Intestinal ischemia-reperfusion (I/R) may cause acute systemic and lung inflammation. However, the detailed mechanism of this inflammatory cascade has not been fully elucidated. Inactive rhomboid protein 2 (iRhom2) is essential for the maturation of TNF-α converting enzyme (TACE), which is required for TNF-α secretion. We evaluated the role of iRhom2 in a mouse model of intestinal I/R using iRhom2 knockout (KO) and wild-type (WT) mice. Lung injury following intestinal I/R was significantly attenuated in iRhom2 KO mice compared with WT mice. After intestinal I/R, lungs from iRhom2 KO mice showed significantly lower myeloperoxidase (MPO) activity and markedly reduced cell apoptosis associated with a decreased level of active caspase 3 and decreased TUNEL staining compared with lungs from WT mice. TNF-α levels were elevated in the serum and lungs of WT mice with intestinal I/R and significantly reduced in iRhom2 KO mice with intestinal I/R. iRhom2 may play a critical role in the pathogenesis of acute lung injury (ALI) after intestinal I/R and thus may be a novel therapeutic target for ALI after intestinal I/R injury. Therefore, a new treatment strategy for intestinal I/R is needed.Intestinal ischemia-reperfusion (I/R) injury develops when the blood flow to the intestines decreases, followed by the reestablishment of the blood supply to the ischemic tissue. Intestinal I/R injury results in intestinal mucosal barrier dysfunction, which may cause severe local and systemic inflammation and subsequent distant organ injury. This injury is potentially serious and can be life threatening; however, the mortality rates in patients with acute intestinal I/R are constant and range from 60–80%5. Although several pathophysiologic mechanisms of ALI in intestinal I/R have been suggested, the detailed molecular mechanism is not fully understood. Inflammatory mediators during intestinal I/R include reactive oxygen species, platelet-activating factor, chemokines and cytokines10. Among these mediators, TNF-α is the most widely studied. High levels of TNF-α are associated with an inflammatory response that leads to many diseases, such as ALI and other chronic lung diseases12. Animal treatments with anti-TNF-α antibodies and experiments in TNF-α receptor 1-deficient animals have revealed a central role for TNF-α in mediating tissue injury and systemic inflammation in intestinal I/R9.Acute lung injury (ALI), a medical condition characterized by widespread inflammation in the lung with an acute onset, is the most serious complication of intestinal I/R injury15. TACE is an essential enzyme responsible for TNF-α release and is required for the cleavage of other ligands, including epidermal growth factor receptor (EGFR) ligands16. Inactive rhomboid protein 2 (iRhom2) was recently identified as an essential regulator of TACE maturation in immune cells17. iRhom2 is predominantly expressed in immune cells, particularly in macrophages, and its expression is significantly up-regulated in response to lipopolysaccharide (LPS) stimulation19. In iRhom2-deficient macrophages, LPS-induced release of TNF-α is significantly inhibited by the failure of TACE maturation. Thus, iRhom2 may be an attractive novel therapeutic target for TNF-α-dependent inflammatory diseases.Soluble TNF-α is cleaved from membrane-bound TNF-α by TNF-α converting enzyme The aim of this study was to evaluate the role of iRhom2 in the development of ALI resulting from intestinal I/R.20.The murine macrophage cell-line, RAW 264.7 , was cultured, as described previously18. iRhom2 gene KO was confirmed in iRhom2 KO mice by PCR genotyping of colonic tissue DNA  were purchased from Orient , and iRhom2 knockout (KO) mice, on a C57BL/6 background, were obtained from Dr Tak W. Mak 20. Twenty-four hours after transfection, the cells were stimulated with LPS (1 µg/ml LPS for 4 h), and the concentration of TNF-α in the culture supernatants was measured using a commercially available ELISA kit .To clarify whether iRhom2 regulates TNF-α secretion in macrophages, RAW264.7 cells were transfected with iRhom2 small interfering RNA (siRNA) before LPS stimulation, and TNF-α secretion by LPS-stimulated RAW264.7 cells was measured by enzyme-linked immunosorbent assay (ELISA). Before LPS stimulation, RAW264.7 cells were transfected with iRhom2 or control siRNA using Lipofectamine RNAiMax (Invitrogen)21. iRhom2 KO and WT mice were randomized into an intestinal I/R group (each n = 10) or a sham laparotomy group (each n = 5). The mice in the intestinal I/R group underwent 1 h of ischemia followed by 3 h of reperfusion. Intestinal I/R was induced by the complete clamping of the superior mesenteric artery (SMA) with a microvascular clip. After 1 h of occlusion, intestinal perfusion was reestablished by removing the clip. Sham-operated mice underwent identical surgical interventions and time courses without SMA clamping. We performed 1 h of intestinal ischemia followed by 3 h reperfusion because a previous pilot experiment showed that this time course provoked the most severe pulmonary inflammation in the WT mice without significant mortality.Intestinal I/R was performed as previously reported22.Histological changes in the intestine and lung were evaluated. Tissue samples, including small intestine and lung tissues, were fixed in a buffered 10% formalin solution, paraffinized and stained with hematoxylin and eosin (H&E). All histopathological assessments were performed in a blinded fashion by a pathologist who was unaware of the study details. The small intestine and lung injuries were graded in accordance with previously described scoring systems22.Myeloperoxidase (MPO) activity, which reflects neutrophil infiltration, in the lung tissues was measured as described previously23. The immunoreactivity of caspase 3 was assessed by determining the percentage of positive cells using a visual scoring system and was classified as 0 (no staining), 1+ (<10%), 2+ (10–30%), 3+ (31–60%) and 4+ (61–100%), as previously reported24.Immunohistochemistry (IHC) staining for active caspase 3, a marker of apoptosis, was measured in the lung tissues as previously described25. The TUNEL assay detects DNA fragmentation that results from apoptosis. The apoptotic index was calculated based on the ratio of TUNEL-reactive cells to the total number of cells in each of three randomly selected, nonadjacent fields (magnification: x 200)26.The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed in the lung tissues as previously describedThe levels of TNF-α and IL-6 in the serum and lung homogenates were measured using a commercially available ELISA kit according to the manufacturer’s instructions.P < 0.05 was considered statistically significant.All data are expressed as the mean ± standard deviation (SD) and were analyzed using GraphPad Prism software version 5.0 . Data were analyzed using a two-way Student’s t-test or one-way analysis of variance (ANOVA). This study protocol was approved by the Institutional Animal Care and Use Committee of Seoul National University (IACUC No. SNU-160612). The experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.The effect of iRhom2 siRNA treatment on LPS-induced TNF-α secretion by macrophages was examined by ELISA. The results indicated that stimulating RAW264.7 macrophages with LPS led to TNF-α secretion. This effect was significantly decreased by siRNA-mediated iRhom2 knockdown Fig. , establiAfter 1 h of ischemia followed by 3 h of reperfusion, the WT mice showed severe lung damage, as indicated by substantial alveolar edema, inflammatory cellular sequestration and hemorrhage, whereas the sham-operated group exhibited normal lung histology Fig. . HoweverCompared with the sham control group, neutrophil infiltration, as measured by MPO activity, increased in the lungs of WT mice subjected to intestinal I/R Fig. . The MPOIHC staining for active caspase 3 and a TUNEL assay were subsequently performed to examine apoptosis in the lungs Fig. . In the Reperfusion after SMA occlusion in both the iRhom2 KO and WT mice caused intestinal injury, characterized by epithelial erosion, inflammatory cell infiltrates and destruction of the villi Fig. . The intWe measured the levels of two inflammatory cytokines, TNF-α and IL-6, in the serum and lung homogenates (Fig. In the present study, we confirmed our hypothesis that iRhom2 played pivotal roles in the remote lung inflammation induced by intestinal I/R injury. Lung injury following intestinal I/R was significantly attenuated in the iRhom2 KO mice compared with the WT mice. iRhom2 deficiency reduced neutrophil activity and cell apoptosis in the lung. Following intestinal I/R, the TNF-α levels increased in the serum and lungs of WT mice. However, TNF-α production during intestinal I/R was markedly decreased in the iRhom2 KO mice. Unlike lung injury, the intestinal injury severity was not different between the iRhom2 WT and KO mice. To the best of our knowledge, the present study is the first to evaluate the role of iRhom2 in intestinal I/R.29. We performed 1 h of intestinal ischemia followed by 3 h of intestinal reperfusion, and this condition was regarded as a model of injury with inflammation characterized by neutrophil infiltration into the intestine and lung that peaked after 3 h without significant mortality. Similarly, in a previous study, 45 min of intestinal ischemia followed by 4 h of intestinal reperfusion was used to induce a mild injury in the lungs without mortality21.The intestinal I/R injury procedure performed in this study is a simple and reproducible model of local and systemic inflammation with significant effects on lung and gut homeostasis31. In addition to tissue damage, marked systemic inflammation is observed, as assessed by increased serum levels of inflammatory cytokines and chemokines31. Similarly, in the present study, intestinal I/R injury induced intestinal and lung injury and up-regulated the expression of inflammatory cytokines such as TNF-α and IL-6.In mice, intestinal I/R injury is followed by severe local intestinal and remote lung tissue pathology, characterized by a marked neutrophil influx, edema formation, hemorrhage, and tissue destruction32. Furthermore, mice with markedly reduced TACE activity show increased susceptibility to acute colitis, probably due to a lack of EGFR signaling. However, since iRhom2 is a myeloid-specific regulator of TACE maturation, iRhom2 blockade might be an effective way to treat inflammatory conditions without inhibiting EGFR signaling. Accordingly, in the present study and in previous studies, iRhom2 KO mice were viable and appeared normal, without morphological defects or growth retardation (Supplementary Figure 34.TNF-α is involved in the pathogenesis of various inflammatory diseases. Since TACE was first discovered to be essential for TNF-α release, TACE blockade has been considered as a treatment for TNF-α-mediated inflammatory diseases. However, blockade of TACE inhibits not only TNF-α secretion but also EGFR signaling. TACE KO mice are not viable and show a phenotype similar to that of mice lacking EGFR signaling18. A previous study showed that other inflammatory cytokines, such as IL-1β, IL-6 and IL-12, were induced normally in iRhom2-deficient macrophages18. Taken together, these results could suggest that iRhom2 and the subsequent release of TNF-α play an important role in mediating systemic inflammation after intestinal I/R. Several studies have established a role for TNF-α in mediating both local and systemic inflammation during I/R injury37. TNF-α induces the expression of adhesion molecules on endothelial cells and the recruitment of inflammatory cells into the lung, and an anti-TNF-α antibody reduces the severity of lung injury37. TACE inhibition reduces the release of soluble TNF-α and the severity of LPS-induced lung injury11. However, the role of TNF-α in intestinal I/R-mediated ALI has been controversial. A previous report showed that neither the neutralization of soluble TNF-α with antibody treatment nor the lack of TNF-α or its receptors diminished lung inflammation upon intestinal I/R21.In the present study, the iRhom2 KO mice showed attenuated lung injury and lower levels of TNF-α in the serum and lungs compared with the WT mice. Consistent with previous studies, in RAW264.7 macrophages, TNF-α secretion was increased by LPS stimulation and significantly decreased by siRNA-mediated iRhom2 knockdown40. Recombinant human TNF-α stimulates a neutrophil respiratory burst and lysozyme release and significantly elevates the neutrophil respiratory response41. In this study, the influx of neutrophils into the lungs in the iRhom2 KO mice was reduced compared with that in the WT mice. Cell apoptosis has been proposed as another possible mechanism in ALI43. Previous studies have reported that TNF-α induces caspase activation in endothelial cells, which results in the induction of apoptosis and endothelial cell dysfunction45. iRhom2 KO mice with ALI exhibited a significantly lower apoptosis intensity than the WT mice, and our findings suggested that iRhom2 deficiency directly and/or indirectly resulted in less cell apoptosis in the lungs. TNF-α has also been shown to induce necroptosis, a non-apoptotic form of cell death, in different cell types46. The precise cell death mechanism involved in intestinal I/R-induced ALI could be characterized in future studies.Neutrophils are known to play a critical role in the pathogenesis of ALI. TNF-α stimulates neutrophils in several disease models47. Recently, several studies have reported that transmembrane TNF-α plays a key role in local inflammation50. In transgenic mice expressing a mutant transmembrane TNF-α, transmembrane TNF-α is sufficient to induce localized tissue injury and chronic inflammatory arthritis52. Moreover, a recent study in a mouse model of T cell-mediated colitis reported that only neutralization of transmembrane TNF-α induces remission of experimental colitis53. In iRhom2-deficient macrophages, TNF-α biogenesis is normal, but TACE proteolytic activity is defective. Therefore, transmembrane TNF-α in the iRhom2 KO mice may cause local intestinal inflammation similar to that of the WT mice.Interestingly, there was no significant difference in the severity of the local intestinal injury between the iRhom2 WT and KO mice. This result may be explained by the function of transmembrane TNF-α in intestinal inflammation. Although the role of transmembrane TNF-α is not fully understood, data suggest that both soluble TNF-α and transmembrane TNF-α are important in the inflammatory response, particularly in local inflammation. Transmembrane TNF-α transmits signals as both a ligand and a receptor in TNF-α-producing cellsIn conclusion, the iRhom2 protein may be associated with the pathogenesis of intestinal I/R. iRhom2 deficiency has a protective role in the systemic inflammation induced by intestinal I/R. Although the detailed molecular mechanisms of iRhom2 and subsequent TNF-α signaling in intestinal I/R remain to be clarified, our data suggest that iRhom2 may be a novel therapeutic target for ALI after intestinal I/R injury.Supplementary Information


HH with VHHs or scFvs directed against abundant surface molecules on myeloid cells and serve to limit the bioavailability of TNF produced by these cells. Such reagents may become prototypes of a novel class of anti-cytokine biologics.Proinflammatory cytokines, such as TNF, IL-6, and IL-1, play pathogenic roles in multiple diseases and are attractive targets for biologic drugs. Because proinflammatory cytokines possess non-redundant protective and immunoregulatory functions, their systemic neutralization carries the potential for unwanted side effects. Therefore, next-generation anti-cytokine therapies would seek to selectively neutralize pathogenic cytokine signaling, leaving normal function intact. Fortunately, the biology of proinflammatory cytokines provides several such opportunities. Here, we discuss various applications of bispecific antibodies targeting cytokines with specific focus on selective TNF neutralization targeted directly to the surface of specific populations of monocytes and macrophages. These bispecific antibodies combine an anti-TNF V Many currently used therapeutic antibodies represent antagonists or inhibitors of signaling cascades that are known to be pathogenic in a particular disease state. Examples include anti-cytokine therapies and immune checkpoint inhibitors, both of which have resulted in major advances in the treatment of autoimmune diseases and cancer. One common problem with many of such inhibitors, when applied systemically, is incomplete discrimination of “pathogenic” signaling from “physiological” signaling, the latter being beneficial for the patient. Therefore, most current therapies have unwanted side effects resulting from collateral damage to beneficial or protective signaling cascades. This problem can be potentially addressed through additional specificity conferred by more sophisticated inhibitory antibodies that target their cognate antigens only in a particular organ or cell lineage.Therapeutic bispecific antibodies have showed efficacy in both experimental animal models and in clinical trials , findingin vivo. Two different inhibitors of TNFRI signaling, each with a second specificity to serum albumin for half-life extension in vivo, are effective in mouse models of Crohn’s disease and arthritis  and CD11b  can be employed.In our studies employing conditional gene targeting, we found that TNF produced by myeloid cells is pathogenic in several experimental mouse disease models –32. AssuCamelidae (HHs) specific to almost any protein. One particular aspect that drew our attention was the usefulness of VHHs in creating bispecific reagents, as two or even three VHHs can be easily combined in a single polypeptide chain by the methods of genetic engineering  binds to the surface of murine macrophages, competes with another anti-F4/80 reagent for this binding Figures  were incTo get a better insight into the fate of hTNF and of MYSTI after its binding to the surface of macrophage, we utilized confocal microscopy, as outlined in Figures HH domains, may be sufficiently long-lived on the surface of cytokine-producing cells, we evaluated these reagents in vivo. In LPS/D-Gal lethal toxicity model, pathogenic TNF is known to be produced by myeloid cells (HH module) failed to do so ; however, differences in molecular weight and avidity should be taken in account when comparing full-length systemic TNF-inhibitors with MYSTI. Additionally, MYSTI was active in anti-collagen antibody transfer arthritis model (data not shown). Another potential target for the “second specificity” is CD11b for which a VHH was recently reported , the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, Council of Europe (ETS 123), and “The Guidelines for Manipulations with Experimental Animals” . All animal procedures were approved by Scientific Council of the Engelhardt Institute of Molecular Biology.MN, K-SA, GE, AK, MD, and SN designed the research and analyzed the data; MN, K-SA, and MD performed the experiments; VM, GE, and ST developed and produced bispecific reagents; all authors contributed to writing the manuscript.GE, AK, and SN are coauthors of the patent application describing the initial version of MYSTI. Other authors declare no conflict of interest.


These data demonstrate that EBV glycoprotein(s)-based VLPs have excellent immunogenicity, and represent a potentially safe vaccine that will be invaluable not only in preventing EBV infection, but importantly, in preventing and treating the 200,000 cases of EBV-associated cancers that occur globally every year.Previous Epstein-Barr virus (EBV) prophylactic vaccines based on the major surface glycoprotein gp350/220 as an immunogen have failed to block viral infection in humans, suggesting a need to target other viral envelope glycoproteins. In this study, we reasoned that incorporating gH/gL or gB, critical glycoproteins for viral fusion and entry, on the surface of a virus-like particle (VLP) would be more immunogenic than gp350/220 for generating effective neutralizing antibodies to prevent viral infection of both epithelial and B cell lines. To boost the humoral response and trigger cell-mediated immunity, EBV nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2), intracellular latency proteins expressed in all EBV-infected cells, were also included as critical components of the polyvalent EBV VLP. gH/gL-EBNA1 and gB-LMP2 VLPs were efficiently produced in Chinese hamster ovary cells, an FDA-approved vehicle for mass-production of biologics. Immunization with gH/gL-EBNA1 and gB-LMP2 VLPs without adjuvant generated both high neutralizing antibody titers  Precn humans –8. Howevn humans , 10. TheAnother major limitation of vaccines currently in pre-clinical and clinical trials is that none of the vaccines has created sterile immunity , perhaps because most of the strategies only target one arm of the immune system, either humoral   or cell-Virus-like particles (VLPs) overcome many of these challenges. VLPs lack the viral genome and typically assemble from one or more viral structural proteins, forming repetitive arrays that resemble a natural virus . ImportaEBV membrane glycoproteins, which are critical for viral entry and infection of host epithelial and B cells , 22, proin vivo neutralization of EBV infection is suboptimal. In support of these observations, in four independent phase I/II clinical trials, vaccination with vector constructs expressing gp350/220 or with the purified recombinant non-splicing variant, gp350, soluble protein did not prevent infection, although acute infectious mononucleosis (AIM) was reduced in young adults [Although neutralizing antibodies (nAbs) directed to EBV envelope glycoproteins are present in humans, prevent neonatal infection, and are generated in response to EBV infection in humans, or to immunization with virus glycoproteins in humans and other animals , 36, 37,g adults , 38, 39.g adults . These og adults , 38. Thi+ and CD8+ T cells are frequently detected in EBV-infected individuals [Much evidence establishes EBV intracellular latency proteins EBNA1 and LMP2 as attractive targets for stimulation of a cell-mediated immune response in a therapeutic EBV vaccine candidate –8, 41–46ividuals , 47, 48,ividuals , 44, 45.ividuals . Adoptivividuals , 51, sugIn this study, we provide evidence that subunit VLPs can incoporate EBV surface glycoproteins, package intracellular antigens, and that the VLPs can be stably produced in the Chinese hamster ovary (CHO) cell line. The polyvalent EB VLPs can stimulate both humoral and T cell-mediated immune responses in wild-type BALB/c mice. To our knowledge, these combinations have not yet been tested in pre-clinical or clinical trials as part of a prophylactic and therapeutic EBV vaccine candidate.VLPs have typically been used to express only surface glycoproteins, for stimulation of the humoral immune response . In addiTo demonstrate that both surface glycoprotein(s) and intracellular proteins can be packaged in a VLP, we generated plasmids encoding genes to be incorporated as part of the EB VLP, surface glycoprotein (gp350/220) and enhanced green fluorescent protein (eGFP), a marker for intracellular expression. First, we synthesized chimeric sequences of EBV gp350/220 surface glycoprotein fused to the NDV fusion (F) protein transmembrane (TM)/cytoplasmic (CT) domains ; eGFP-NP; or eGFP-NP, gp350/220-F, and M  expressed eGFP-NP protein Figure . VLP-proTo generate a colony of stable VLP-producing cells, CHO cells transfected with pCAGGS-gp350/220-F, pCAGGS-eGFP-NP, pCAGGS-M and pCI-puro were selected with 10 μg/ml of puromycin 48 hours post-transfection. Five colonies of stable cells were randomly selected, expanded, and sorted by flow cytometry using eGFP and anti-gp350/220 mAb 72A1. Sorted stable cells were expanded and VLPs were purified as above. Confocal microscopy analysis of the stable cells for eGFP expression, and immunoblot analysis of VLPs purified from stable cells, revealed that stable cells produced VLPs equivalent to those produced in the initial transfection, up to passage 10, without any remarkable loss in quantity of gp350/220 in purified VLPs Figure . These rAlthough the major EBV envelope glycoprotein gp350/220 has been proposed as a potential vaccine antigen, attempts to develop a potent gp350/220-based vaccine in the past four decades have had limited success , 52. We cis-linked sequences (amino acids 1-325) to MHC class I is deleted. The fragments were individually cloned into pCAGGS and cDNA fidelity was verified by sequencing.To demonstrate the efficacy of packaging both viral surface glycoproteins (gp350/220 or gH/gL) and an intracellular latency protein (EBNA1) inside a VLP, we used a similar strategy as above to generate both gp350/220-EBNA1  and gH/gTo demonstrate that chimeric gH-F and gL-HN form gH/gL complexes at the surface of the transfected cells, similar to full length gH/gL or gH alone, we transfected CHO cells with the relevant pCAGGS plasmids. At 48 hours post-transfection, cells were scraped, stained with anti-gH (E1D1) Figure  or anti-To assemble and produce the gH/gL-EBNA1 VLPs, equal amounts of cDNAs (8 μg/T-175 flask) of pCAGGS-gH-F, pCAGGS-gL-HN, and pCAGGS-tEBNA1-NP chimeras were co-transfected into CHO cells, together with pCAGGS-M Figure . VLPs weFollowing the success of producing VLPs that express both surface glycoprotein and intracellular protein, we developed another VLP packaging EBV glycoprotein, gB, with full length LMP2. LMP2 is recognized by T cells and is immunogenic in the control of post-transplant lymphoproliferative diseases. We reasoned that it would be an excellent vaccine component when combined with gB, which was recently shown to also play an integral role in EBV entry . To consTo assemble and produce the gB-LMP2 VLPs, we co-transfected equal amounts of pCAGGS-gB-F, and pCAGGS-LMP2-NP chimeras into 40 T175 flasks seeded with CHO cells, together with pCAGGS-M Figure . SupernaTo test the efficacy of individual EB VLPs to generate antibodies and induce T-cell responses, nine groups (n=5/treatment) of 6–8-week-old female BALB/c mice were immunized intraperitoneally three times  with 10 μg of purified gH/gL-EBNA1 VLPs, gB-LMP2 VLPs, or gp350/220 VLPs in 0.5ml of TNE buffer . Purified UV-inactivated EBV (10 μg) was used to immunize mice as positive control; TNE buffer served as negative control. To test whether there are additive or synergistic effects of using two or more immunogens in the vaccine, we immunized groups of 5 mice with two or three individual VLPs . Mice were tail vein-bled to obtain serum at two and four weeks after primary immunization and again once after both secondary (Day 29) and tertiary (Day 54) boost immunizations Figure . Mice weTo demonstrate the ability of VLPs to induce EBV subunit-specific antibody responses, we used enzyme linked immunosorbent assay (ELISA) to assess antibody titers against gH/gL, gB, gp350/220, and UV-inactivated EBV (positive control) in collected sera. First, lysates from lytically induced AGS-EBV-eGFP cells, which express all viral glycoproteins, were used as binding targets. Sera collected at Days 14, 18, 33, 46, 68, and 97  were used to determine the presence of IgG-specific antibodies Figure . All VLPin vitro, we quantitated sera collected at Days 14, 18, 33, 46, 68, and 97.  of sera from TNE-immunized mice (negative control), fluorescence dropped from 19.1%  to 10% in HEK-293 cell line. Similar results were observed in Raji cells. In contrast, when AGS-EBV-eGFP was pre-incubated with serially diluted sera from mice immunized with gH/gL-EBNA1 VLPs, gB-LMP2 VLPs, gp350/220 VLPs, or UV-inactivated EBV, infection was reduced in a dose-dependent manner compared to TNE-immunized sera Figure . Purifiein vitro better than gp350/220 VLPs in both Raji and HEK-293 cells.When we performed the neutralization assay in HEK-293 epithelial cells, sera from mice immunized with UV-inactivated EBV at the 1:2.5 dilution effectively neutralized 94.3% of EBV infection, followed by sera from mice immunized with gH/gL-EBNA1 VLPs (77.9%), gB-LMP2 VLPs (76.8%), and gp350/220 VLPs (54.9%) Figure . OverallSurprisingly, sera from mice immunized with a combination of two VLPs , or with all three VLPs, did not significantly reduce EBV infection compared to the single glycoprotein immunogens, in either of the cell lines used. In fact, pre-incubation of EBV with sera from mice immunized with both gB-LMP2 and gH/gL EBNA1 somewhat increased infection of HEK-293 cells.5) from sacrificed mice in vitro with 1 μg/mL synthetic peptide antigens derived from EBNA1(HPVGEADYFEY), LMP2(CLGGLLTMV), or Promix EBV-peptide pool (PeproTech). After overnight culturing, we used ELISA to test supernatants for release of IFN-γ, an indicator of T-cell activation. Splenocytes from mice immunized with gH/gL-EBNA1 VLPs or gB-LMP2 VLPs generated significantly higher IFN-γ in response to EBNA1  known to mediate viral entry , and proteins important for establishment of viral latency (EBNA1 or LMP2), allowed us to generate unique VLPs that can serve as polyvalent prophylactic and therapeutic EBV vaccine candidates. To our knowledge, the incorporation of select EBV surface glycoproteins and packaging of viral T-cell antigens inside the VLPs is unique to our study; we consider it a critical advancement in the development of a safe and effective polyvalent EBV vaccine, compared to previously reported candidates , 20, 42.in vitro [in vivo during natural infection (perhaps due to masking by the immunodominant gp350/220), immunization of animals with recombinant gH or gH/gL can boost immunogenicity and generate antibodies capable of blocking EBV infection [Because of gp350/220's critical role as the attachment protein in viral entry, and observations that mAb directed against the protein (72A1) blocks EBV infection in vitro , to datein vitro , 52. Howin vitro . Althougnfection , 58, 59.nfection , 61, cytnfection , and Kapnfection . Furthernfection , 64.Importantly, a recent report that rabbits immunized with 25 μg of gH/gL or gB, and enhanced with aluminum hydroxide as an adjuvant, generated superior nAb responses  to gp350/220 supports our findings . AlthougSurprisingly, sera from mice immunized with a mixture of both gH/gL-EBNA1 and gB-LMP2 VLPs had a contrasting outcome: supporting rather than blocking infection of epithelial cells. Whether this is true in natural infection, or whether co-expression of the three glycoproteins together on the surface of a single VLP  will have a different outcome remains to be explored. Pooled sera from mice immunized with both gp350/220 and gB-LMP2 VLPs or with gB-LMP2, gp350/220, and gH/gL-EBNA1 VLPs did not provide a better neutralization outcome than the use of single immunogens in both cell lines tested. Only pooled sera from mice immunized with both gp350/220 and gH/gL-EBNA1 VLPs achieved an additive effect in the HEK-293 cell line, suggesting that a strategy combining these two immunogens might achieve sterile immunity, particularly if it is enhanced by the use of adjuvants, as for the case of three FDA-approved human papillomavirus vaccines.In summary, our study demonstrates that EB VLPs can be stably produced in CHO cells, which grow to high density in suspension . CHO celIn future studies, we aim to improve our current vaccines by generating a polyvalent vaccine candidate that combines gp350/220-gH/gL-EBNA1-LMP2 as a single VLP. It might also be important to consider including gp42 as part of the proposed pentameric VLPs, as nAbs to gp42 have been detected in human sera , and intThe B95-8 cell line (marmoset B cell line) harboring recombinant Epstein-Barr virus (EBV) expressing an enhanced green fluorescence protein (eGFP) reporter at the LMP2A locus (B95-8-eGFP)  was a ge°C for subsequent experiments.HEK-293 (human embryonic kidney), Raji (EBV-positive Burkitt lymphoma), CHO, ELL-0 (chicken embryo), and HB168 (72A1 murine hybridoma) cell lines were all purchased from the American Type Culture Collection. B95-8-eGFP, Raji, and HB168 cells were cultured in RPMI media. HEK-293, CHO, and ELL-0 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM). AGS-EBV-eGFP cells were cultured in Ham's F-12 media. Both B95-8-eGFP and AGS-EBV-eGFP media were supplemented with 50μg/ml of neomycin (G418). All media contained 1% L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), and 2% penicillin-streptomycin unless otherwise specified. Lytic induction and purification of virus from B95-8-eGFP and AGS-EBV-eGFP cell lines harboring EBV-eGFP was performed as described . PurifiePrimary antibodies: Mouse monoclonal 72A1 (anti-gp350/220) was purified from HB168, a producer cell line, as described . Rabbit Secondary antibodies: Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG , goat anti-rabbit, goat anti-rat, or rabbit anti-goat antibodies for immunoblot or ELISA were purchased from Bio-Rad. Alexa Fluor (AF) 488- or AF594-conjugated goat F(ab’)2anti-mouse IgG (H+L) for flow cytometric analyses or confocal microscopy were purchased from Life Sciences Tech.Construction of pCI-puro has been previously described . All othFull-length EBV gp350/220 (BLLF1) from the BamHI L fragment of EBV genome strain B95-8  was amplThe rationale for fusion of type 1 membrane protein to NDV F protein and type 2 membrane protein to NDV HN has been described . Chimeracis-linked sequences to MHC class I); LMP2-NP chimera, full-length EBV LMP2 (aa 1-497).Full-length NDV-NP (aa 1-489) was synthesized and fused to a 29-aa (PATLSLPPDHPNSSARSPPP DPGAAGAGA) linker sequence . To conscDNA sequences of genes encoding full-length: gp350/220 (aa 1-907), gH (aa 1-707), gL (aa 1-137), gB (aa 1-790), gB-His (aa 1-742), eGFP (aa 1-241), EBNA1 (aa 1-641), and LMP2  following manufacturer's recommendations. To assess formation of the gH/gL complex, pCAGGS-gH-F and pCAGGS-gL-HN or pCAGGS-gH and pCAGGS-gL plasmids were co-transfected in combination into CHO cells.2 flasks; supernatant from transfected cells was collected and VLPs were purified as previously described [For VLP preparation, equal amounts (8 μg/plasmid) of the relevant plasmids were co-transfected into 80% confluent CHO cells seeded in T-175cmescribed , 20. VLPCells transfected with plasmids expressing eGFP  or untransfected CHO cells (negative control) were imaged using filters set for FITC, with an excitation wavelength of 488 nm resulting in emission at 507 nm, as described .5 CHO cells seeded in a six-well tissue culture plate. At 48 hours post-transfection, 10 μg/ml of puromycin was used to select transfected cells. Individual colonies were cloned and expanded, followed by flow cytometric sorting using anti-gp350/220 antibody (mAb 72A1) and eGFP. Cells were seeded and passaged 10 times and VLP production was assessed in each passage by immunoblot as described below.To generate a stable CHO cell line producing gp350/220-eGFP VLPs, 1.0 μg/well of pCAGGS-gp350/220-F, pCAGGS-eGFP-NP, pCAGGS-M, and pCIpuro plasmids were co-transfected into 1×10To assess surface expression of relevant protein(s) and protein complexes by flow cytometry, transfected cells were harvested at 48 hours post-transfection and stained with relevant primary and secondary antibodies. Flow cytometric analysis was performed on an LSRII benchtop FC  and data was analyzed using CellQuest Pro Version 4.0.1 (BD Biosciences) and/or FlowJo Cytometry Analysis software  as described . All theTo assess expression of relevant protein(s) and protein complexes using silver stain and immunoblot, purified VLPs, NDV, and B95-8-eGFP, untransfected CHO cells (negative control), or cells transfected with various pCAGGS plasmids were lysed in RIPA buffer (Boston Bioproducts) containing complete protease inhibitor cocktail (Roche Applied Science). Lysates were incubated on ice for 30 min, and then centrifuged for 5 min at 18,407 rcf in a microcentrifuge. The protein content of the lysates was determined by Bradford assay using Coomassie Brilliant Blue (Sigma). Lysates were boiled for 5 min in Laemmli SDS sample buffer (Boston Bioproducts) under non-reducing or reducing conditions. A known quantity of protein lysate was separated on a 4–12% gradient polyacrylamide gel. Pierce silver stain kit (Thermo-Fisher) was used to detect proteins following manufacturer's recommendations. All the experiments were independently repeated at least three times unless otherwise specified. Immunoblot analyses were performed by transferring proteins from the gel to a PVDF membrane using iblot (Thermo-Fisher). Membranes were pre-incubated with 5% non-fat dry milk (LabScientific) for 30 min and detected with relevant primary and secondary antibodies as previously described , 85.Nine groups (n=5/treatment) of 8–10-week-old female BALB/c mice were immunized intraperitoneally three times  with 10 μg of purified gp350/220-F, gH/gL-EBNA1, or gB-LMP2 VLPs, or with a combination of 10 μg of each of two or three of the VLPs, in 0.5 ml of TNE buffer . Purified UV-inactivated EBV (B95-8-eGFP) and gp350/220-F VLPs served as positive controls; TNE buffer alone served as negative control. To assess the short, mid, and long-term immunogenic nAb responses, mice were tail vein-bled to obtain serum at two and four weeks after primary immunization and again once after both secondary (Day 29) and tertiary (Day 54) boost immunizations. On Day 97, mice were sacrificed and terminal bleed (~1 mL) was collected for use in determination of antibody titer and EBV neutralization assays. Splenocytes were collected from sacrificed mice for determination of EBNA1- and LMP2-specific T-cell responses as described .2 flasks. Triton X lysis buffer was used to extract lysates from induced AGS-EBV-eGFP cells or transfected ELL-0 cells (48 hours post-transfection). Dilutions of the cell extracts were adjusted so that the amount of glycoprotein targets was comparable from experiment to experiment, as determined by Western blotting and Bradford assay. Untransfected ELL-0 or uninduced AGS-EBV-eGFP cell extracts served as negative controls. ELL-0 cells provided less non-specific background in the ELISA assay.To assess the EBV IgG-specific antibody titer for each immunized mouse, enzyme linked immunosorbent assay (ELISA) was performed as previously described  using va405) with ELISA reader . The highest dilution yielding an OD405 two times higher than that of TNE-treated mice without dilution was designated as the antibody endpoint titer. Anti-gp350/220 (mAb 72A1) was used as a standard for the ELISA assays. Three independent replicates of the experiments were performed at least three times.To perform ELISA, 96-well microtiter plates (Nunc-Immuno Plate with Maxisorp surface treatment) were coated with 50 μl/well of lysates expressing relevant viral glycoproteins (1 μg/ml) in a phosphate-buffered saline (pH 7.2) overnight at 4°C. After blocking the plates with 2% bovine serum albumin (BSA), serial dilutions (in PBS) of sera from Days 14, 18, 33, 46, 68, and 97  were bound for 2 hours at room temperature. Antibody binding was detected using HRP-conjugated goat anti-mouse IgG secondary antibody at room temperature for 1 hour. The plates were washed five times with PBS and the chromogenic substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)  was added. The reaction was stopped using ABTS Peroxidase Stop Solution containing 5% sodium dodecyl sulfate (SDS) in water. To determine the antibody titer, optical density was read at 405nm , and serum from TNE only-treated mice served as positive and negative controls, respectively. Plates were incubated at 37°C and the number of GFP+ cells was determined by flow cytometry 48 hours post-infection. All dilutions were performed in triplicate and the assays repeated at least two times for Raji cells. Due to sera limitation, neutralization in HEK-293 cells was performed only twice, that is why statistical analysis was performed in Raji cells.Pooled sera from Day 97  of mice immunized with gp350/220-F, gH/gL-EBNA1, or gB-LMP2 VLPs, or with a combination of 10 μg of each of two or three of the VLPs, UV-inactivated EBV, or TNE buffer alone were complement heat-inactivated at 56°C and diluted in RPMI without FBS. Sera were diluted by 1:2.5, 1:5, and 1:10 and incubated with 5μl of purified AGS-EBV-eGFP  for 1 hour at room temperature. To represent EBV infection of B and epithelial cells, AGS-EBV-eGFP-incubated with sera from immunized mice were used to infect 1×105) from sacrificed immunized mice were stimulated in vitro with 1 μg/mL synthetic peptides derived from EBNA1(HPVGEADYFEY), LMP2(CLGGLLTMV), or Promix EBV-peptide pool (PeproTech). After overnight culturing at 37°C, supernatants were tested for IFN-γ release, an indicator of T-cell activation, by ELISA, as described above. SIINFEKL ovalbumin was used as a negative control and concavalin A and IL1β were used as model antigens [To quantitate T-cell activation in immunized mice, splenocytes  of mice were analyzed using unpaired two-tailed t-tests for independent groups. Statistical significance of the tests was based on a p-value equal to or lower than 0.05.


The TNF-α biological inhibitors have significantly improved the clinical outcomes of many autoimmune diseases, in particular rheumatoid arthritis. However, the practical uses are limited due to high costs and the risk of anti-drug antibody responses. Attempts to develop anti-TNF-α vaccines have generated encouraging data in animal models, however, data from clinical trials have not met expectations. In present study, we designed a TNF-α epitope-scaffold immunogen DTNF7 using the transmembrane domain of diphtheria toxin, named DTT as a scaffold. Molecular dynamics simulation shows that the grafted TNF-α epitope is entirely surface-exposed and presented in a native-like conformation while the rigid helical structure of DTT is minimally perturbed, thereby rendering the immunogen highly stable. Immunization of mice with alum formulated DTNF7 induced humoral responses against native TNF-α, and the antibody titer was sustained for more than 6 months, which supports a role of the universal CD4 T cell epitopes of DTT in breaking self-immune tolerance. In a mouse model of rheumatoid arthritis, DTNF7-alum vaccination markedly delayed the onset of collagen-induced arthritis, and reduced incidence as well as clinical score. DTT is presumed safe as an epitope carrier because a catalytic inactive mutant of diphtheria toxin, CRM197 has good clinical safety records as an active vaccine component. Taken all together, we show that DTT-based epitope vaccine is a promising strategy for prevention and treatment of autoimmune diseases. TNF-α is a pleiotropic pro-inflammatory cytokine playing pivotal roles in both physiological and pathological processes . The priBlockade of TNF-α activities with monoclonal antibodies  as well as a receptor-immunoglobulin fusion protein (e.g. Etanercept) –5 signifActive immunization against TNF-α has been intensively investigated as an alternative approach to address these limits . So far,et al have shown that a cyclic TNF-α epitope peptide (aa 80–96) conjugated to KLH elicits a stronger neutralizing antibody response than the linear counterpart, which suggests that stabilizing the conformation of the peptide epitope is a key criterion in the vaccine design [Since TNF-α is a potent cytokine, a whole molecule immunogen need inactivated either by chemical modification such as formaldehyde treatment  or site e design . Here, w2 according to American Veterinary Medical Association Guidelines. The mice were inspected on a daily-basis for signs of ill health and distress. We euthanized the animals at the end of the experiments since there was no mice that reached humane endpoints including weight loss by 20% or more, severe lameness, dyspnea, ruffled fur, weakness, dehydration, or a hunched appearance, blistering at the injection site [For immunization, we used 54 BALB/c mice , 6 mice per experimental group. For vaccine efficacy in CIA mouse model, we used 19 DBA/1J mice , 9 mice in the DTNF7 group, and 10 mice in the DTT control group. All mice were purchased from SLAC Laboratory Animal Centre  and kept in specific pathogen-free conditions in Shanghai Jiao Tong University Animal Center. All animal studies were performed in accordance with institutional guidelines and with approval by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University. Euthanasia was carried out using COion site .Various number of amino acid residues at position 89–96 on diphtheria toxin T-domain  corresponding to aa 290–297 of DT were replaced by the TNF-α epitope using Discovery Studio 3.5. The resulting epitope-scaffold proteins denoted as DTNF proteins were named DTNF1 to DTNF7. In DTNF1 to 6, amino acid residues corresponding to DTT aa 95–96, aa 94–96, aa 93–96, aa 92–96, aa 91–96, aa 90–96 were replaced with TNF-α epitope VSRFAISYQEKVNLLSA (aa 80–96), respectively. In DTNF7, aa 89–96 were replaced with TNF-α epitope VSRFAISYQEKVNLLSAV (aa 80–97). The inter residue distances (TD and DD) were calculated in Swiss-Pdb Viewer 4.0.1 based on the X-ray crystal structures of mouse TNF-α and Diphtheria Toxin . TD (Å) stands for the interatomic distance between Valine 80 to Serine 96 or Valine 97 of TNF-α. DD (Å) stands for the interatomic distance between end residues of the replaced segment. The interatomic distance is calculated using the coordinates of the nitrogen atom of the alpha-amino group in N-terminal end residue and the carbon atom of the alpha-carboxy group in C-terminal end residue.The proteins were expressed and purified to homogeneity according to Hatem A. Elshabrawy . The DNA50 μg of purified DTT or DTNF proteins mixed with aluminum hydroxide adjuvant (InvivoGen) were injected into Female BALB/c mice (6–7 weeks old) intradermally followed by three boost injections with 30 μg, 20 μg, 20 μg proteins plus aluminum hydroxide adjuvant on day14, 28, 42. Blood samples were draw by retro-orbital sinus puncture after anesthesia by isoflurane inhalation on day 49. Anti-TNF-α and Anti-DTT Ab titer and IgG subclasses were measured by ELISA.0.5 mg/ml DTNF proteins were degassed and the protein unfolding events were recorded on a VP-Capillary differential scanning calorimeter  between 35 and 100°C at a scan rate of 1°C /min and under a constant pressure of 30 psi. The data were analyzed using Origin according to manufacturer’s instruction.The CD spectra (195–250 nm) were recorded on JASCO J-815 in 1-mm quartz cell at 25°C. Protein concentrations were 0.230 mg/ml.The tertiary structure of DTNF proteins were modeled in Modeller9V7 based on the X-ray crystal structure of DT and the mouse TNF-α mentioned above, and validated by Procheck . MD simuCIA was induced and the arthritis score were assessed according to Wei Tang . BrieflySera were diluted 4-fold and the concentration of cytokines  were measured using Luminex multiplex technology (Bio-Rad Laboratories) according to manufacturer’s instruction.P ≤ 0.05,**P ≤ 0.01, or ***P ≤ 0.001 was considered significant.The means ± SEM or means ± SE were calculated in Prism 6.0 (GraphPad) from three to ten replicates. Statistical comparisons were performed using t-test, where *et al have shown that a neutralizing monoclonal antibody 3B10 binds TNF-α at position 81–88 [Nagahira K on 81–88 . When coon 81–88  whereas E.coli as a highly soluble protein and the CD spectrum of the purified DTT shows a typical helical conformation with two minima at 208 and 222nm  could be a good candidate for both conformational stabilization and adjuvanting. Firstly, the function of T-domain is engaged in translocation of the catalytic domain of DT across the endosomal membrane in low pH-dependent manner . Thus thnd 222nm . This woAs shown in E. coli and purified to homogeneity. All of them are highly soluble at the concentration greater than 10 mg/mL in PBS pH 7.4, 25°C. Their circular dichroism spectra are characteristic of α-helical conformation similar to that of DTT  is composed of T-domain and receptor binding R-domain, and has been shown an effective vaccine carrier . HoweverCollagen-induced arthritis (CIA) is the most commonly used animal model to human rheumatoid arthritis, a chronical inflammatory autoimmune disease attacking joint. So far, TNF-α immunogens have only shown phylactic effects in CIA mouse model. DTNF7 is the first vaccine to show therapeutic effect after collagen immunization and the onset of the arthritis. The plasma level of TNF-α is extraordinarily high in the CIA model mice (2000 pg/ml) compared to that of TNF-α transgenic mice (9 pg/ml) . ConsequAlmost all known cytokines are present in the RA synovial fluid, among which TNF-α is the first cytokine proved to be effective therapeutic target . In rece


The aim of this work was to study the contribution of NF-κB pathway during UC and CAC, as well as the immunomodulatory effect of all-trans retinoic acid (AtRA). We analyzed the expression of NOS2, TNF-α, TLR4, and NF-κB, in colonic mucosa. We also studied NO/TNF-α modulation by LPS in colonic mucosa pretreated with AtRA. A marked increase in TLR4, NF-κB, TNF-α, and NOS2 expression was reported in colonic mucosa. The relationship between LPS/TLR4 and TNF-α/NO production, as well as the role of NF-κB signaling, was confirmed by ex vivo experiments and the role of LPS/TLR4 in NOS2/TNF-α induction through NF-κB pathway was suggested. AtRA downregulates NOS2 and TNF-α expression. Collectively, our study indicates that AtRA modulates in situ LPS/TLR4/NF-κB signaling pathway targeting NOS2 and TNF-α expression. Therefore, we suggest that AtRA has a potential value in new strategies to improve the current therapy, as well as in the clinical prevention of CAC development and progression. Colitis associated cancer (CAC) is the colorectal cancer (CRC) subtype that is associated with bowel disease such as ulcerative colitis (UC). The data on role of NF- Colorectal cancer (CRC) is one of the most common lethal cancers worldwide . There aκB activation in the intestinal mucosa [α) can further enhance NF-κB activation in various cell types [α is a multifunctional cytokine involved in apoptosis, cell survival, inflammation, and immunity acting via two receptors (TNF receptor p55 or TNF-Rp75) [Although many reports have documented the critical link between inflammation and development of colon cancer, our knowledge of the underlying mechanisms remains incomplete. Different TLR ligands have been implicated in various experimental tumor models and are known to play different roles. While some TLRs contribute to antitumor responses, others conversely promote tumor growth and facilitate the evasion of immune surveillance . TLR4 sil mucosa  and indul mucosa , 17. Amoll types . TNF-α iNF-Rp75) , 20.α play important roles in regulating inflammatory response. Generation and secretion of a high NO concentration by infiltrating cells and resident activated macrophages may lead to perpetuation of local tissue damage [α [γ or IL-17A and correlates with histological damage [κB signaling in TNF-α/iNOS induction in UC-associated carcinogenesis are still not fully understood and derived from preclinical studies, whereas data from human CAC are rare.Proinflammatory mediators and cytokines such as nitric oxide (NO) and TNF-e damage , 22. Theamage [α . The actamage [α . In our l damage , 11. Thul damage . These ol damage . HoweverGreat progress has been made in the development of chemotherapy, as well as targeted therapies for advanced different forms of CRC. Meanwhile, many cases show that tolerance develops to such treatments . HoweverAccumulative researches indicated that retinoids were associated with the prevention and amelioration of numerous chronic diseases and cancers –32. RetiκB has attracted our attention. In this way, we investigate the contribution of TLR4/NF-κB pathway in UC and CAC Algerian patients, as well as the effect of AtRA on TNF-α and NOS2 expression.AtRA has been shown to exert immunomodulatory and anti-inflammatory functions in various cell types –38. In oThirty-five Algerian patients with ulcerative colitis, UC , and 9 patients with colitis associated cancer, CAC , were enrolled in this study. UC and CAC patients were diagnosed by standard endoscopic and histological examination in the Department of Gastroenterology, Maillot Hospital, Algiers, Algeria, Service of Oncology, Public hospital Rouiba Algiers Algeria and Anatomic Pathology service, Mustapha Pacha Algiers Algeria.n = 16) were obtained from adult volunteer donors. Each patient has given a written informal consent for the study required by the ethic committee of the national agency of research development in health (ATRSS) which supported our project.The histopathological examination confirmed the diagnosis of UC active stage and cancer. Healthy controls (α and nitric oxide (NO) determination.Blood samples collected from healthy donors and patients were centrifuged at 2,000 rpm for 10 min to obtain plasma. All plasma samples were stored at −45°C until TNF-n = 12) and with CAC (n = 6). Biopsies from normal mucosa (n = 6) were also used as negative controls. Colonic biopsies were immediately placed in the transport medium Hanks' balanced salts solution, pH 7.4, supplemented with antibiotics. Cultures of colonic mucosa were pretreated with all-trans retinoic acid (AtRA) at 10-7 M for 6 h and stimulated with lipopolysaccharide . To confirm the engagement of NF-kB signaling pathway on TNF-α and NO production, colonic biopsies were stimulated with LPS (10 μg/mL) and SN50, an inhibitor peptide of NF-κB, AP-1, and STAT pathways (50 μM/mL). The cultures were incubated at 37°C in an atmosphere of 5% CO2 up to 24 hours. Supernatants were then collected for NO (nitrite) and TNF-α measurement. The estimation of total protein per well was performed using Bradford method and nitrite or TNF-α contents of each well were expressed as μM/mg or pg/mL of total protein, respectively [Multiple colonic biopsies were taken from patients who underwent colonoscopy in the Department of Gastroenterology, Maillot Hospital , Anatomic Pathology Service, Mustapha Pacha , and Service of Oncology, Public Hospital Rouiba, . Our study included biopsies from inflamed mucosa of patients with UC in active stage (ectively .α levels were determined by using enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions . The absorbance was read on an ELISA reader at 450 nm (LABSYSTEM). These assays detected only human cytokines. The results are expressed as picograms per milliliter relating to a standardized dose curve of the relevant recombinant TNF-α.Plasma TNF-−2) concentration, being the end product of NO oxidation, was determined by the method described by Touil-Boukoffa and others (1998). Briefly, nitrite (NO−2) was quantified by spectrophotometry (at 543 nm) in samples  after reaction with Griess reagent. The NO−2 concentration was determined by extrapolation from a NaNO2 standard curve and expressed as μM/mL [The stable nitrite . After reverse transcription into cDNA with a Reverse Transcription Kit (Bio-Rad), qPCR was then performed on MyiQ single color RT-PCR detection system with SYBR Green Super Mix (Bio-Rad) and gene-specific primers were summarized in n = 10; CAC, n = 9) and were fixed for 24 hours in buffered formaldehyde solution (10% in PBS) at room temperature. The biopsies were dehydrated by graded ethanol and embedded in paraffin (solidification point 60–62°C). Tissue sections (thickness 3 μm) were deparaffinized with toluene, stained with hematoxylin and eosin (H&E). Digital images were captured with digital camera at ×400 resolution. Each colonic segment was evaluated based on the Geboes histology score. Scores can range from 0 to 5.4, with higher scores indicating more severe histological inflammation.Multiple colonic biopsies were taken from patients  were cut from paraffin embedded tissues and mounted on positively charged super frost slides. Tissues were deparaffinized and rehydrated through graded alcohols. All sections were incubated in 3% hydrogen peroxide (10 min) to blockade endogenous peroxidase activity. Nonspecific binding was blocked by incubation (2 hours) in PBS containing 5% skim milk. The sections were subsequently incubated overnight at 4°C in monoclonal mouse anti-TLR4   containing 5% skim milk). Binding of the primary anti-TLR4 was detected with biotinylated rabbit anti-mouse immunoglobulin horse radish peroxidase- (HRP-) conjugated streptavidin (1 : 500). Immunoreactive complexes were detected using DAB system . Slides were counterstained briefly in hematoxylin (Sigma Aldrich) and mounted in Eukit (Sigma Aldrich). Slides were observed using a standard microscope (Zeiss) and pictures were taken using a digital camera at ×100 resolution. The intensity of staining of each section was evaluated subjectively by three separate observers in 4 fields nonoverlapping using the following designations: 0–10% of cells stained, score 0; 11–25% of cells stained, score 1; 26–50% of cells stained, score 2; 51–100% of cells stained, score 3. Those scoring 0-1 were considered to be negative, and those scoring 2-3 were considered to be positive.Sections   and washed again with blocking buffer. Specific protein signals were visualized using Western Lightning H Plus, ECL, Enhanced Chemiluminescence Substrate kit .The biopsies were lysed with phosphate buffered containing 1% Triton X-100 and 1% protease inhibitor mixture (Sigma Aldrich). Protein concentrations were determined using a Bio-Rad Protein Assay  according to manufacturer's instructions. SDS-PAGE was performed according to the Laemmeli procedure using gradient precasts gels  (Invitrogen). Proteins were transferred on to PVDF membrane  and probed with primary Ab   and the rabbit anti-μm) were saturated by incubation (2 hours) in PBS containing 5% skim milk and then permeabilised with 0.1% Triton X-100. A rabbit IgG1 monoclonal antibody (diluted 1/100), a mouse IgG1 monoclonal antibody (diluted 1/100), a rabbit IgG1 monoclonal antibody (diluted 1/100), and a rabbit IgG1 monoclonal antibody (diluted 1/50) were used as primary antibody for NF-κB, Ikk, NOS2, and TNF-α detection, respectively. Fluorescein isothiocyanate- (FITC-) conjugated IgG was used as secondary antibody. Slides were covered with Kaiser's glycerin-PBS and observed using a standard microscope (Zeiss) and pictures were taken using a digital camera at ×100 resolution. The intensity of staining of each section was evaluated subjectively by three separate observers in 4 fields nonoverlapping using the following designations: 0–10% of cells stained, score 0; 11–25% of cells stained, score 1; 26–50% of cells stained, score 2; 51–100% of cells stained, score 3. Those scoring 0-1 were considered to be negative, and those scoring 2-3 were considered to be positive.Colonic mucosa sections  and circulating TNF-α production by Griess method and ELISA, respectively, revealed that NO and TNF-α levels are increased in all groups of patients in comparison to the healthy controls (P < 0.001) (α levels are higher in sera of patients with CAC than in sera of patients with active UC (374.7 ± 16.31 pg/mL versus 291.48 ± 19.03 pg/mL) (μM versus 43.91 ± 7.28 μM) (P > 0.05) (In order to assess the involvement of TNF-< 0.001) . Interes3 pg/mL) . In cont > 0.05) .α levels in plasma of active UC . A similar observation was observed in CAC patients, who presented linear positive correlation between NO levels and TNF-α  (As shown in  < 0.01) .α assessed in vivo, we decided to determine the possibility of upregulation of NOS2 and TNF-α/TNFR expression in inflamed colonic mucosa of UC and CAC patients. Therefore, we analyzed the expression of messenger-RNAs (mRNAs) encoding TNF-α and TNF receptors (TNFRp55 and TNFRp75) in inflamed colonic mucosa from UC and CAC patients. The NOS2 (iNOS) mRNA expression was also analyzed by QPCR in these patients.Considering the high levels of circulating NO and TNF-α, TNF receptors, and NOS2 transcripts reveals that messenger-RNA transcript levels of the indicated genes are elevated in inflamed colonic mucosa from all groups of patients as compared with normal colonic mucosa. Moreover, TNF-α mRNA expressions are higher in colonic mucosa of patient's with CAC than in colonic mucosa of patients with active UC (P < 0.01). A similar observation was noted in CAC patients, who presented significantly higher TNFRp75 mRNA levels compared with active UC patients (~2-fold P < 0.01). In contrast, no statistically significant difference in TNFRp55 and NOS2 mRNA expression was detected between active UC and CAC patients (P > 0.05) (As shown in  > 0.05) .α protein expression was upregulated in inflamed colonic mucosa of all cohorts of patients in comparison with the controls  characterized by crypt destruction. Inflammatory cells infiltration into mucosa in UC patients  was alsoα in inflamed colonic mucosa of UC and CAC patients, we also decided to evaluate the percentage of mixed leukocytes (inflammatory cell infiltrate) in areas with higher or low expression of iNOS and TNF-α.Considering the overexpression of iNOS and TNF-P < 0.01). A similar observation was noted in areas with higher expression of TNF-α when compared with areas of low TNF-α expression (P < 0.01). Our finding showed that the overexpression of iNOS (NOS2) and TNF-α in inflamed colonic mucosa was associated with histological damage.Interestingly, the percentage of mixed leukocytes is higher in areas of iNOS overexpression than in areas with low iNOS expression in UC and CAC patients  , 26, 28.κB and iKK protein expression was assessed in inflamed colonic mucosa from the same patients by immunofluorescent staining.Colon specimens from patients with UC or CAC were examined for TLR4 mRNA expression by Q-PCR. We also evaluated TLR4 protein expression in inflamed colonic mucosa by Western blot analysis and immunohistochemical staining. In addition, NF-P < 0.001), especially in CAC patients (P < 0.01)  .In our current study, we also observed with interest that TLR4 protein expression was upregulated in colonic mucosa of patients compared with the controls, especially in patients with CAC Figures  and 8.κB and iKK protein expression is highly increased in colonic mucosa of UC patients in comparison with controls  in the absence or presence of SN50 (50 μM/mL). NO and TNF-α production was analyzed after 24 hours of incubation.To further confirm the relationship between LPS/TLR4 and TNFα production by inflamed colonic mucosa from patients with active UC compared with unstimulated biopsies (P < 0.01). These findings were also observed in the culture with colonic mucosa of CAC patients (P < 0.001) (μM/mL) inhibited LPS induction of NO and TNF-α production in inflamed colonic mucosa cultures in all groups of patients (P < 0.01) and TNF-α  compared with colonic mucosa stimulated with LPS alone (P < 0.05) (As shown in < 0.001) . Our stupatients . The SN5PS alone . The sam < 0.05) .κB and TNF-α/NOS2 induction in colorectal tumors, we focused our attention to elucidate the immunomodulatory effect of all-trans retinoic acid in NOS2 and TNF-α expression in inflamed colonic mucosa cultures. We used RT-PCR to study NOS2 and TNF-α mRNAs in colonic mucosa pretreated with AtRA (10−7 M) and stimulated with LPS. We also evaluated NO and TNF-α production by these colonic mucosae using Griess method and ELISA, respectively. Finally, we examined the ability of AtRA to modulate NOS2 protein expression in colonic mucosa from UC and CAC patients in response to LPS.Having demonstrated the link between TLR4/NF-α mRNA expression in inflamed colonic mucosa cultures of all patients (P < 0.05) and TNF-α  mRNA expression compared with colonic mucosa stimulated with LPS alone  .Our study showed that AtRA inhibited LPS induction of NOS2 and TNF-patients . Moreove Figures , resp..  Figures .  FigureFinally, we have investigated whether AtRA inhibits NO production directly by modulating iNOS expression. Interestingly, we report in our work that the pretreatment with AtRA decreased significantly NOS2 expression in colonic mucosa stimulated with LPS in all groups of patients . Indeed,κB provide a mechanistic link between inflammation and cancer [κB activation and induced the expression of many proinflammatory molecules including NO synthase 2 (iNOS) [κB/TNF-α signaling to promote tumor growth in experimental models of colitis associated cancer [It is possible that overproduction of inflammatory mediators, including proinflammatory cytokines, during IBD may facilitate the development and progression of colorectal cancer , 42–44. d cancer –48. Acti2 (iNOS) , 50. A gd cancer –53, wherα and NO in plasma of patients with UC and CAC. Our findings suggest that TNF-α is involved in upregulation of NOS2 induction in patients with UC and CAC. We also evaluated the expression of mRNAs of TNF-α, TNFR , and NOS2 (iNOS) in colonic mucosa of patients with UC and CAC. Interestingly, we have noticed significant levels of mRNA transcripts of the indicated genes in all groups of patients. In addition, immunofluorescence analysis showed that TNF-α and NOS2 expression was also upregulated in colonic mucosa of all cohorts of patients in comparison with the controls. Our findings are in agreement with previous data showing the overexpression of TNF-α [α and nitric oxide have been suggested to be an important mediators involved in the initiation of intestinal inflammation and perpetuation of local tissue damage [α is a crucial mediator of the initiation and progression of colitis associated colon carcinogenesis [β-catenin pathway, resulting in the development and growth of colitis associated neoplasms [In our study, we observe with interest the positive correlation between TNF-of TNF-α  and NOS2of TNF-α  in the iα, TNFR p, p75, anof TNF-α . Both TNe damage , 46. Popogenesis . Furtherogenesis . The TNFogenesis . The exceoplasms , 60.Previous works indicated that inducible nitric oxide synthase (iNOS) is expressed by several human gastrointestinal neoplasms including gastric cancer , colonicα expression in colonic mucosa of the same patients. It can be argued that NO are clearly implicated in the mucosal injury observed during CAC pathogenesis.Our results show that histologic examination of biopsies obtained from CAC patients arising in several ulcerative colitis reveals a severe mucosal degeneration, crypt loss, and destruction of epithelium in addition to cellular infiltrates. These observations correlate with the high NOS2 and TNF-In our study, we also demonstrate that TLR4 transcript is highly expressed in inflamed colonic mucosa of UC patients and CRC patients arising in chronic ulcerative colitis. In addition, our Western blotting and immunohistochemistry analysis showed the upregulation of TLR4 in colonic mucosa of UC and CAC patients. This finding is in agreement with previous data showing that patients with active UC had significantly more TLR4-positive epithelial cells than controls , 67. In α/NOS2 induction in chronic colitis [Collectively, these data raise a clear link between TLR4 and CAC development. TLR4 triggers elevated production of prostaglandin E2, influences epidermal growth factor receptor signaling (EGFR), and increases TNF- colitis .κB and iKK protein expression is highly increased in colonic mucosa in all groups of patients. In fact, some studies have shown the overexpression of NF-κB in the inflamed intestinal mucosa in UC and CAC patients and murine model of CAC [LPS has been shown to bind directly to the TLR4/MD2 receptor complex that initiates the intracellular signaling cascade in a MyD88-dependent or MyD88-independent manner , 73. LPSl of CAC , 75.κB activation or inhibition on TNFα/NOS2 induction are confirmed by ex vivo experiments. Our results showed that SN50 inhibited LPS induction of NO and TNF-α production in inflamed colonic mucosa cultures in all groups of patients. Although SN50 peptide could also competitively inhibit many transcription factors such as STAT and AP-1, entering nucleus, it mainly and firstly inhibits the NF-κB pathway after LPS activation.In our study, the effects of NF-α inhibition in the animal model prevents the development of CAC via blockade of TNF receptor 1 (TNFR1) signaling in infiltrating hematopoietic cells such as neutrophils and macrophages [α potently induces NF-κB activation but can also be a NF-κB-target gene [α through NF-κB activation could influence CAC development.The pivotal role of NF-kB signaling in tumor progression was provided in the AOM-DSS model for colitis associated colorectal cancer by Greten et al. , which srophages . TNF-α pget gene , 77. TheSeveral hematopoietic and nonhematopoietic lineages within the gastrointestinal tract, including macrophages, dendritic cells, and lymphocytes T share the capacity to synthesize retinoic acid , 79. A nα, β, and γ: the RARs and the retinoid X receptors (RXRs) that are expressed in lymphoid cells and act as transcription factors to regulate cell signaling, differentiation, and tumor suppression [β was downregulated in mice bearing mutations in the Apc tumor suppressor gene (ApcMIN mouse), human FAP adenomas, and human sporadic colon carcinomas [β may lead to alteration of cell growth and differentiation in the colon and rectum, thus contributing to the progression of colorectal cancer. Several series of preclinical models of colon carcinogenesis and in vitro system of a retinoid sensitive/resistant human colon carcinoma cells lines suggested that retinoid mediated autoinduction of the endogenous RAR β gene may play a crucial role in mediating the biological effects of retinoids [The biological effects of AtRA are mainly mediated by two families of nuclear retinoic acid receptors (RARs), each consisting of three receptor subtypes designated by pression . In addircinomas , 87. Theetinoids , 89. A setinoids . Howeverα and iNOS expression in colonic mucosa cultures stimulated with LPS. Our current studies demonstrated that AtRA decreased TNF and NOS mRNA expression in inflamed colonic mucosa stimulated with LPS and also inhibited TNF-α and NO production by some colonic mucosa. Our finding suggests that AtRA modulates LPS-TLR4 signaling targeting TNF-α and nitric oxide synthase 2 expression in UC and CAC. These results are in agreement with previous studies investigating the inhibitory effect of AtRA on NOS2 expression in human colon cancer cell lines [In this sense, we aimed to explore the mechanisms by which AtRA regulates TNF-ll lines –93.α [α production could be the result of direct inhibition of cytokine gene expression and could therefore indirectly decrease NOS2 gene expression.Several studies reported that AtRA could downregulate NOS2 gene expression by direct mechanisms mediated by RARa binding to the putative RARE in the promoter or the NOS2 gene . AtRA coα . In addiα , 99. Conα –102, ourκB signaling in UC and CAC pathogenesis through NO synthase 2 (NOS2) and TNF-α induction and (ii) AtRA downregulating NO synthase 2 and TNF-α expression targeting the LPS/TLR4/NF-κB pathway. Therefore, we suggest that AtRA has a potential value in new strategies to improve the current therapy and probably in the clinical prevention of CAC development and progression.Overall, our present study strongly supports (i) the pivotal role of TLR4/NF-


Kidney-Oriented UNderstanding of correcting CILiopathies) consortium, several groups and specialties united to perform scientific groundwork with the aim to develop genetic and therapeutic personalized care for NPH patients. At the start of this consortium, a genetic diagnosis for NPH was available for only 30–40% of patients, which improved to 50–60% during the course of the 4-year KOUNCIL project. Other major accomplishments of the consortium were (1) the establishment of a Dutch renal ciliopathy patient database with genotype and phenotype data; (2) composition of a proteomics-based integrated network of protein modules disrupted in NPH; (3) the development of non-invasive, urine-based assays that allow functional assessment of genomic variants in NPH and of therapeutic efficiency of drugs; and (4) chemical screening toward the identification of compounds that delay or prevent disease progression in NPH, which resulted in four potential medical interventions for NPH. In conclusion, the KOUNCIL consortium effectively channeled complementary approaches to broaden our understanding of NPH pathogenesis, resulted in 54 publications, improvement of genome diagnostics for NPH patients, awareness in the nephrology and clinical genetics communities for NPH, and new avenues for patient management.Nephronophthisis (NPH) is the most common monogenic cause of renal failure in children. Treatment options are limited to dialysis and transplantation. Therapeutics to significantly delay or prevent end-stage renal disease (ESRD) in children are currently not available. In the Dutch-Anglo KOUNCIL ( PS256100) is an autosomal recessive, genetically heterogeneous disorder that results in chronic renal disease in children and young adults  consortium was founded to perform scientific groundwork for the improvement of personalized care for NPH patients. Our consortium augmented the individual expertise of the collaborating workgroups toward reaching the challenging goals. The consortium included research groups with established expertise in renal ciliopathy research and diagnostics, including clinical geneticists, pediatric nephrologists, molecular biologists, cell biologists, and bioinformaticians  identify novel genes associated with NPH and related disorders by using next generation sequencing (NGS) approaches, (2) gain insights in genotype-phenotype relations through the setup and analysis of a renal ciliopathy database, (3) increase our understanding of the composition and function NPH-related protein modules in the ciliary proteome and explore mutational effects by state-of-the-art proteomics, and (4) initiate the development of targeted therapies to delay or prevent renal failure in NPH patients by drug screening in zebrafish and 3D culturing of cells derived from patients' urine samples.By bringing together the three university medical centers in our project we maximized patient inclusion. This was an important aspect of our study as NPH and -related disorders are individually rare. Combining our efforts has led to significant conclusions and high impact findings. The interactions within and between the different groups and work packages have been very open and effective, and by connecting different disciplines in the KOUNCIL project, a robust workflow could be implemented in the clinic that results from this synergy. We believe that without the close and successful collaboration within the consortium, the results would have been less optimal.http://www.agoraproject.nl) (http://www.kouncil.nl). Clinical data including renal and extrarenal phenotypic information was obtained and manually curated for inclusion in the renal ciliopathy database , and we hope to follow up on that initiative.The success of research consortia relies heavily on patient numbers, diagnostic accuracy, and deep-phenotyping of patients. In KOUNCIL, we used the AGORA data- and biobank protocols to systematically recruit and include patients (ject.nl) . Furthere Figure . So far,WDR60 , as a candidate-selected approach. From the second approach, four individual drugs and one drug combination significantly rescued the NPH phenotype in zebrafish embryos with acceptable toxicity profiles. These drugs warrant further To further advance therapy development, a major effort in KOUNCIL was the standardization of the use of URECs in molecular and cellular diagnostics . The 3D Our project advanced medical care for kidney patients and their families. First, NGS-based methods that were implemented in DNA diagnostics at the start of our project almost doubled the diagnostic yield for NPH-RC. Secondly, NGS-based DNA diagnostics for renal ciliopathies allow clinicians to make the NPH diagnosis at a much earlier stage, which resolves the need for invasive renal biopsies for diagnostic purposes. Third, a solidified work flow has been implemented for renal ciliopathy patients in our dedicated multidisciplinary outpatient clinics for kidney diseases in the Radboud university medical center, Nijmegen, The Netherlands and the University Medical Center Utrecht, Utrecht, The Netherlands. In London, UK, multi-disciplinary clinics for Bardet-Biedl Syndrome (BBS) patients are provided. NPH-RC patients are seen by a nephrologist, ophthalmologist, geneticist, dietician, general pediatricians, endocrinologist (diabetes type II), and a clinical psychologist. The aim is to provide a “one stop” visit to ensure that patients receive specialized and expert attention and management. This should result in a major change in how NPH and BBS is managed, with a focus on diagnosis, early intervention and appropriate health management. These multidisciplinary clinics form a role model for other hospitals worldwide and will hopefully be more widely implemented in the near future.The fourth improvement in patient care results from the setup of a renal ciliopathy database, which increased our insight into the relation between genotypes and phenotypes of NPH-RC patients. In the future, the database can be connected to other databases and converted to an interactive, international online registry. This will in turn allow for more refined insights in genotype-phenotype correlations and prognosis . FinallyKOUNCIL and KOUNCIL collaborations revealed several new renal ciliopathy genes and molecular mechanisms of disease, which significantly improved the diagnostic yield for NPH and related disorders as well as advanced knowledge of ciliopathy pathophysiology. In combination with the phenotypic characteristics sampled in our coded renal ciliopathy database, the power of genotype-phenotype correlations for NPH is just emerging and will require further prospective validation. Continued genetic screening and ongoing deep phenotyping of NPH-RC patients will further dissect these correlations, ultimately improving patient counseling. Understanding the impact of allelic variants from patients on disease manifestations demands understanding of the extensive molecular connectivity of cilium-directed pathways; to this end, we have developed a proteomics-based approach to evaluate the assembly defects of the ciliary transport particles (IFT) in a subset of ciliopathy patients. Although ciliopathies represent a class of rare pediatric diseases, through the work of KOUNCIL, we have found novel insights into the molecular mechanisms of chronic renal disease progression widespread in the general population. Preliminary work suggesting that cilia are key repressors of cellular fibrosis in the kidney, will direct and further streamline future efforts to develop therapeutic intervention with a focus on fibrosis treatment.It should be noted that development of methodologies independent of animal experimentation are a priority for most scientific bodies today. We have generated a robust protocol growing renal cells from urine that is non-invasive to patients, yet is patient-specific, and is amenable to screening . We showThe KOUNCIL consortium, consisting of pediatric nephrologists, clinical geneticists, molecular biologists and bioinformaticians, provided a unique, coherent inter- and transdisciplinary team to combat juvenile kidney failure. The resulting five PhD theses from three centers in two countries implicated the relevance of our consortium for the next generation of renal clinicians and scientists, and will continue to have a high impact on the renal field.This consortium aimed to improve the understanding of genetics and biology of renal ciliopathies with innovative NGS and biochemical techniques, and set the first important steps toward therapeutic approaches to delay the progressive degenerative defects in the kidney. The results of this consortium project have already significantly impacted the clinical management of NPH-RC by improvements in diagnosis, prognosis, and accuracy of genetic risk assessment. Our project has provided us with optimal preparations for the development and refinement of future personalized therapies, and is a message of hope for NPH patients for whom there is currently no satisfactory treatment.KR, RG, and HA performed the writing of the manuscript; ML, MO, and RR provided figures; RG, PB, RR, HA, and NK were work package leaders in the KOUNCIL consortium. All authors provided feedback on the revised 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. The handling Editor declared a shared affiliation, though no other collaboration, with two of the authors: RR and MO.


A complex remodelling of the TNFα-TNFα receptor system thus takes place in patients treated with anti-TNF-α drugs, that involves either the production of anti-drug antibodies or the modulation of monocyte phenotype or inflammatory activity.TNF-α has a central role in the development and maintenance of psoriatic plaques, and its serum levels correlate with disease activity. Anti-TNF-α drugs are, however, ineffective in a relevant percentage of patients for reasons that are currently unknown. To understand whether the response to anti-TNF-α drugs is influenced by the production of anti-drug antibodies or by the modulation of the TNFα-TNFα receptor system, and to identify changes in monocyte phenotype and activity, we analysed 119 psoriatic patients who either responded or did not respond to different anti-TNF-α therapies , and measured plasma levels of TNF-α, TNF-α soluble receptors, drug and anti-drug antibodies. Moreover, we analyzed the production of TNF-α and TNF-α soluble receptors by peripheral blood mononuclear cells (PBMCs), and characterized different monocyte populations. We found that: i) the drug levels varied between responders and non-responders; ii) anti-infliximab antibodies were present in 15% of infliximab-treated patients, while anti-etanercept or anti-adalimumab antibodies were never detected; iii) plasma TNF-α levels were higher in patients treated with etanercept compared to patients treated with adalimumab or infliximab; iv) PBMCs from patients responding to adalimumab and etanercept produced more TNF-α and sTNFRII  Althoral area –7. The mral area .Among these cytokines, TNF-α is a crucial pathogenetic driver. TNF-α binds two different cell-membrane receptors, TNF receptor (TNFR) type 1, also known as p55, and TNFR2, also known as p75. Soluble forms of these receptors, namely soluble TNFR1 (sTNFR1) and soluble TNFR2 (sTNFR2), have also been identified and likely act as endogenous TNF-α inhibitors. Increased levels of sTNFR1 have been found in serum from patients with psoriasis . TNF-α mSeveral TNF-α blockers, including neutralizing antibodies and soluble TNF-α receptors, have been developed, and are currently used in clinical practice. Adalimumab, etanercept and infliximab represent the first-generation inhibitors targeting TNF-α, and are largely used in clinical settings. Adalimumab is a fully human monoclonal antibody, while infliximab is a chimeric human/mouse monoclonal antibody containing almost 75% human-derived amino acids and almost 25% mouse-derived amino acids. Etanercept is a fusion protein where a dimer of the extracellular domains of human TNFR2 is fused to the Fc portion of human IgG1 .Anti-TNF-α drugs provide remission in a great proportion of patients, reducing inflammation and decreasing the “Psoriasis Area and Severity Index” (PASI) –15. Howeth 2014 and ended on April 20th 2015. Blood samples were collected during the follow-up visits of patients receiving biological drugs. For patients treated with etanercept or adalimumab, blood samples were obtained three months after the last drug injection, while for patients treated with infliximab, blood samples were obtained two months after the last drug infusion. The study was approved by the local Ethical Committee of Modena (identification code number 296/13) and by the Institutional Review Board of the Dept. of Surgery, Medicine, Dentistry and Morphological Sciences of the University of Modena and Reggio Emilia, and was conducted according to the Declaration of Helsinki principles. Signed consent forms were collected from all patients.A total of 119 patients with psoriasis and psoriatic arthritis were enrolled at the Department of Dermatology of the University of Modena and Reggio Emilia . Of these, 48 were treated with adalimumab (standard dosage for psoriasis), 30 with etanercept (standard dosage for psoriasis), 41 with infliximab (standard dosage for psoriasis). Twenty healthy controls  were also enrolled. Patients’ demographic and clinical characteristics are shown in Twenty ml of venous blood were collected from each patient into ethylenediamine-tetraacetic acid (EDTA) tubes. Samples were centrifuged and plasma was stored at -80°C. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient according to standard procedures and stored in liquid nitrogen until use.The plasma levels of TNF-α, sTNFRI, sTNFRII were quantified by Quantikine ELISA kit , following instructions provided by the manufacturer. In particular, the following kits were used: human TNF-α Quantikine HS ELISA kit , human sTNFRI/TNFRSF1A Quantikine ELISA Kit , and human sTNFRII/TNFRSF1B Quantikine ELISA Kit .The plasma levels of etanercept, anti-etanercept antibodies, adalimumab, anti-adalimumab antibodies, infliximab and anti-infliximab antibodies were quantified by the following ELISA kits: shikari Q-ETA, shikari S-ATE, shikari Q-ADA, shikari S-ATA, shikari Q-INFLIXI and shikari Q-ATI .PBMCs were thawed and plated in RPMI-1640 complete medium  with or without 50 ng/mL LPS  for 16 hours. Supernatant was collected and used for the quantification of TNF-α, sTNFRI and sTNFRII by human TNF-α Quantikine ELISA kit , human sTNFRI/TNFRSF1A Quantikine ELISA Kit and human sTNFRII/TNFRSF1B Quantikine ELISA Kit.PBMCs were thawed according to standard procedures . Monocytp-value <0.05 was considered significant [Kruskal-Wallis test with Dunn’s Multiple Comparison Test was used to compare TNF-α, sTNFRI and sTNFRII plasma levels in patients from different groups. Mann-Whitney test was used to compare drug concentration among different groups. All statistical analyses were performed by using Prism 5.0 software ; a nificant . PrincipWe studied 119 patients receiving biological drugs, whose plasma was screened for TNF-α, sTNFRI and sTNFRII, drug and anti-drug antibodies levels. Among patients treated with adalimumab , 30 were R (62.5%) and 18 were NR (37.5%); among patients treated with etanercept , 20 were R (66.7%) and 10 were NR (33.3%); among patients treated with infliximab , 29 were R (70.8%) and 12 were NR (29.2%).-1 (range: 0.002–9.89), and a significant difference was observed between R and NR . Concerning etanercept, the mean concentration of the drug was higher in NR (3.91±0.01 μg mL-1) than in R  in R, and 1.63 μg mL-1 in NR (range: 0.04–7.14 μg mL-1), and no significant differences were observed between R and NR  patients treated with infliximab . No sign-1 .-1, 942.2±31.77 ng mL-1 and 2,587±75.85 ng mL-1. TNF-α concentration in plasma from patients treated with etanercept was significantly higher compared to those treated with adalimumab or infliximab. Patients responding to etanercept had higher levels of TNF-α compared to patients responding to adalimumab or infliximab. No differences were detected between R and NR to the different treatments. Levels of sTNFRI or sTNFRII were similar among all groups of patients  and can be divided into three different subsets on the basis of CD14 and CD16 expression. Classical monocytes express CD14 and do not express CD16 , non-classical monocytes express CD14 and high levels CD16 , and intermediate monocytes express high levels of CD14 and CD16  [Monocytes represent a heterogeneous cell population that express MHC class II molecules  . Differei.e. classical) monocytes, was higher in patients responding to adalimumab than in patients not responding to the same drug. A slight decrease in the percentage of classical monocytes, although not significant, was observed in patients not responding to infliximab. Concerning HLA-DR, its expression was slightly higher in non-classical and intermediate monocytes from patients treated with infliximab or adalimumab compared to patients treated with etanercept.Given the importance of monocytes in inflammatory diseases, we identified these cells and their subsets in PBMCs from patients treated with different anti-TNF-α drugs. As shown in The hallmark of psoriasis is an extensive inflammation affecting the skin, mainly mediated by Th1/Th17 cytokines, with a key role of TNF-α , 27. ForWe found that 15.8% of patients treated with infliximab had detectable levels of ADAs, most of them being associated with low plasma infliximab concentrations. The presence of anti-infliximab antibodies (ATIs) has also been described in patients with Crohn’s disease (CD) or rheumatoid arthritis (RA), with a prevalence ranging from 12% to 44% in CD, and 6–61% in RA , 26, 28.in vitro, in the presence or in the absence of LPS. When stimulated with LPS, PBMCs from patients responding to etanercept or adalimumab produce almost 4-fold TNF-α together with sTNFRII than PMBCs from patients receiving infliximab. PBMCs from patients not responding to infliximab produced higher levels of TNF-α and sTNFRII, whereas PBMCs from patients treated with etanercept and not responding to this therapy, produced lower levels of sTNFRI. This could partially explain the higher TNF-α levels observed in these patients’ plasma. Previous studies suggested that etanercept and infliximab strongly affect gene expression in PBMCs, and that effects of etanercept were different from infliximab in the modulation of proinflammatory genes [Concerning plasma levels of TNF-α, sTNFRI and sTNFRII, the main differences involved TNF-α levels. Firstly, we found that TNF-α plasma levels do not correlate with the subjects’ age. This was quite unexpected, as in the healthy population TNF-α plasma levels increase with age . In psorry genes , 32. Ouri.e., all CD14+ cells, as well as the percentage of classical monocytes in patients treated with adalimumab was significantly lower if compared to patients treated with etanercept and infliximab. However, the stratification of patients according to PASI did not reveal any difference in the percentage of monocyte subsets between R and NR to the different treatments. The loss of monocytes in patients treated with adalimumab could be caused by three, non-mutually exclusive phenomena, i.e., redistribution and homing of cells, increased differentiation , or net loss of cells due to apoptosis. If on the one side, it is difficult to analyze monocyte redistribution to tissues other than blood, or to detect osteoclastic differentiation, on the other side, it is possible to hypothesize that, as already demonstrated using in vitro models [We found that anti-TNF-α drugs also modify monocyte subsets in patients with psoriasis. Circulating monocytes derive from the bone marrow and mediate both regulatory and effector functions in adaptive and innate immunity. In response to several stimuli, monocytes can migrate into tissues where they can differentiate into phagocytes, dendritic cells and osteoclasts . Three mo models , these cIn summary, our study suggests that a complex remodelling of the TNF-α/sTNFR system occurs in patients with psoriasis treated with anti-TNF-α drugs. Other studies are, however, needed to further characterize functional changes of monocytes in patients treated with different drugs, and that could devote their attention to other early, crucial aspects of inflammatory phenomena modulated by these drugs. Therefore, the correlations between plasma levels of TNF-α, its soluble receptor, the phenotype of monocytes and their ability to produce cytokines must be investigated also in other human diseases that benefit of he treatment with molecules that block TNF-α.