construction of chimeric VLPs depends on the nature of the VLPs. Although both non-enveloped and enveloped VLPs can be produced by genetic manipulation, the surface chemical modification and protein transfer technology represent alternatives for the production of non-enveloped and enveloped VLPs, respectively with minimum genetic modifications. In addition, VLPs containing PAMP can be used in the development of self-adjuvating vaccines, mitigating the reliance on the use of adjuvants which is often associated with some undesired side effects.a’ determinant of HBV were invented as new therapeutic and preventive vaccine candidates, respectively (in vitro and in vivo, paving way to the development of low cost, broad spectrum HPV prophylactic vaccines. On the other hand, the VLP-based therapeutic CC vaccines were developed to target E7 and E6 proteins expressed by the HPV-associated CC. These VLP-based vaccines could represent a curative treatment to patients with pre-existing HPV-associated CC and they can also be engineered to induce a desired immune response by fusing with specific immunostimulatory molecules via genetic engineering approaches. For instance, MHC class II restricted T cells epitope was incorporated into the RHDV VLPs to favor the proliferation and activation of T cells which in turn enhance CTL immune responses (In treating or preventing HBV-related diseases, VLPs carrying fragments of antibodies against HBsAg and the ‘ectively . Furtherectively . To combesponses .Besides oncovirus-related cancers, other forms of cancer without a known etiology agent can also potentially be treated with VLP-based therapeutic vaccines by inserting tumor antigens into the viral envelop or capsid proteins followed by antigen presentation on the target tumor side. Highly aggressive pancreatic cancer may be potentially treated with Trop2 TAA incorporated into the envelope of SIV VLPs, although co-administration with gemcitabine may be required for optimal protection . SimilarMorphologically, EVs resemble viral particles, and the former are important carriers in intercellular signaling, vaccines and drug delivery systems. Similar to VLPs, EVs were employed as a platform for cancer vaccine development owing to their unique features in harboring and displaying tumor antigens. Nevertheless, the challenges faced by EV-based carriers include safety, immunogenicity and efficacy, which must be addressed properly in the future. In fact, due to the natural occurrence of EVs in the cells, most of the studies about EV-based carriers in the past were mainly focused on drug delivery system instead of vaccine development . HoweverPre-existing immunity of the immunized individuals and cost of vaccine production are the two major drawbacks of using VLPs as a platform in vaccine development. Pre-existing immunity against VLPs could interfere the efficacy of any subsequent vaccination involving the same VLP via CIES. Although the effect of CIES can be alleviated by several approaches, a careful selection of VLPs could avoid CIES. On the other hand, vaccine production must be cost effective and scalable. Ideally, to eradicate a preventable disease, a vaccine must be affordable for everyone in the globe, especially the developing countries where the diseases are actually most prevalent.In conclusion, efficacy of the VLP-based vaccine is highly dependent on the selection of tumor antigens and the design of VLPs. In order to produce an effective VLP-based vaccine, the genetic factor of an individual tumor has to be taken into consideration during the selection of the tumor antigen because mutations of the tumor cells vary considerably from person to person even they are diagnosed with the same kind of cancer . In the


RNA interference mediated by small interfering RNA (siRNA) provides a powerful tool for gene regulation, and has a broad potential as a promising therapeutic strategy. However, therapeutics based on siRNA have had limited clinical success due to their undesirable pharmacokinetic properties. This study presents pH‐sensitive nanoparticles‐based siRNA delivery systems (PNSDS), which are positive‐charge‐free nanocarriers, composed of siRNA chemically crosslinked with multi‐armed poly(ethylene glycol) carriers via acid‐labile acetal linkers. The unique siRNA crosslinked structure of PNSDS allows it to have minimal cytotoxicity, high siRNA loading efficiency, and a stimulus‐responsive property that enables the selective intracellular release of siRNA in response to pH conditions. This study demonstrates that PNSDS can deliver tumor necrosis factor alpha (TNF‐α) siRNA into macrophages and induce the efficient down regulation of the targeted gene in complete cell culture media. Moreover, PNSDS with mannose targeting moieties can selectively accumulate in mice liver, induce specific inhibition of macrophage TNF‐α expression in vivo, and consequently protect mice from inflammation‐induced liver damages. Therefore, this novel siRNA delivering platform would greatly improve the therapeutic potential of RNAi based therapies. The disulfide linkages could react with intracellular reductive molecules, such as glutathione (GSH), which is an important antioxidant and can reduce disulfide bonds by serving as an electron donor. Crosslinked siRNA nanoparticles were prepared by crosslinking the siRNA bearing a terminal sulfanyl group with the polymer chains through the disulfide linkages.FigureComparing to the intracellular reductive environment, microenvironmental pH variation is one of useful stimuli for altering the property of siRNA carriers and promoting the release of siRNA.The unique siRNA‐crosslinked structures of PNSDS allow it to improve siRNA stability, loading efficacy, cellular uptake, endosomal escape, and biological activity, endowing it with ability to mediate efficient gene silencing. siRNA is chemically crosslinked into the compact nanoparticle without the aid of any polycationic agents, and thus reduce the risk of nonspecific protein absorption, immunogenicity, and cytotoxicity. In addition, the tethering and tunable release of siRNA from PNSDS has also been achieved by the incorporation of acid labile acetal linkages near siRNA cross‐links, allowing PNSDS to release siRNA in response to the intracellular pH reduction. Furthermore, PNSDS has low cytoxicity, long blood circulation time, and minimal non‐specific interactions with serum proteins and non‐target tissues because of its positive‐charge‐free feature and protective PEG shell respectively. Finally, PNSDS is easy to be modified with targeting moieties, facilitating the target delivery of siRNA and inducing specific inhibition of gene expression in vivo. Therefore, the PNSDS would be an ideal siRNA delivery nanocarrier for efficient siRNA silencing.22.1FigureThe most common materials chosen for the crosslinked nanoparticles is polyethyleneglycol (PEG) due to its excellent biocompatibility, low toxicity, and non‐adhesion toward protein and cells, and ability to decrease renal clearance. In this research, eight‐armed PEG functionalized with azide moieties (AAPEG) was clicked to cyclooctyne‐containing siRNA crosslinkers via copper‐free strain‐promoted azide‐alkyne cycloaddition (SPAAC),2.2FigureTo investigate the potency of PNSDS to facilitate the siRNA delivery, tumor necrosis factor alpha (TNF‐α) siRNA was chosen to fabricate PNSDS, and its ability to alleviate inflammation‐induced cell damages was evaluated. As shown in the An ideal nanoparticle based siRNA delivery system is expected to have some common structural features including a small particle size (10–200 nm) and high amount of siRNA loading. Scanning electron microscopy (SEM) was performed to examine the morphology and size of PNSDS. Figure PNSDS is formed by crosslinking of siRNA with acid labile acetal linkages on the surface of AAPEG, which is designed to be stable in the physiological environment with pH 7.4, but allows to release siRNA from PNSDS in response to acidic intracellular environment (pH 4.5–6.5). Although extensive crosslinking of PNSDS prevents nuclease degradation of siRNA, it also suppresses the release of siRNA from complexes. Therefore, pH sensitive hydrolysis of acetal linkages in PNSDS is a critical factor for the successful delivery of siRNA. To verify the hypothesis, the hydrolysis kinetics of AAPEG was measured at pH 5.0 and 7.4. Figure 2.3FigureThe above results have demonstrated that siRNA can be easily incorporated into AAPEG and form well‐defined nanocomplexes. We next examined whether PNSDS can carry siRNA into cells. Macrophage cells (RAW 264.7), in which nucleus was stained with 4′,6‐diamidino‐2‐phenylindole, were incubated with PNSDS carrying FAM labeled siRNA, and the uptake and intracellular location of siRNA were investigated by fluorescent microscopy. −1 , PNSDS loading TNF‐α siRNA (PNSDS‐T), PNSDS loading scramble siRNA (PNSDS‐S) as a negative control. And then, all cell groups were treated with 100 ng mL−1 of LPS for 20 h to stimulate TNF‐α production, which was quantified using an enzyme linked immunosorbent assay (ELISA). FigureNext, we investigated whether the PNSDS could deliver functional siRNA into macrophages and inhibit gene expression. siRNA targeting TNF‐α was incorporated into the PNSDS and their ability to inhibit TNF‐α production in macrophages stimulated with lipopolysaccharide (LPS) was investigated.2.5To achieve the therapeutic applications of siRNA, the delivery systems not only need to protect siRNA against serum nucleases, prevent renal clearance, stimulate endosomal disruption, enhance cellular uptake, and release siRNA into cytoplasm, but also need to selectively localize and target siRNA to the diseased tissue. Therefore, an ideal nanoparticle based siRNA delivery system should have the multifunctional properties, which can be accomplished by optimizing the cross‐linking density, surface‐active, stimuli‐responsive constituents, and targeting moieties associated with specific diseases. We have demonstrated that well‐dispersed spherical PNSDS could be easily prepared via SPAAC methods. We further utilized SPAAC strategy to functionalize the PNSDS with the mannose moieties, which can improve its ability to target macrophages and to minimize aggregation with serum proteins. Mannose moieties were introduced in PNSDS via mixing cyclooctyne functionalized mannose with PNSDS‐T or PNSDS‐S to block the residual azides on AAPEG, leading to mannose modified PNSDS (M‐PNSDS‐T or M‐PNSDS‐S).d‐galactosamine , was chosen as the animal model, because LPS can stimulate macrophages to release inflammatory mediator TNF‐α, which plays a major role in the development of liver failure.−1 of siRNA via the intraperitoneal injection. 24 h later, these mice were subsequently challenged with an intraperitoneal injection of LPS/d‐GalN. The therapeutic efficacy of PNSDS for acute liver failure was then evaluated by measuring the expression levels of TNF‐α in the serum. Figured‐GalN, mice pretreated with M‐PNSDS‐T had a twofold decrease in TNF‐α level after challenged with LPS/d‐GalN. In contrast, neither free TNF‐α siRNA nor M‐PNSDS‐S had inhibitory effect on the production of TNF‐α. Additionally, liver tissue was isolated from these mice and the expression of TNF‐α mRNA in liver was determined by gel electrophoresis to determine the specificity of PNSDS mediated gene suppression. Figure We further investigated the potential of M‐PNSDS‐T to mediate TNF‐α gene silencing and treat the inflammatory liver diseases, which is one of major causes of mortality still increasing year‐on‐year. Acute liver failure, induced in mice by LPS and d‐GalN challenge. For example, mice pretreated with M‐PNSDS‐T exhibited significantly lower serum ALT than mice with the pretreatment of free TNF‐α siRNA or M‐PNSDS‐S. In addition, histological examination on HE‐stained liver sections further confirmed the remarkable therapeutic efficacy of PNSDS. In comparison of healthy liver with normal histology, liver tissue in LPS/d‐GalN + M‐PNSDS‐T group, LPS/d‐GalN + M‐PNSDS‐S group, and LPS/d‐GalN + free TNF‐α siRNA group showed different levels of injury  and liver histopathological examination. Figure y Figure d. Mice r3In summary, we have developed a novel pH‐responsive nanoparticle based siRNA delivery system, termed the PNSDS, for selective delivery of siRNA both in vitro and in vivo. PNSDS has a unique crosslinking structure, which allows it address all challenges of siRNA delivery including the perspectives of physicochemical properties of siRNA, pharmacokinetics and biodistribution, and intracellular trafficking. We show that PNSDS is capable of delivering TNF‐α siRNA into macrophage cells, and induces efficient and specific down regulation of the expression of TNF‐α. In addition, mannose functionalized PNSDS exhibits efficient TNF‐α siRNA gene silencing in vivo, and displays remarkable anti‐inflammatory effects against hepatic injury. Furthermore, PNSDS can be simply prepared by crosslinking of AAPEG with siRNA crosslinker carrying concyclooctyne moieties via SPAAC, indicating that PNSDS provides an efficient and modular platform for systemic delivery of various siRNA by utilizing different siRNA sequence. Therefore, the PNSDS provides a clinically suitable, safe, and effective siRNA delivery system, and can be broadly used in RNAi therapeutics for disease prevention and treatment.4Materials, characterization, detailed synthetic procedure of AAPEG and targeting group, and primer sequences for RT‐PCR are described in the Supporting Information.Synthesis of Cyclooctyne Modified siRNA: 4 × 10−3m (10 μL) of siRNA modified with an amine group at both ends in DEPC water was reacted with 30 × 10−3m (30 μL) of 4‐nitrophenyl chloroformate activated cyclooctyne, a functional group for copper free click, dissolved in dimethyl sulphoxide (DMSO). After 4 h incubation at room temperature, the residual and byproduct was eliminated through dialysis in deionized water for overnight. The resulting product was stored at −20 °C for further use. The complete and successful reaction of siRNA with cyclooctyne was confirmed by gel shit assay and mass spectrum.General Procedure for the Preparation and Characterization of siRNA Crosslinked Particles: 1 μL of this duplex solution (10 × 10−6m) was mixed with 2.5 μL of AAPEG (200 × 10−6m), and kept at room temperature for 10 min. The successful incorporation of siRNA to the nanocomplexes (PNSDS) was confirmed by agarose gel and characterized for size and zeta potential using dynamic light scattering , and also characterized by SEM . Mannose conjugated nanoparticles (M‐PNSDS) was formed by simply mixing PNSDS with cyclooctyne modified mannose (compound 4 shown in the Supporting Information) and the size was characterized by DLS .Hydrolysis of AAPEG: A stock solution of AAPEG (2.2 mg per 40 μL) in phosphate‐buffered saline (PBS) solution (pH = 8.0) was prepared and 2.5 μL (3.35 × 10−3m) AAPEG was added to a 700 μL PBS solution at either pH 5.0 or 7.4, in a spectrophotometer cuvette. The hydrolysis of the acetal was monitored at 37 °C by measuring the absorbance of at 280 nm.The Release of siRNA from PNSDS: Gel electrophoresis was performed to determine if free siRNA could be released from the acid degradable nanocomplexes. A small amount of PNSDS was hydrolyzed for 2 h at pH 5.0 and the resulting solution was run on a 1% agarose gel at 100 V for 15 min. After that, a photograph was taken with a gel imaging system.Fluorescent Microscopy of siRNA Delivered with PNSDS: This study investigated the intracellular distribution of siRNA delivered by PNSDS to macrophages, using fluorescent microscopy. RAW264.7 macrophages were maintained at 37 °C under a humidified atmosphere of 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), supplemented with penicillin (100 U mL−1) and streptomycin (100 mg mL−1). The macrophages  were incubated with PNSDS with FAM‐siRNA as cross linker for 4 h. Cells were washed three times with ice cold PBS, then fixed with 4% formaldehyde for 10 min, washing again with PBS for two times, adding 5 μg mL−1 of DAPI solution to the fixed cells for 5 min, the cells were washed three times with PBS and imaged with fluorescent microscopy.Delivery of TNF‐α siRNA with the PNSDS In Vitro: RAW264.7 macrophages  in DMEM with 10% FBS were incubated with either PNSDS TNF‐α siRNA, PNSDS‐scrambled siRNA or free TNF‐α siRNA for 4 h . The cells were stimulated with 100 ng mL−1 LPS for another 20 h to induce TNF‐α. The amount of extracellular TNF‐α production was determined using an ELISA assay kit following the manufacturer's instructions in the kit.Cytotoxicity of the AAPEG and PNSDS (MTT Reduction Assay): An MTT reduction assay was performed to measure the cytotoxicity of the AAPEG and PNSDS. The macrophages  were treated with the AAPEG and PNSDS at various concentrations (0.075–3 mg mL−1) for 24 h. After pouring out the medium, the cells were then treated with 100 mL of MTT (1 mg mL−1 in PBS) and incubated for another 4 h. The medium was removed, the cells were lysed by adding 150 μL of DMSO, and the absorbance of the purple formazan was recorded at 520 nm using a microplate reader . Percentage cell viability was calculated by comparing the absorbance of the control cells to that of AAPEG and PNSDS treated cells respectively.M‐PNSDS Induced In Vivo RNAi against Liver Injury: Male C57BL/6 mice were intraperitoneally injected with free siRNA, M‐PNSDS containing TNF‐a siRNA or Scr siRNA at a siRNA dose of 50 μg kg−1 (4 mice per group), and with untreated mice serving as a control group. Twenty‐four hours post administration, LPS/d‐GalN (12.5 mg kg−1 and 1.25 g kg−1) were intraperitoneally injected. Blood was collected 2 h later to determine the serum TNF‐α level by ELISA .−1 (4 mice per group), and i.p. challenged with LPS/d‐GalN as described above. Six hours later, blood was collected and serum was isolated via centrifugation. Serum ALT levels were determined by clinical laboratory of XinQiao Hospital ). Mice were then sacrificed; liver was harvested, cut into small pieces, washed with saline, and homogenized with RNAiso Plus  reagent to extract the total RNA. cDNA was synthesized from 1 μg total RNA with PrimeScriptTM II1st Strand cDNA Synthesis kit  according to the manufacturer's suggested protocol. The reaction condition was 42 °C for 60 min, 95 °C for 5 min. Then the synthesized cDNA was amplified. Primers used for the amplification of TNF‐α and the GAPDH gene were as in Table S1 (shown in supporting information). PCR was performed under the following conditions: 95 °C for 30 s, 55 °C for 30 s, and then 72 °C for 1 min (30 cycles). After that, 0.8% agarose gel was used to analyze the levels of the TNF‐α mRNA, the GAPDH as reference gene to normalize the data. For histological evaluation, mouse liver was harvested 6 h post LPS/d‐GalN stimulation, fixated in paraffin, cross‐sectioned, and stained with hematoxylin/eosin. Images were acquired by microscopy .In another experiment, mice were administered with free siRNA, M‐PNSDS containing TNF‐a siRNA or Scr siRNA at a siRNA dose of 50 μg kgAs a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


It is unstable at physiological concentrations and slowly converts into an inactive form. Here, we investigated the mechanism of this process by using a Förster resonance energy transfer (FRET) assay that allowed monitoring of monomeric subunit exchange in time. We observed continuous exchange of monomeric subunits even at concentrations of TNF high enough to maintain its bioactivity. The kinetics of this process closely corresponds with the appearance of monomeric subunits and disappearance of trimeric TNF in time at ng/ml concentrations as monitored by high-performance size-exclusion chromatography (HP-SEC). Furthermore, of the five therapeutic TNF inhibitors that are currently used in the clinic, three  were found to completely inhibit the monomer exchange reaction and stabilize TNF trimers, whereas golimumab and certolizumab could not prevent monomer exchange, but did slow down the exchange process. These differences were not correlated with the affinities of the TNF inhibitors, measured with both surface plasmon resonance (SPR) and in fluid phase using fluorescence-assisted HP-SEC. The stabilizing effect of these TNF inhibitors might result in prolonged residual TNF bioactivity under conditions of incomplete blocking, as observed  Tumor necrosis factor (TNF) is a homo-trimeric cytokine that plays a key role in mediating inflammation24Interestingly, it has been reported that (soluble) TNF is non-stable at physiological concentrations (pg/mL-ng/mL) and slowly converts into inactive, presumably monomeric TNF, both in buffer and serum67TNF is also an important mediator of a number of inflammatory auto-immune disorders including rheumatoid arthritis, Crohn’s disease, and psoriasis910Fig. 112Here, we studied the mechanism of dissociation of TNF into monomeric subunits. We adapted a Förster resonance energy transfer (FRET) assay that was previously used to monitor antibody subunit exchange in human IgG4 molecules (Fab arm exchange)t1/2 = ca. 3–5 and 20–50 hours; In order to investigate the putative dissociation and re-association of TNF subunits, we made use of a FRET assay. TNF was fluorescently labeled with either DyLight-488 or DyLight-594 such that on average ca. 2 dye molecules were attached to TNF . Both spNext, we investigated the dissociation of TNF trimers. TNF-488 was incubated at 3 ng/mL for various times at 37 °C and analyzed using high-performance size-exclusion chromatography (HP-SEC), monitoring the elution of TNF by in-line fluorescence detection. Disappearance of trimeric TNF in time was observed, accompanied by the appearance of monomeric TNF . HoweverNext, we investigated the influence of therapeutic TNF inhibitors on the stability of TNF. Monomer exchange was evaluated by monitoring the ‘dilution’ of mixed and equilibrated TNF488/TNF594 after addition of unlabeled TNF in the presence or absence of an excess of TNF inhibitor. This approach was chosen because binding of several TNF inhibitors to TNF was influenced by the fluorescent labeling . Under tThe differential stabilizing activity of the different TNF inhibitors may be caused by differences in affinity. To investigate this possibility, affinities were measured by two methods. First, binding of TNF to TNF blockers captured onto a biosensor chip was monitored by surface plasmon resonance (SPR). This method has previously been used by others to measure the affinities of several of the TNF blockers, but not all five of them together, and with sometimes widely different results between studies151617181920Kd. Bioactivity was analyzed immediately or after three days incubation at 37 °C. As expected, adalimumab Fab’ reduced TNF bioactivity due to its competition with cell bound TNF receptors. However, after incubation for three days at 37 °C, additional loss of TNF bioactivity was almost completely prevented . Recombinant therapeutic TNF blockers used in this study are adalimumab , infliximab , golimumab , certolizumab pegol , and etanercept . The first three TNF blockers are full-length IgG1 kappa antibodies, certolizumab pegol is a Fab’ fragment conjugated to a polyethylene glycol (PEG) group, and etanercept is a receptor-Fc construct.Full-length IgG1 certolizumab was made by cloning constructs coding for VH and VL of certolizumabF(ab’)2 and Fab’ fragments were generated using pepsin, and subsequent reduction and alkylation with dithioerythritol and N-ethylmaleimide respectively, as described beforeTNF, Fab and Fab’ fragments were fluorescently labeled with DyLight 488 or DyLight 594 amine reactive dye (Pierce/Thermo Scientific). Unreacted dye was removed by repeated dilution/concentration using Amicon Centriprep centrifugal filter devices  until no dye could be detected anymore in the filtrate. The average degree of labelling (DOL) ranged from 0.3 - 3.Frel) is determined by dividing emission at 620 nm by emission at 588 nm).Kinetics of the exchange reaction of TNF monomers were monitored in real-time using a previously developed FRET assay for the monitoring of non-covalent protein subunit exchangeStability of TNF was assessed by incubating TNF-488 at 0.3–3 ng/mL in PBS-I for various times between 0 and 5 days, alone or in the presence of unlabeled TNF (30 or 300 ng/mL), adalimumab, certolizumab pegol, or etanercept (1 μg/mL) in the dark at 37 °C. Samples were subsequently analyzed applying 500 μl to a Superdex 200 HR 10/300 column  at 0.5 ml/min using a thermostatted autosampler (4 °C), which was connected to an ÄKTAexplorer HPLC system . Elution profiles of TNF-488 were monitored by measuring the fluorescence (excitation/emission 488/525 nm) with a Prominence RF-20Axs on-line fluorescence detector .Affinities were determined as described previouslyN-hydrosuccinimide/1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydro-chloride (NHS/EDC) at a flow rate of 15 μL/min. Anti-TNF monoclonal antibodies were dissolved at 5 μg/ml in PBS containing 0.05% polysorbate-20 (PBS-T), and passed through the cells at 30 μL/min yielding ca. 100 response units (RU) of bound antibody. Subsequently, binding of TNF (50–300 ng/mL) dissolved in PBS-T was measured at a flow rate of 30 μL/min for 600 s followed by monitoring dissociation for another 600 s. After each run, immobilized ligand was regenerated by removing bound analyte with 5 μL of 0.1 M phosphoric acid. Adsorptions obtained in the reference channel without bound anti-TNF antibody was subtracted from the adsorptions in the other cells. Association and dissociation kinetics were fitted using the models provided with the Biacore analysis software.Surface Plasmon Resonance measurements were performed using a Biacore T-200 instrument  at 25 °C. Mouse anti-human IgG  was immobilized at a concentration of 10 μg/ml in 10 mM sodium acetate, pH 5.0, on a CM5 sensor chip using 2 after which cell viability was measured using the MTT-reduction method. MTT  was added to the cell cultures in a final concentration of 0.83 mg/ml and incubated for four hours, after which SDS  was added to a final concentration of 5% for overnight incubation. Adsorption was measured at 595 nm and as reference 670 nm using a plate reader .TNF bioactivity was determined using a WEHI bioassay. Per well 40.000 WEHI-164 cells were plated in IMDM (BioWhittaker) containing 5% FCS (Bodinco), 100 U/ml penicillin, 100 μg/ml streptomycin (both from Gibco), 1 μg/ml Actinomycin D and 50 μM β-Mercapto-ethanol (both from Sigma). TNF  was incubated in presence or absence of adalimumab Fab’ (13.3 or 26.6 ng/ml) or adalimumab (20 ng/ml) and either used immediately or incubated for three days at 4 °C or 37 °C. Subsequently, TNF, TNF-adalimumab Fab’ or TNF-adalimumab samples were 1:1 added to the WEHI-164 cells, resulting in 0–10 000 pg/ml TNF, 6.6 ng/ml or 13 ng/ml adalimumab Fab’ and 10 ng/ml adalimumab as a final concentration. WEHI-164 cells were incubated for 24 h at 37 °C and 5% COHow to cite this article: van Schie, K. A. et al. Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange. Sci. Rep.6, 32747; doi: 10.1038/srep32747 (2016).


Here, we investigated whether a PAFR antagonist would modulate the immune response in vivo. Mice were subcutaneously injected with OVA or OVA with PAFR-antagonist WEB2170 on days 0 and 7. On day 14, OVA–specific IgG2a and IgG1 were measured in the serum. The presence of WEB2170 during immunization significantly increased IgG2a without affecting IgG1 levels. When WEB2170 was added to OVA in complete Freund’s adjuvant, enhanced IgG2a but not IgG1 production was also observed, and CD4+ FoxP3+ T cell frequency in the spleen was reduced compared to mice immunized without the antagonist. Similar results were observed in PAFR-deficient mice, along with increased Tbet mRNA expression in the spleen. Additionally, bone marrow-derived DCs loaded with OVA were transferred into naïve mice and their splenocytes were co-cultured with fresh OVA-loaded DCs. CD4+ T cell proliferation was higher in the group transferred with DCs treated with the PAFR-antagonist. We propose that the activation of PAFR by ligands present in the site of immunization is able to fine-tune the adaptive immune response.We have previously shown that the Platelet-Activating Factor Receptor (PAFR) engagement in murine macrophages and dendritic cells (DCs) promotes a tolerogenic phenotype reversed by PAFR-antagonists treatment  PAF is a potent mediator of inflammatory events such as increased vascular permeability, smooth muscle contraction, inflammatory cells migration, and wound healing, among other effectsPAF has also been shown to induce striking functional changes in macrophages. Analysis from our laboratory showed that in human and murine macrophages, the engagement of PAFR by oxidized low-density lipoprotein or apoptotic cells induced an anti-inflammatory profile, and this required the association of PAFR with the scavenger receptor CD36 in plasma membrane lipid rafts. Blocking PAFR with selective antagonists clearly reversed the macrophages towards a pro-inflammatory profile782 production without affecting the IL-12. Moreover, DCs treated with PAFR-antagonists in vitro induced higher CD4+ T cell proliferation in an antigen-specific lymphocyte proliferation assay.Dendritic cells are a heterogeneous group of antigen presenting cells with a determinant role in the activation and suppression of immune responses, by altering the balance of the effector or regulatory T cells (Treg cells)14wild type (WT) mice treated with PAFR antagonist or in PAFR-deficient (PAFR-KO) mice. We also carried out an investigation to find out if PAFR antagonist treatment would increase the T cell priming activity of DCs in vivo. To this purpose, the immune response to OVA was transferred into naïve mice by injection of mature OVA-loaded BM-DCs. We found that the blockage of PAFR during immunization affected antibody production and that the transference of DCs previously treated with PAFR antagonist during maturation in vitro promoted an enhanced T cell response. We propose that PAF or PAF-like moieties, present at the site of immunization, have a damping effect on the immune response and suggest that PAFR-antagonists could be used as adjuvants in immunization protocols.In this study, we suggested that the activation of PAFR in DCs, by ligands generated during LPS-induced maturation, shifts them towards a “regulatory” phenotype6 in OVA-WEB and 5.7 ± 0.7 × 106 cells in the OVA group (P ≤ 0.05). OVA-specific IgG1 and IgG2a antibodies were measured in the serum as surrogate markers of Th2 and Th1 responses, respectively. While IgG1-OVA specific antibody levels were similar for both groups studied, it was found that IgG2a-OVA concentration was significantly higher in OVA-WEB compared to OVA group. Total IgE was also assessed but no differences were observed between OVA or OVA-WEB groups (BALB/c mice were injected subcutaneously with OVA (OVA group) combined or not with the PAFR antagonist WEB 2170 (OVA-WEB group). On day 7, they were challenged with OVA or OVA plus WEB 2170 and at day 14, blood, draining lymph nodes (DLNs), and splenocytes were collected. One interesting observation was that the DLNs of OVA-WEB mice were consistently larger than those of the OVA group, and their number of total cells followed this pattern, being 8.8 ± 0.5 × 10B groups .recall response of splenocytes from both groups re-stimulated ex vivo with OVA (100 μg/mL). After 72 h, the supernatants of splenocytes from the OVA-WEB group presented significantly higher levels of total IgG2a and IgE than the OVA group. The IgG1 concentration was not significantly different between the groups  and challenged on day 7 with OVA in incomplete Freund’s adjuvant. wild type (WT) and PAFR-KO mice. On the other hand, the Th1-related IgG2a was increased in the PAFR-KO mice, supporting our previous findings using PAFR antagonist.In order to confirm this effect of PAFR activation in the immune response, we immunized PAF receptor-deficient mice (PAFR-KO) with OVA in CFA, using the same protocol described above and assessed IgG1 and IgG2a OVA-specific antibodies, and total IgE at day 14. To further understand this shifted response in the PAFR-KO mice, we performed a qPCR for Tbet, the master-regulation transcription factor for Th1 immune response, in total splenocytes of WT and PAFR-KO mice. As shown in naïve PAFR-KO mice had significantly less CD4+ T cells expressing FoxP3 than the naïve WT  and to the PAFR-KO CFA/OVA group. Thus, the absence of PAFR activation during the immunization process impairs the differentiation of the Treg cell population, indicating yet a new mechanism of potentiating the immune response.In the search of the T regulatory cell population (Treg cells) in the spleen of these mice, it was observed that naïve WT . When im+ T cell proliferation in vitroIn a previous study, we found that the engagement of the PAFR in DCs by endogenous PAF induced a tolerogenic phenotype, as the blockage of the receptor by antagonists during DCs maturation increased their capacity to induce CD4in vitro would boost their capacity to transfer immunity in vivo, bone marrow-derived DCs were loaded with OVA and treated with PAFR antagonist during maturation in vitro. After extensive washing to remove the antagonist, these cells were then transferred into naïve mice through two intraperitoneal injections, seven days apart. As control, a group of mice was injected with OVA-loaded DCs without the antagonist treatment. On day 14, OVA specific IgG1 and IgG2a antibodies were found in the serum of recipient mice, indicating that they were successfully immunized by DCs transference. When the transferred DCs were previously treated with the PAFR antagonist (DC(WEB)-OVA), high levels of OVA-specific IgG2a were also found, although no changes in the antibody production were observed in comparison with the DC-OVA group , whereas IgG2a was clearly enhanced when mice were immunized with or without CFA. Evidence of PAF affecting antibody production is rare. There is one work showing that PAF added to B lymphoblastoid cells in culture enhanced IgG productionex vivo splenocytes re-stimulation from mice that were immunized in the presence of WEB 2170.Our experiments with PAFR-KO mice immunized with CFA/OVA strengthened the findings obtained in the experiments where mice were treated with the PAFR antagonist. Upon immunization they produced higher levels of antigen-specific IgG2a than the WT while IgG1-OVA levels were similar between the two groups. Altogether, these data suggested that the absence of PAFR signaling during immunization potentiates antibody production favoring the IgG2 isotype production. This last assumption was supported by the augmented Tbet expression in PAFR-KO splenocytes: the Tbet transcription factor is related to Th1 cell differentiation, which controls the production of IFN-γ  in T cells, but its expression is also required for IgG2a IFN-γ-mediated class switch recombination in B cells21+ FoxP3+ T cells) frequency in the spleen of naïve PAFR-KO mice was surprisingly lower in comparison to the WT mice. Tregs control the intensity of the immune response, and can be induced in vivo by adjuvants in order to restrain their inflammatory effectWe also found that the Treg population  or PAFR-deficient (PAFR-KO) mice were obtained from the Department of Immunology’s Animal Facility at the University of São Paulo, and were housed under specific pathogen-free conditions. Experiments were performed following the guidelines for animal use and care approved by the Institutional Animal Care and Use Committee of the Institute of Biomedical Sciences at the University of São Paulo. PAFR-deficient mice were originally provided by Dr. Satoshi Ishii and Dr. Takao Shimizuth day, blood, spleen, and the inguinal draining lymph nodes were collected. After blood collection, samples were left at 4 °C for 30 min, followed by centrifugation . Serum samples were transferred to new tubes and stored at −80 °C.Mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg)/xylazine (10 mg/kg), and were immunized subcutaneously in the flanks of the lower back, with 5 μg of chicken ovalbumin , according to the following protocols: a) OVA in saline (200 μL); b) OVA in 100 μL of complete Freund’s adjuvant  and 100 μL saline or c) OVA in 30 μL of aluminium hydroxide gel , and 170 μL saline. At day 7, mice were boosted by the subcutaneous injection of OVA in saline, OVA in incomplete’s Freund adjuvant (Sigma-Aldrich), or OVA in alum, respectively. In each protocol, a group of mice received OVA together with the PAFR antagonist WEB 2170 6 cells/100 μL. Three to five million cells were transferred to naïve mice via intraperitoneal injection. This process was repeated after 7 days. At day 14 of the first cell transference, the mice were euthanized by CO2 exposure, and their serum and spleen were collected.Bone marrow cells were collected from the femurs of BALB/c mice and cultured in RPMI 1640  medium, supplemented with 10% heat-inactivated fetal bovine serum , and antibiotic-antimycotic . BM-DCs differentiation was induced by 20 ng/mL of granulocyte–macrophage colony-stimulating factor . After 6 days, the cells were harvested, pulsed with 100 μg/mL of chicken ovalbumin , and treated with 50 μM of the PAFR antagonist WEB 2086ex vivo stimulation, splenocytes were plated at 106 cells/mL, and incubated with 100 μg/mL of OVA. Supernatants were collected after 72 h and stored at −80 °C until use. For proliferation assay, the splenocytes were labeled with CFSE , and co-cultured with fresh OVA-loaded BM-DCs generated as described above in a 4:1 ratio (splenocytes:BM-DCs) for 72 h. After that, cells were stained at 4 °C for 30 min with anti-mouse CD4-APC (L3T4)  antibody. For Treg detection, splenocytes were stained with the Mouse Regulatory T Cell Staining Kit #1  according to the manufacturer’s instructions. Data was acquired using a FACSCanto II flow cytometer (BD Biosciences), and analyzed by the FlowJo software . For both proliferation and Treg detection assays, doublets were excluded, and analyses were conducted on CD4+ gated cells.Spleen and draining lymph nodes were collected and passed through a 70 μm nylon cell strainer to remove connective tissues. The cells obtained were incubated with ACK Lysis Buffer (GIBCO), to lyse the red blood cells. For ex vivo stimulated cells were collected and the total antibody profile was determined by a Cytometric Bead Array , using a FACSCanto II Flow Cytometer. Data were analyzed by FlowJo software. Positive events for each isotype tested were gated and analyzed as Median Fluorescence Intensity (MFI) in a semi-quantitative way (higher MFI meaning higher amount of antibody detected).Supernatants of IL-4 and IFN-γ were assayed by OptEIA ELISA (BD Biosciences). Absorbance was evaluated at 450 nM in the microplate reader SpectraMax 190 and was analyzed by SoftMax Pro 4.0 software . Values were expressed as pg/mL, which were deduced from standard curves of recombinant cytokines run in parallel.2SO4. Absorbance was acquired at 450 nM in the microplate reader SpectraMax 190 and was analyzed by SoftMax Pro 4.0 software. Absorbance sample values were considered after subtracting values of wells incubated with fresh serum from naïve mice in the same dilutions or incubated with assay diluent. Absorbance values for diluents were close to those obtained with control serum at the dilution used for immune serum. Data are shown in optical density (OD) units.To determine OVA-specific IgG1 and IgG2a antibodies, plates were coated overnight at 4 °C with OVA (20 μg/mL) in phosphate-buffered saline (PBS). Plates were washed three times with wash buffer (PBS containing 0.05% Tween-20), and blocked with assay diluent  for 1 h. Mice serum samples were diluted in assay diluent and added to plates after three more washes. Following a 2 h incubation at room temperature (RT), plates were washed five times, and anti-IgG1 (BD Pharmingen) or anti-IgG2a (Invitrogen) conjugated with peroxidase were used as secondary antibodies. Plates were incubated for 1 h RT and washed seven times. TMB Substrate Reagent Set (BD Biosciences) was added and plates were left for 30 min RT in the dark. Colorimetric reaction was stopped by the addition of 2 NH−ΔΔCT method as previously describedSpleens were collected and passed through a 70 μm nylon cell strainer to remove connective tissue, and red blood cells were lysed with ACK Lysis Buffer (GIBCO). Total RNA was isolated using the Trizol Reagent , according to the manufacturer’s instructions. cDNA was generated from the total RNA, using the RevertAid First Strand cDNA Synthesis Kit . Real-time PCR was performed with the StepOnePlus Real-Time PCR System , using SYBR Green , and specific primers for Tbet , Gata3 , Rorc  and Gapdh . Relative gene expression was calculated using the 2t test, using GraphPad Prism 5.0 software . P < 0.05 values were considered as significant.Results were presented as mean values ± SEM. Statistical differences between mean values were determined by the one-way ANOVA, followed by the Newman-Keuls test or Student’s How to cite this article: Koga, M. M. et al. Boosting Adaptive Immunity: A New Role for PAFR Antagonists. Sci. Rep.6, 39146; doi: 10.1038/srep39146 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Osteoarthritis (OA), an inflammatory form of arthritis, is characterized by synovial inflammation and cartilage destruction largely influenced by two key proinflammatory cytokines—interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α). Notably, levels of visfatin (a proinflammatory adipokine) are elevated in patients with OA, although the relationship of visfatin to IL-6 and TNF-α expression in OA pathogenesis has been unclear. In this study, visfatin enhanced the expression of IL-6 and TNF-α in human OA synovial fibroblasts (OASFs) in a concentration-dependent manner and stimulation of OASFs with visfatin promoted phosphorylation of extracellular-signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), while ERK, p38, and JNK inhibitors or siRNAs all abolished visfatin-induced increases in IL-6 and TNF-α production. Moreover, transfection with miR-199a-5p mimics reversed visfatin-induced increases in IL-6 and TNF-α production. Furthermore, we also found that visfatin-promoted IL-6 and TNF-α production is mediated via the inhibition of miR-199a-5p expression through the ERK, p38, and JNK signaling pathways. Visfatin may therefore be an appropriate target for drug intervention in OA treatment. Osteoarthritis (OA), a common chronic inflammatory disorder of the synovial joint, is characterized by articular cartilage degradation, cartilage remodeling/degeneration, subchondral sclerosis and osteophyte formation ,2,3. OA IL-6 involves several biological functions and plays a key role in hematopoiesis formation , the innVisfatin, a growth factor for B lymphocyte precursors, is a pro-inflammatory adipokine found in the liver, skeletal muscles and bone marrow and produced by visceral white adipose tissue, which mimics the effects of insulin . Serum vIL-6 and TNF-α mRNA expression in a concentration-dependent manner , ERK, p38 MAPK and JNK are involved in the regulation of inflammatory cytokine expression ,21. We tα levels A–C. In aα levels A–C, wherα levels D. Thus, IL-6 and TNF-α mRNAs harbor potential binding sites for miR-199a-5p  and a vector containing mismatches in the predicted miR-199a-5p binding sites (IL-6-3′-UTR-mut and TNF-α-3′-UTR-mut)  F. We fouUTR-mut) G. In addUTR-mut) H. CollecIt is well established that a complex cytokine network contributes to chronic inflammation of the synovial membrane and the development of disease and cartilage degradation in OA . HoweverThe MAPK signaling pathway helps to regulate gene expression levels . Here, wIL-6 and TNF-α genes, thereby negatively regulating visfatin-mediated IL-6 and TNF-α expression. These findings provide insight into potential miRNA-based strategies for visfatin-mediated IL-6 and TNF-α production.miRNAs have been investigated for their role in gene regulation . By bindIn conclusion, our investigations into the signaling pathway involved in visfatin-induced increases in IL-6 and TNF-α expression in human synovial fibroblasts reveal that visfatin inhibits miR-199a-5p expression through the ERK, p38 and JNK signaling pathways . These fWe obtained control miRNA, miR-199a-5p mimic and Lipofectamine 2000 from Life Technologies , rabbit polyclonal antibodies for P-ERK, ERK, P-p38, p38, P-JNK, JNK, TNF-α, IL-6 and β-actin; ERK, p38, JNK and control siRNA were purchased from Santa Cruz Biotechnology , recombinant human visfatin was purchased from PeproTech . All other chemicals were purchased from Sigma-Aldrich .Human synovial fibroblasts were obtained from synovial tissue collected from generally healthy OA patients aged 50–75 years undergoing knee replacement surgery. Written approval  was obtained from the Institutional Review Board of China Medical University Hospital, Taichung, Taiwan, and also from the patients, prior to sample collection. OASFs were isolated, cultured, and characterized as previously described ,34. In vWe pretreated human OASFs with various inhibitors for 30 min or transfected the OASFs for 24 h with miRNA mimic or siRNAs prior to visfatin administration. IL-6 and TNF-α in the medium was assayed using IL-6 and TNF-α enzyme immunoassay kits , as according to the manufacturer’s procedure.® one-step PCR Master Mix  to perform real-time quantitative PCR (RT qPCR) analysis; we added 100 ng of total cDNA per 25 µL reaction with sequence-specific primers and Taqman® probes. We purchased sequences for target gene primers and probes from Applied Biosystems ; qPCR assays were carried out in triplicate using the StepOnePlus sequence detection system  [Total RNA was extracted from OASFs using a TRIzol kit . Reverse transcription was performed using oligo(dT) primer ,36. We uCA, USA) .U6 gene as an endogenous control. The relative gene expression level was calculated using the comparative CT method.For the miRNA assay, we used the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) to synthesize cDNA; reactions were incubated at 16 °C for 30 min, then at 42 °C for 30 min, followed by inactivation at 85 °C for 5 min. All reactions were run using the StepOnePlus sequence detection system. Relative quantification analysis of gene expression was performed with the The cell lysates were resolved by SDS-PAGE ,38 and tIL-6 and TNF-α contains a miR-199a-5p binding site. DNA fragments containing wild-type (wt)-IL-6-3′-UTR, mutant-type (mut)-IL-6-3′-UTR, wt-TNF-α-3′-UTR and mut-TNF-α-3′-UTR were purchased from Invitrogen . The fragments were subcloned into the luciferase reporter vector pmirGLO-control , upstream of the vector’s promoter. These plasmids were transfected into cells using Lipofectamine 2000. Cell extracts were prepared and used to measure luciferase and β-galactosidase activity.The three prime untranslated region (3′-UTR) of human t-test were used to test for any significant difference in the means. The difference was denoted significant when the p-value was less than 0.05.The data are expressed as the mean ± SEM. Statistical analysis was performed using GraphPad Prism 4 software . One-way analysis of variance (ANOVA) and the unpaired two-tailed Student’s