Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine expressed by macrophages, monocytes, and T cells, and its expression is triggered by the immune system in response to pathogens and their products, such as endotoxins. TNF-α plays an important role in host defense by inducing inflammatory reactions such as phagocytes and cytocidal systems activation. TNF-α also plays an important role in bone metabolism and is associated with inflammatory bone diseases. TNF-α binds to two cell surface receptors, the 55kDa TNF receptor-1 (TNFR1) and the 75kDa TNF receptor-2 (TNFR2). Bone is in a constant state of turnover; it is continuously degraded and built via the process of bone remodeling, which results from the regulated balance between bone-resorbing osteoclasts, bone-forming osteoblasts, and the mechanosensory cell type osteocytes. Precise interactions between these cells maintain skeletal homeostasis. Studies have shown that TNF-α affects bone-related cells via TNFRs. Signaling through either receptor results in different outcomes in different cell types as well as in the same cell type. This review summarizes and discusses current research on the TNF-α and TNFR interaction and its role in bone-related cells. Skeletal health, architecture, and homeostasis are functions of the constant process of bone remodeling . Bone-reOsteoclasts, descendants of hematopoietic stem cells, are responsible for bone resorption , and disOsteoblasts are derived from mesenchymal stem cells . Their aOsteocytes are terminally differentiated osteoblasts entombed in a mineralized bone matrix ,17. TheyTNF-α interacts with two receptors: the 55 kDa tumor necrosis factor receptor-1 (TNFR1), also called CD120a and p55, and the 75 kDa tumor necrosis factor receptor-2 (TNFR2), also called CD120b and p75 . TNFR1 aThis review outlines the current knowledge on the interaction of TNF-α with TNFR1 and TNFR2 in bone-related cells.The tumor necrosis factor receptor superfamily (TNFRSF) is characterized by the ability to bind tumor necrosis factor superfamily (TNFSF) ligands ,28. The The expression of TNFR2 is restricted compared to the ubiquitous expression of TNFR1. TNFR2 is expressed on endothelial cells, mesenchymal stem cells, and immune cells, such as macrophages, monocytes, T cells, B cells, and NK cells ,45,46,47RANKL is an essential factor for osteoclast differentiation ,59 and bTNF-α plays a central role in inflammatory osteoclast formation and affects cells involved in osteoclast formation, including macrophages, stromal cells, and T cells expressing RANKL, which enables their osteoclastogenic potential ,67. Our Several reports showed that the compression force in orthodontic tooth movement (OTM) in mice induces the expression of TNF-α ,78,79,80Analysis of bone marrow cells derived from mice differentially expressing TNFR1 or TNFR2 revealed that TNFR1 induced osteoclast formation results are comparable to those obtained in wild-type mice, and marrow from mice expressing only TNFR2 could not stimulate osteoclast formation . MoreoveUnderstanding TNFR-TNF-α signaling goes hand in hand with understanding the signaling pathways involved in osteoclast differentiation by M-CSF and RANKL. M-CSF is one of the early cytokines involved in the differentiation of monocytes to osteoclasts and the survival of osteoclasts. Osteoclast precursors express the M-CSF receptor c-Fms, and M-CSF, and in turn, activates the mitogen-activated protein kinase (MAPK) pathways, PI3K, and Akt .Differentiation involves the expression of RANK on the surface of osteoclast precursors, which are activated by RANKL, leading to the induction of a plethora of effectors. The downstream signals include MAPK pathways, such as family-mediated JNK, p38, NF-κB, and the AP-1 family, such as c-Fos and TRAF . NF-κB- Previous studies examined whether TRAF6 is required for TNF-α-induced osteoclast formation using osteoclast precursors from TRAF6-deficient mice. The results showed that TNF-α cannot induce osteoclast formation and suggested that TRAF6 was required for TNF-α-induced osteoclast formation in vitro . TRAF6 iTNF-α signaling induces different biological reactions depending on the receptor activated, either TNFR1 or TNFR2, because each receptor mediates distinct intracellular signals. A study in TNFR1- or TNFR2-deficient mice for TNF-α-induced osteoclast formation in vitro revealed that TNFR1 enhanced osteoclast formation and TNFR2 inhibited osteoclast formation . TNF-α iTNF-α has been shown to support osteoclast survival  by activTNF-α was indicated to induce actin ring formation, which is a required osteoclast formation in vitro . PreviouThe signaling pathways activated via TNFR1 and TNFR2 have been intensively studied in a variety of other cell types; few studies have examined TNF-α-signaling in osteoclast precursors/osteoclasts. Further studies are needed to elucidate the relationship between the signaling downstream of the interaction of TNF-α with TNFRs in osteoclast differentiation.Osteoblasts differentiate from multipotent mesenchymal stem cells (MSCs) capable of producing adipocytes, chondrocytes, fibroblasts, and skeletal muscle cells ,115,116.Reports on the expression of TNFRs by osteoblasts have yielded inconsistent results. While studies have agreed on the expression of TNFR1, TNFR2 has been considered by some authors as “not detected” in MG63 osteosarcoma cells and in non-transformed osteoblasts made from mesenchymal stem cells using unamplified gene screening . While TTNF-α inhibits osteoblast differentiation in cells from multiple cell sources: fetal calvarial cells, the MC3T3 pre-osteoblastic cell line, and murine marrow stromal cells ,129. InhTNFR1 is the receptor responsible for TNF-α-mediated osteoblast differentiation inhibition, while TNFR2 is dispensable in this process. While wild-type or TNFR2-deficient MSCs cultured with TNF-α exhibit the complete inhibition of osteoblast differentiation, nodular formation, and mineralization as well as decreased alkaline phosphatase activity and osteocalcin expression, TNFR1-deficient MSCs are completely unresponsive to TNF-α and exhibit no effects on osteoblast differentiation. Researchers reasoned that cytotoxicity might be why TNF-α inhibits osteoblast differentiation—because reducing the pool of precursor cells led to a decrease in the number of available osteoblasts. However, MC3T3 apoptotic cell death was not the reason why TNF-α blocks differentiation to osteoblasts, because treatment with caspase inhibitors did not reverse the inhibitory effects of TNF-α . TNFR1 sThe protective effect of TNFR2 on osteoblast differentiation is puzzling as TNFR2 has been proposed to assist TNFR1 signaling through ‘’ligand passing,’’ in which TNFR2 increases the concentration of TNF-α around TNFR1 due to the kinetic properties of TNF-α association and dissociation from TNFR2 . ConversTNF-α inhibits osteoblast differentiation distal to the signaling of known osteogenic inductive proteins  and by directly affecting Runx2 activity at multiple levels. First, TNF-α regulates Runx2 at the transcriptional level by decreasing mRNA expression of the two major Runx2 isoforms , and second, by reducing mRNA half-life and stability, which translates into a reduction in the Runx2 nuclear content as well as a reduction in Runx2 binding to promoter regions ,130. IntVarious post-transcriptional and post-translational mechanisms regulate Runx2 expression and activity. BMP-2 regulates Runx2 via stabilizing its binding to Smads and preventing its degradation by Smurf1/2 ,136. TNFIn line with the established role of TNF-α in inhibiting osteoblast differentiation, TNF-α also decreases Osx mRNA expression and Osx promoter activity in M3CT3 cells; however, it does not affect the brief half-life of Osx mRNA as it does with Runx2 mRNA. TNF-α inhibition of Osx promoter activity and mRNA expression is due to the activation of the MEK/ERK pathway, while p38 and JNK are not involved in this process . NF-κB aTNFR1 and TNFR2 activate the NF-κB pathway using TRADD and the TRAF1/TRAF2 complex. Furthermore, signaling through TRAF1/2 to activate NF-κB promotes cell survival, for the most part, while the case is not the same for TNFR1 signaling through TRADD, which promotes survival or apoptosis . Which oAfter reducing Runx2 and osterix mRNA expressions, TNF-α suppresses alkaline phosphatase and α1(I) procollagen gene expressions. The inhibition of skeletal-specific matrix proteins, osteonectin, and osteopontin is only seen with TNF-α but not with other inhibitory cytokines, such as IL-1β, which are also reported to inhibit osteogenic differentiation .TNF-α apoptotic effects on osteoblasts depend on the cell differentiation stage. While cells expressing low levels of Runx2 show inhibition of differentiation, mature osteoblasts expressing high Runx2 levels are susceptible to apoptosis by TNF-α signaling through TNFR1 . TNF-α iTransient caspase activation, in particular, caspases 2, 3, and 8 by BMP-4 is important for osteoblast differentiation, and the inhibition of these caspases leads to reduced ALP and PTH activity in MC3T3 cells. Paradoxically, triggering the caspase cascade signal is important for apoptosis; however, the critical difference in whether apoptosis or differentiation occurs is in the level of differentiation of the cells when treated with TNF-α. These results reconcile the finding that caspase activation is essential for osteoblast differentiation with the lack of apoptosis that is seen in not fully differentiated cells. Cells that have already assumed the osteoblastic phenotype are susceptible to TNF-α-induced caspase activation and apoptosis. In contrast, cells that have not yet differentiated and that are still pre-osteoblastic require the activation of the caspase pathway to assume the mature osteoblastic phenotype .TNFR activation in osteoblasts generally inhibits bone formation and stimulatory bone resorption. Downstream activation of TNFRs inhibits transcription and translation of genes favoring bone formation, including α-collagen, alkaline phosphatase, osteocalcin, insulin-like growth factor 1, parathyroid hormone receptor, platelet-derived growth factor receptor, and N-cadherin genes. TNF-α induces transcriptional activation of genes in osteoblasts favoring indirect bone resorption, such as IL-6, tPA, uPA, PAI-1, gelatinases, MMPs, TIMPs, NF-κB, ICAM-1, CSF-1, GM-CSF, and G-CSF genes ,158,159.The intracellular domains of TNFR1 and TNFR2 share no homology; however, TNFR1 and TNFR2 activate nearly the same pathways via different mechanisms. Upon activation, both receptors are inert and signal through TRAFs. The main difference between the two receptors is the presence of a DD only in TNFR1. The DD associates with the silencer of the death domain (SODD); upon activation of TNFR1, SODD frees the cytoplasmic domain to associate with the TRADD protein, which then recruits TRAFs to propagate the downstream signal of the receptor. The main pathways activated by TNFRs are the NF-κB, MAPK family, G protein family, and caspase pathways. Some differences are present in the selectivity and efficiency of either receptor to activate these pathways; however, their functions overlap considerably. The downstream activation of both receptors is required for the full effects seen in osteoblasts compared with effects observed upon activation of just one of the two receptors ,161.While TNF-α is generally inhibitory of bone formation, TNF-α shows a dual function in fracture healing. In the early stages of fracture healing, a brief but necessary inflammatory stimulus recruits osteoblast progenitors cells capable of forming osteoblasts. This observation was demonstrated in two models of fracture repair—marrow ablation and simple transverse fractures—in which TNFR-deficient mice experienced delayed healing. On day three after model establishment in wild-type mice, young osteoblasts appeared in the marrow space, while only granulation tissue appeared in the marrow cavity in TNFR-deficient mice. In the marrow ablation model in TNFR-deficient mice, type I collagen and osteocalcin mRNA expressions were reduced to 50% of levels in wild-type mice. There was a complete absence of the initial intramembranous bone formation on the periosteal surfaces in the fracture repair model. In TNFR-deficient mice, endochondral bone formation was delayed but the inhibition of osteogenesis was not observed; the intramembranous bone formation was initially completely inhibited .In bone resorption induction, TNF-α is a RANKL synergist, and in bone marrow stromal cells, it induces RANKL expression through activating p38 MAPK; inhibiting p38 signaling arrests its expression. TNF-α also induces other MAPK family members, including JNK and ERK . TNF-α iThe effects of TNF-α on osteoblasts obtained from different cell sources converge towards a common outcome. Fetal calvarial cells and murine marrow stromal cells differentiation to osteoblasts is halted when cultured with TNF-α. Additionally, MC3T3 cell line treatment with TNF-α exhibits an inhibition of differentiation via decreasing Runx2 and Osx mRNA expressions and increased apoptotic changes. MSCs, in addition to experiencing inhibition of differentiation, experience reduced nodular formation and mineralization, decreased alkaline phosphatase and osteocalcin expression when signaling through TNFR1. They also experience apoptotic changes once their differentiation to the osteoblastic phenotype takes place, as MSCs apoptosis was shown to be facilitated by Runx2 expression. Cells from the C2C12 line experience apoptosis with TNF-α treatment which can be overcome with BMP-2 and BMP-4, and activation of the NF-κB pathway. Primary human osteoblasts and the MG63 osteoblastic cell line also experience apoptotic changes when treated with TNF-α due to increased Fas expression.Osteocytes form from a group of osteoblasts that have become encapsulated in their secreted matrix and represent the terminally differentiated stage of the osteoblast. These cells are mechanosensing cells in the bone and perform other regulatory functions, including bone remodeling and maintaining mineral homeostasis . OsteocyOsteocytes express both TNFRs, and the effects of TNF-α on osteocytes are similar to those on osteoblasts. TNF-α promotes osteocyte apoptosis and steers osteocyte control over bone remodeling in a negative net balance. In addition, and per osteocyte involvement in propagating inflammation, TNF-α is involved in osteocytic expression and the withholding of signals that exacerbate inflammation ,165.Osteocyte apoptosis is a function of two distinct processes. The first preserves the integrity and strength of bone by removing senile osteocytes, which promotes self-renewal of bone tissue ,167. TheTNF-α increased apoptosis in the MLO-Y4 osteocytic cell line from baseline to 13%–18% (depending on the apoptotic test used), which can be attenuated using the CD40 ligand ,177. AnoOsteocytes express both TNFR1 and TNFR2. A wealth of investigators has established that TNF-α is mostly correlated with osteocytic-induced bone loss, either directly via RANKL expression in diseases of inflammatory bone loss, such as rheumatoid arthritis and periodontitis ,179,180,TNF-α works directly on osteocytes to increase their osteoclastogenic ability via RANKL expression; osteocytes treated with TNF-α induce the formation of osteoclasts in vitro from TNFR-deficient osteoclast precursors. In mice lacking TNFRs, osteocyte RANKL expression, osteoclast formation, and the OTM distance are significantly decreased compared with observations in wild-type mice. TNF-α signaling in the osteocyte activates the NF-κB pathway and signals RANKL mRNA expression through ERK1/2 and p38 MAPKs .2+) intracellular levels by MLO-Y4, and reduced the F-actin content to 63% of the control values [TNF-α also inhibited pulsatile fluid flow-stimulated NO production and decreased pulsatile fluid flow-stimulated (Cal values . This isl values , and ostl values . WhetherInterestingly, TNF-α can induce osteocytic bone resorption independent of RANKL. Kogianni et al. demonstrated that osteocyte apoptotic antibodies induced bone resorption in vitro and in vivo. The co-administration of the TNF-α neutralizing antibody with osteocyte apoptotic antibodies reduced bone resorption, an effect not seen with OPG or when osteocytes are treated with TNF-α neutralizing antibody . TNF-α aOsteocytes seem to be either involved in propagating inflammation or are heavily affected by inflammation. TNF-α signaling in osteocytes relative to its functions in other bone cells is not completely understood and needs further clarification. TNF-α is a ubiquitous inflammatory cytokine, and its effect on osteocytes is worth further study. TNF-α signaling is also vital for initiating fracture healing, and osteocytes supply cancellous bone with the RANKL needed for bone remodeling in the mature skeleton. How TNF-α signaling in the osteocyte affects local and systemic inflammatory conditions should be examined in further research. Whether osteocytes have potential therapeutic value needs more attention.TNFR1 and TNFR2 signaling pathways activated by TNF-α have been investigated in osteoclasts, osteoblasts, and osteocytes. Finally, we summarize how these cells interact to progress bone resorption, as shown in The well-documented role of TNF-α in bone inflammatory diseases is to promote bone resorption and contribute to disease progression. TNF-α directly induces osteoclast formation via TNFR1 but not TNFR2 and indirectly induces osteoclast formation by TNF-α-induced RANKL expression in osteoblasts and osteocytes. In this way, TNF-α induces bone resorption events from multiple directions. TNFR1 and TNFR2 signaling pathways activated by TNF-α have been investigated in osteoclasts, osteoblasts, and osteocytes, with both receptors capable of activating MAPKs phosphorylation and activating the NF-κB pathway through different adaptor proteins. TNFRs signaling may induce apoptotic or survival signals depending on the type of cell involved, and the receptor activated, while signaling in osteoclasts is generally proliferative, and signaling in osteoblasts and osteocytes is inhibitory. Few studies have examined the effects of the TNF-α and TNFRs interaction in bone-related cells. Further studies are required to elucidate the relationship between TNF-α and TNFRs.


TNF-α)-308G/A gene polymorphism with circulating TNF-α levels and EDS among 326 participants. CAD patients with OSA (apnea–hypopnea-index (AHI) ≥ 15 events/h; n = 256) were categorized as having EDS (n = 100) or no-EDS (n = 156) based on the Epworth Sleepiness Scale score with a cut-off of 10. CAD patients with no-OSA  were included as a control group. The results demonstrated no significant differences regarding the distribution of the TNF-α alleles and genotypes between CAD patients with vs. without OSA. In a multivariate analysis, the oxygen desaturation index and TNF-α genotypes from GG to GA and GA to AA as well as the TNF-α-308A allele carriage were significantly associated with the circulating TNF-α levels. Moreover, the TNF-α-308A allele was associated with a decreased risk for EDS  independent of age, sex, obesity, OSA severity and the circulating TNF-α levels. We conclude that the TNF-α-308A allele appears to modulate circulatory TNF-α levels and mitigate EDS in adults with CAD and concomitant OSA.Obstructive sleep apnea (OSA) is common in patients with coronary artery disease (CAD), in which inflammatory activity has a crucial role. The manifestation of OSA varies significantly between individuals in clinical cohorts; not all adults with OSA demonstrate the same set of symptoms; i.e., excessive daytime sleepiness (EDS) and/or increased levels of inflammatory biomarkers. The further exploration of the molecular basis of these differences is therefore essential for a better understanding of the OSA phenotypes in cardiac patients. In this current secondary analysis of the Randomized Intervention with Continuous Positive Airway Pressure in CAD and OSA (RICCADSA) trial , we aimed to address the association of tumor necrosis factor alpha ( Coronary artery disease (CAD) is the most common type of heart disease and is characterized by a poor prognosis and a high risk of morbidity and mortality . The traObstructive sleep apnea (OSA) is a common disorder characterized by repeated upper-airway collapse during sleep, resulting in intermittent hypoxia, fragmented sleep, fluctuations in blood pressure and increased sympathetic nervous system activity . The preIncreased inflammatory activity plays a crucial role in the development of atherosclerotic plaques and CAD . MoreoveTNF-α is a pro-inflammatory cytokine that plays an important role in the immune system and the development of autoimmune and infectious diseases as well as atherosclerosis and CAD . TNF-α iThe manifestation of OSA varies significantly between individuals in clinical cohorts, and not all OSA patients demonstrate the same set of symptoms; i.e., excessive daytime sleepiness (EDS) and/or increased levels of inflammatory biomarkers. The further exploration of the molecular basis of these differences is therefore essential for a better understanding of the characteristics, prognosis and cardiovascular outcomes in CAD patients with OSA.TNF-α (position-308G/A), where allele A at this position (TNF-α-308A) is suggested to be associated with the higher occurrence of OSA [TNF-α-308A allele and obesity is also reported [Several potential genetic risk factors for OSA have been studied using SNP (single nucleotide polymorphism) analysis, among which TNF-α has received special attention ,20. There of OSA  as well e of OSA ,23,24,25reported ,21, wherreported .TNF-α-308G/A gene polymorphism modulates circulating TNF-α levels as well as EDS independent of OSA severity in patients with CAD.We have previously conducted studies in the Randomized Intervention with CPAP in Coronary Artery Disease and Sleep Apnea (RICCADSA) trial with the primary goal of addressing the impact of CPAP on cardiovascular outcomes in revascularized CAD patients with OSA ,28. In tTNF-α-308G/A polymorphism study , and written informed consent was obtained from all patients. An additional ethics approval was obtained for the molecular analysis of the biomarkers . The RICCADSA trial was registered with ClinicalTrials.gov (NCT 00519597) as well as with the national researchweb.org .® Portable Digital System device . As explained previously [2). Apnea was defined as at least 90% cessation of airflow, and hypopnea as at least a 50% reduction in nasal pressure amplitude and/or in thoraco-abdominal movement for at least 10 s [2 exceeding 4% from the immediately preceding baseline was also recorded, and the oxygen desaturation index (ODI) was determined as the number of significant desaturations per hour.The HSAT was conducted with the Emblettaeviously , the HSAast 10 s . The totThe ESS questionnaire  was asse2 were categorized as obese, and abdominal obesity was defined as a waist-to-hip ratio (WHR) ≥ 0.9 for men and ≥0.8 for women [As previously described , the antor women .Blood samples were collected after an overnight fasting using ethylenediaminetetraacetic acid and serum tubes in the morning (07:00–08.00 am). Tubes were centrifuged and the plasma/serum samples were aliquoted and stored at −70 °C. TNF-α levels were analyzed in undiluted plasma samples using commercially available MILLIPLEX MAP (based on Luminex technology) human serum adipokine assay kits according to the manufacturer’s instructions . The minimum detectable concentration (assay sensitivity) for TNF-α was 0.14 pg/mL. The TNF-α concentration in all samples (undiluted) was within the standard curve, ranging from 0 to 10,000 pg/mL. As previously described , the intVenous blood samples were drawn into EDTA-containing tubes for DNA isolation. Genomic DNA was extracted from the whole blood using the PAXgene Blood DNA Kit . The quality and concentration of DNA samples were determined using a nano drop photometer  and DNA samples were stored at –80 °C until further use.TNF-α gene promoter region (-308G/A) was performed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). The DNA fragment harboring the TNF-α-308G/A SNP Rs1800629 was PCR-amplified using the forward primer 5′-AGGCAATAGGTTTTGAGGGCCAT-3′ and reverse 5′-TCCTCCCTGCTCCGATTCCG-3′ (product size 107 bp), as reported previously [The genotyping of SNP Rs1800629 in the eviously . The totThe amplicons were digested by 1 U of NcoI restriction enzyme  according to the manufacturer’s protocol. Restriction enzyme reactions were incubated at 37 °C for 4 h, and the enzyme was subsequently inactivated at 65 °C for 20 min. The expected fragment sizes after restriction enzyme digestion were 87 bp and 20 bp in the presence of the G allele (restriction site present) and 107 bp in the presence of the A allele (restriction site absent). The band pattern and intensity of all the produced bands were determined using the Fragment Analyzer 5200 (Agilent) by the DNF-905 dsDNA reagent kit , according to the manufacturer’s instructions see .To ensure the accuracy of the amplified PCR fragment as well as the produced RFLP genotypes, PCR products of 16 randomly selected samples were subjected to Sanger sequencing (Eurofin Genomics) . The obtTNF-α-308G/A gene polymorphism were tested by the Kruskal–Wallis test for continuous variables and by the Chi-square test for the categorical data. The Hardy–Weinberg equilibrium was tested by using the formula (p2 + 2pq + q2 = 1) for genotype distribution across all CAD subgroups. A univariate linear regression analysis was performed to test the association between circulating TNF-α levels and age, sex, ESS, BMI, WHR, AHI, ODI, OSA and comorbidities as well as TNF-α genotypes  and TNF-α alleles , respectively. Two distinct multivariate linear regression models were conducted to associate circulating TNF-α levels with the variable TNF-α genotypes and TNF-α alleles. Both multivariate models included the same significant covariates based on the univariate analysis as well as the variables age, BMI and sex in order to adhere to the recent guideline [TNF-α alleles were entered into the multivariate model. All statistical tests were two-sided, odds ratios (ORs) with 95% confidence interval (CI) were reported, and a p-value < 0.05 was considered significant. Statistical analyses were performed using SPSS® 26.0 for Windows® .For descriptive statistics, variables were reported as a median with interquartile ranges (IQRs) for continuous variables, and as a percentage for categorical variables. The Shapiro–Wilk test was used to test the normality assumption of the current data for all variables. The baseline differences between the three groups stratified by the uideline . MoreoveThe study population consisted of 326 participants . The comparison of the amplified PCR fragments with RFLP genotyping and with Sanger sequencing in 16 random samples showed consistent results.n = 186), were slightly younger (p = 0.039), had a higher proportion of current smokers (p = 0.042) and lung disease (p = 0.041) and were slightly less frequent users of statins (p = 0.028) at baseline compared to the patients who participated in the current study. Gender, BMI, obesity, OSA, AHI, ODI, ESS score and EDS as well as circulating TNF-α levels were similar . In total, the genotype distribution of the study population across the subgroups did not deviate from the Hardy–Weinberg equilibrium.As illustrated in y cohort B. The TNIn the entire study population, the median values of the circulating TNF-α levels were higher among CAD patients with OSA (5.05 pg/mL (IQR 3.43–6.94)) compared to 4.62 pg/mL (IQR 3.06–6.31) in CAD patients with no-OSA, but the difference was not statistically significant. The corresponding values were 5.05 pg/mL (IQR 3.23–6.93) in the sleepy OSA phenotype compared to 5.15 pg/mL (IQR 3.67–6.99) in the non-sleepy OSA phenotype (n.s).TNF-α genotypes from GG to GA and GA to AA as well as the TNF-α-308A allele were significantly associated with the circulating TNF-α levels  independent of age, sex, obesity, OSA severity and the circulating TNF-α levels in the multivariate model , where the -308A allele occurred in 28% of OSA subjects compared to 18% in the healthy controls [n = 207), where the -308A allele frequency in the OSA cohort was 28%, and the frequency was 13% in the control group [n = 179) [n = 111) [TNF-α-308G/A polymorphism has been shown to be significantly associated with OSA [To the best of our knowledge, this is the first study addressing the association of controls . Similarol group . Neutraln = 179) , and 13%n = 111) . Howeverwith OSA ,36.TNF-α-308A allele was found among 23% of the patients with OSA vs. 27% in the no-OSA group. One explanation for the higher -308A allele frequency in the no-OSA group in our study might be the confounding effect of other comorbidities such as obesity, hypertension and diabetes mellitus and/or CAD per se, given that those individuals without OSA were not healthy controls. Similar to OSA, CAD is an inflammatory condition mediated by the activity of pro-inflammatory cytokines including TNF-α [In the current cohort, the ng TNF-α .TNF-α gene polymorphism in CAD pathogenesis has been extensively investigated with inconsistent results. Some researchers have reported that TNF-α-308G/A polymorphism is implicated in CAD development [TNF-α-308G/A polymorphism with the development of CAD according to a recent meta-analysis [The role of elopment ,38, wherelopment . It is sanalysis .Circulating TNF-α levels have been postulated to be elevated in individuals with OSA compared to controls ,40. In aAs mentioned above, circulating levels of inflammatory markers predict future cardiovascular events in general and cardiac populations ,9. It haTNF-α-308A allele is suggested to promote a two-fold increase in TNF-α transcription activity [TNF-α-308 genotypes from GG to GA and GA to AA. TNF-α is a mediator in the sleep regulatory system, and the fragmented sleep pattern associated with OSA is believed to increase the circulatory levels of TNF-α [TNF-α-308A allele on the circulating TNF-α levels as well as its protective role against EDS in patients with CAD and concomitant OSA. Moreover, interactions of several SNPs in the promoter region of TNF-α may collectively affect the expression level, and thus the subjects in the present cohort could be carriers of SNPs that also affect the gene activity, which were not covered by the current study. It is worthy of note that there are also other recent genetic studies that have found polymorphisms in the IL-18 receptor gene region, which was associated with SpO2 at the genome-wide significance level [The activity . We founof TNF-α ,40. Thusce level .Given that only 326 of 511 participants in the original clinical trial were included in the current study, one may argue that the exclusion of participants might have led to biased estimates. However, the baseline characteristics of the patients that were not included in the current study did not differ from the included patients except for slight variations in age, current smoking, lung disease and statin use at baseline, which probably do not have any clinical importance in the context of the outcomes of the current study. In the literature, there have been reports suggesting that statins may influence sleep quality , and theThe current study has certain limitations. Firstly, the power estimate for the entire RICCADSA cohort was conducted for the primary outcome and not for the secondary outcomes assessed in this study for the comparison of genotypes and alleles across the subgroups. However, the total number of the participants was comparable with the number of participants reported in the aforementioned studies. Secondly, EDS was categorized based on the ESS threshold, which may not be precise in a CAD population, and the clinical reproducibility of ESS has been shown to be variable when administered sequentially . NeverthTNF-α-308G/A gene polymorphism and OSA severity in terms of ODI were independently associated with circulating TNF-α levels in the current CAD cohort. The TNF-α-308A allele appears to modulate circulatory TNF-α levels and mitigate EDS independent of age, sex, obesity and OSA severity in adults with CAD.In brief,


Abnormal aggregation of the microtubule-associated protein Tau is closely associated with tauopathies, including Alzheimer’s disease and chronic traumatic encephalopathy. The hexapeptide 275VQIINK280 (PHF6*), a fibril-nucleating core motif of Tau, has been shown to play a vital role in the aggregation of Tau. Mounting experiment evidence demonstrated the acetylation of a single-lysine residue K280 in the PHF6* was a critical event for the formation of pathological Tau amyloid deposits. However, the underlying mechanisms by which K280 acetylation affects Tau aggregation at the atomic level remain elusive. In this work, we performed replica exchange molecular dynamics simulations to investigate the influence of acetylation of K280 on the aggregation of PHF6*. Our simulations show that acetylation of K280 not only enhances the self-assembly capability of PHF6* peptides but also increases the β-sheet structure propensity of the PHF6*. The inter-molecular interactions among PHF6* peptides are strengthened by the acetylation of K280, resulting in an increased ordered β-sheet-rich conformations of the PHF6* assemblies along with a decrease of the structural diversity. The residue-pairwise contact frequency analysis shows that K280 acetylation increases the interactions among the hydrophobic chemical groups from PHF6* peptides, which promotes the aggregation of PHF6*. This study offers mechanistic insights into the effects of acetylation on the aggregation of PHF6*, which will be helpful for an in-depth understanding of the relationship between acetylation and Tau aggregation at the molecular level. Tau, a microtubule-associated protein first discovered in 1975, was found to be critical for the assembly and stabilization of microtubules . Recent The physiological functions of Tau are regulated by various post-translational modifications (PTMs), including phosphorylation, acetylation, glycation, nitration, O-GlcNAcylation, oxidation, ubiquitination, SUMOylation and methylation. Approximately 35% of the amino acid residues in Tau are susceptible to modification post-translationally . Many PTin vitro and in vivo studies have showed the close relationship between abnormal Tau phosphorylation and aggregation  of the repeat region. Acetylated K280 is detected in neuronal and glial inclusions in many tauopathies , but not in normal brains . Acetylace model . The resce model , while tce model . Therefoce model . Kim et ce model . Despitece model . PHF6* o275VQIINK280. To investigate the effect of acetylation at residue K280 on the conformational ensemble of PHF6* oligomers, we simulated two different systems: the wild type PHF6* system (named PHF6* system) and the K280 acetylated system (named Ac-PHF6* system). Each system consists of twelve peptide chains with random coil conformation for each chain, which are the final conformations generated in a 50 ns molecular dynamics (MD) simulation at 450K in water starting from fully extended peptides. To eliminate the effects of terminal charges, the peptides in two systems were capped by the ACE (CH3CO) group at the N-terminus and the NH2 group at the C-terminus, as done experimentally and computationally by Levine et al. , where H  is the histogram of two selected reaction coordinates, x and y. Here, x and y refer to the number of H-bonds and radius of gyration (Rg) of PHF6* oligomers, respectively. A contact is defined when two carbon atoms or a carbon atom and another heavy atom of two non-sequential residues lie within 0.54 nm, or any other two heavy atoms of two non-sequential residues come within 0.46 nm . The secondary structures of peptides were identified using the dictionary secondary structure of protein (DSSP) program , and the 0.46 nm .Before analyzing the simulation data, the convergence of the two REMD simulations was examined by comparing the following several parameters within two different time intervals (300–350 and 350–400 ns) for the PHF6* and Ac-PHF6* systems at 310 K. These parameters include the average probability of secondary structure  over all residues, secondary structure (coil and β-sheet) probability of each residue, the probability density function (PDF) of the number of H-bonds and Rg of PHF6* oligomers, and the end-to-end distance of each peptide chain in the oligomers. As shown in 16-22  over all residues, and the residue-based β-sheet and coil probability in two systems. As shown in 16-22 , Aβ30-362  can form closed or open β-barrel structures. In order to examine the effects of acetylation on the formation of barrel-like structures, we calculated the probability of different sizes of β-barrel in PHF6* and Ac-PHF6* systems. As shown in After comparing the secondary structure difference between two systems, we further performed an RMSD-based cluster analysis using a Cα-RMSD cutoff of 0.45 nm to investigate the three-dimensional (3D) conformations of PHF6* oligomers in two systems. The conformations in the PHF6* and Ac-PHF6* systems are separated into 493 and 119 clusters, respectively. The larger cluster number in PHF6* system reflects that the structural diversity of PHF6* oligomers in PHF6* system is more pronounced than that in Ac-PHF6* system. The top nine most-populated clusters and their populations are shown in To explore the influence of acetylation on the whole conformational space of the PHF6* oligomers, we plotted the PMF (or free energy surface) as a function of two reaction coordinates, the number of H-bonds and Rg of PHF6* oligomers in two systems. The locations of the first nine clusters are labeled on the PMF plot. As seen in We also calculated the coil and β-sheet probabilities as a function of cluster index to investigate the dominant secondary structure probability of the most-populated conformations. As seen in 25-35 (It is interesting to monitor the extent of solvation of the peptide and look at the role of water in the REMD simulations . To this25-35 .In order to explore the underlying mechanism by which K280 acetylation has a higher probability to form β-sheets, we calculated the PDF of the number of inter-chain H-bonds in two systems. As seen in 192-212, and found the oxygen atoms on the phosphate group are involved in the formation of multiple key H-bonds that making the structure more stable (We then plotted residue-residue H-bonds formation maps in the PHF6* and Ac-PHF6* systems to further investigate the effect of K280 acetylation on the H-bond interactions. As shown in e stable . In addiTo explore the effect of K280 acetylation on pairwise residue-residue interactions of PHF6* oligomers, we plotted the MC-SC and SC-SC contact number maps in the PHF6* and Ac-PHF6* systems. As seen in In summary, we have investigated the self-assembly of the fibril-nucleating core motif PHF6* of Tau protein and the effect of K280 acetylation on PHF6* oligomerization, by performing two 400 ns atomistic REMD simulations starting from a random state. Our simulations show that K280 acetylation markedly increases the β-sheet probability of PHF6* and enhances the formation of larger β-sheet-rich oligomers. The conformations in the PHF6* system display a high diversity, while K280 acetylation reduces the structural diversity and self-assemblies into some ordered β-barrels and bilayer β-sheets. The analyses of SASA and end-to-end distance show that after K280 acetylation, PHF6* peptides assemble into a more compact state and have a larger propensity to adopt extended β-sheet conformations. Moreover, we found that the acetylation of K280 significantly alters the H-bond and contact network and strengthens the residue-residue interactions, especially at the acetylation site. Overall, this study reveals the mechanisms that K280 acetylation affects Tau aggregation at the atomic level, and may provide valuable information for developing a PTM-modifying therapeutic strategy for tauopathies.


Background: About a third of patients with inflammatory bowel disease do not respond to anti-tumour necrosis factor (anti-TNF) therapy, which is challenging. Aim: To review the current data on the two main strategies when facing primary non-response to an anti-TNF agent in inflammatory bowel disease: changing to a second anti-TNF (switching) or to a drug with another mechanisms of action (swapping). Methods: We performed a bibliographic search to identify studies reporting on efficacy of any biologic treatment after primary anti-TNF non-response. Results: The efficacy of a second anti-TNF is lower when the reason to withdraw the first one is primary failure. Nevertheless, switching to another anti-TNF even after primary failure may still be effective in some patients. Both vedolizumab and ustekinumab have generally been shown to be less effective in anti-TNF exposed patients. However, despite primary anti-TNF failure, patients may respond to vedolizumab or ustekinumab in a limited but considerable number of cases. The cause for swapping (primary vs. secondary anti-TNF failure) seems to have limited effect on vedolizumab efficacy. Primary anti-TNF non-response seems to be a clearer predictor of treatment failure for ustekinumab. Unfortunately, the two main strategies to treat specifically a patient with primary non-response to an anti-TNF agent—switching to a second anti-TNF or swapping for vedolizumab/ustekinumab—have not been properly compared. Conclusion: The data reviewed in the present study clearly emphasise the imperative need to carry out head-to-head randomised trials in patients exposed to anti-TNF agents in general, and specifically in those with primary non-response to these agents. Inflammatory bowel diseases (IBD)—ulcerative colitis (UC) and Crohn’s disease (CD)—are chronic idiopathic inflammatory diseases affecting the gastrointestinal tract. Anti-tumour necrosis factor (anti-TNF) agents have revolutionised the treatment of IBD. Their use avoids the need for steroid therapy, promotes mucosal healing, reduces hospitalisations and surgeries, and therefore, dramatically improves the quality of life of IBD patients .However, approximately one-third of patients do not respond to anti-TNF induction therapy (known as primary non-responders) ,3,4. In In patients who lose their response to an anti-TNF drug, it is well established that a switch to another anti-TNF agent in an attempt to regain remission is an accepted option ,8. To daSome studies have shown that previous anti-TNF therapy is associated with heightened probability of primary treatment failure of subsequent anti-TNF therapy . PatientFor many years, anti-TNF agents were the only type of biologics available for IBD treatment. However, new drugs that target different inflammatory pathways have been approved for IBD in recent years: two biologics  or to a drug with another mechanism of action such as vedolizumab, ustekinumab or tofacitinib (swapping).A bibliographic search was designed to identify studies reporting on efficacy of any of these regimens with biologics or small molecules after primary non-response to an anti-TNF agent. An electronic search was performed in PubMed up to April 2021 using the following algorithm: (“inflammatory bowel disease” OR “ulcerative colitis” OR Crohn) AND  AND (vedolizumab OR ustekinumab OR tofacitinib). In addition, a complementary electronic search with the following algorithm was used: (“inflammatory bowel disease” OR “ulcerative colitis” OR Crohn) AND  AND (vedolizumab OR ustekinumab OR tofacitinib) AND . In addition, the reference lists from the selected articles were reviewed to identify additional studies of potential interest. Only studies conducted in humans were included, whereas animal models were excluded. Available data from both clinical trials and “real life” studies were included. Pharmacokinetic studies  were excluded. Studies evaluating the response in patients with post-operative recurrence of CD were also excluded. Articles published in any language were included.The mechanism underlying primary non-response to an anti-TNF drug and subsequent response to another anti-TNF drug is unknown. In view of the similar structure and function shared by infliximab and adalimumab (or any other anti-TNF agent), one might predict that patients who do not have a primary response to one of these agents will not respond to the other. Thus, the lack of efficacy of the anti-TNF agent in these patients could be due to specific disease characteristics in which TNF does not play a pivotal role , suggesting that other pathways could be implicated in the pathogenesis of the disease. In fact, in a patient with primary non-response to one medication, the routine clinical practice is to move out of that mechanism of action.However, in some studies, a considerable proportion of primary non-responders to infliximab had a sustained clinical benefit with adalimumab (or vice versa), indicating that the primary failure to a biologic anti-TNF agent does not necessarily translate across the class of therapy. It has been suggested that differences in efficacy between anti-TNF agents may be due to differences in pharmacokinetic properties or tissue penetration .Only a few studies with small sample size have analysed the effectiveness of the sequential use of a second anti-TNF if the reason for withdrawal of the first drug was primary failure. The rates of remission in these studies were highly variable, ranging from 0% to more than 50%. Studies evaluating the efficacy of a second anti-TNF agent after primary failure with a first one, are summarised in The efficacy of a second anti-TNF agent after secondary—instead of primary—failure with a first one, considering these same studies (to avoid possible biases as much as possible), is also summarised in The aforementioned results are in agreement with a previous meta-analysis, which concluded that the efficacy of a second anti-TNF in CD patients largely depends on the cause for switching: the remission rate was higher when the reason to withdraw the first anti-TNF was intolerance (61%), compared with secondary (45%) or primary failure (30%) .In summary, the efficacy of a second anti-TNF depends on the cause for switching, being lower when the reason to withdraw the first anti-TNF is primary failure.Two large studies, the CHOICE trial  and the The first study specifically designed to evaluate the effectiveness of a second anti-TNF agent after primary failure of the first one (including CD patients who did not achieve remission with the first agent) was recently published . The firThe largest study to date (including 1122 IBD patients) in which the strategy of switching to a second anti-TNF after intolerance, primary failure, or secondary failure to the first one has been evaluated, has recently been published . PrimaryIn summary, some patients  may still benefit from switching to another anti-TNF agent even if the reason to withdraw the first anti-TNF was primary non-response, suggesting that primary failure to anti-TNF therapy may not necessarily be a class-effect phenomenon.Probably due to historical reasons , the experience with the switch from a subcutaneous (sc) agent to infliximab is more limited. It has been hypothesised that intravenous (iv), weight-based TNF dosing could offer advantages over sc, fixed-dose TNFs and may be effective despite primary non-response to previous sc fixed-dose TNFs. Viola et al. investigated IBD outcome after a switch from sc to the iv agents, and showed that patients who switched due to loss of response did numerically, but not statistically, better than patients who switched due to primary failure . In anotInterestingly, it has been shown that several patients who experienced primary failure with a second anti-TNF finally responded to a third one. This suggests that these agents , which have different structural, pharmacokinetic and pharmacological properties, may also differ in their modes of action . A recenOne step further, a small case series suggests that even a fourth anti-TNF drug may have some clinical potential in refractory CD patients with prior triple anti-TNF agent failure . Russi eFinally, the benefit of anti-TNF (infliximab) reintroduction after successive failure of infliximab and adalimumab has recently been assessed . PatientSome studies in IBD patients have shown that previous anti-TNF therapy is a risk factor for failure of treatment with vedolizumab ,54,55,56Patients with anti-TNF-failure represent a highly treatment-experienced population with clinical features that could explain, at least in part, the lower/slower inductive efficacy. For example, compared with TNF-naïve patients, those with prior TNF antagonist treatment failure had, in most of the studies, longer duration of disease and more complicated disease, including prior surgery and histories of fistulas and extraintestinal manifestations.Two post hoc analyses from the GEMINI studies reported efficacy of vedolizumab in UC and CD patients based on prior anti-TNF history ,54. In CFurthermore, the response to vedolizumab seems to be faster in the anti-TNF-naïve population . In UC, Recently, a clinical prediction model and tool for vedolizumab therapy in CD was developed with GEMINI 2 participants and then validated in the VICTORY cohort. One of the predictors included in this tool was the absence of prior exposure to anti-TNF. This model suggested that the ideal positioning of vedolizumab in CD patients should be prior to anti-TNF exposure . However, these results are similar for anti-TNF users, where response rates are also better in patients naïve to biologic agents . FinallyIn summary, vedolizumab has generally been shown to be more effective in anti-TNF-naïve than in anti-TNF exposed patients.The information from the pivotal studies on the efficacy of vedolizumab, specifically in patients with primary anti-TNF failure, is quite limited, and is summarised in In summary, despite primary non-response to an anti-TNF drug, IBD patients may respond to vedolizumab in a considerable number of cases.A recent meta-analysis showed no difference in treatment response to vedolizumab for both anti-TNF primary and secondary non-responders . The risIn summary, the cause for swapping (primary vs. secondary anti-TNF failure) seems to have limited effect on the efficacy of vedolizumab treatment. However, a lower efficacy of vedolizumab in patients with primary anti-TNF failure cannot be ruled out.Several studies have compared anti-TNF and vedolizumab in anti-TNF experienced patients, although most of them do not provide specific data regarding the type of failure of the anti-TNF treatment . These studies are summarised below.The first study directly comparing an anti-TNF drug and vedolizumab in a head-to-head approach, which was not a randomised trial, was performed by Allamneni et al. in 2018  and showMevius et al.  showed tp = 0.002 for vedolizumab vs. adalimumab, p = 0.001 for vedolizumab vs. golimumab). Cox survival analysis demonstrated that patients treated with vedolizumab had reduced probability of treatment discontinuation compared to those treated with adalimumab and golimumab. Macaluso et al.  comparedIn the VARSITY study, a Phase 3, double-blind, randomised, trial conducted at 245 centres in 34 countries, vedolizumab and adalimumab were compared in patients with UC . The genMacaluso et al.  performeHelwig et al.  evaluateHupé et al.  retrospeRundquist et al.  comparedBohm et al.  retrospeIn summary, most of the studies that have compared anti-TNF and vedolizumab treatment in anti-TNF experienced patients (without considering the type of failure to the anti-TNF treatment), have suggested better results with vedolizumab than with a second anti-TNF drug. However, this advantage of vedolizumab could not be confirmed in a few studies.Studies comparing second-line anti-TNF agents and vedolizumab specifically in patients with primary failure to anti-TNF treatment are exceptional and are summarised below.Papamichael et al.  studied More recently, Favale et al.  retrospeIn summary, studies comparing second-line anti-TNF agents and vedolizumab specifically in patients with primary failure to anti-TNF treatment are exceptional, and the number of patients included is very low. Therefore, no conclusion can be drawn about the best strategy (switch or swap) in the specific scenario of anti-TNF primary non-responders.Some studies, including Phase 3 clinical trials , have shown that previous anti-TNF therapy is a risk factor for treatment failure with ustekinumab ,82 whereIn summary, ustekinumab, as any other biologic, probably achieves a higher efficacy in anti-TNF-naïve than in anti-TNF exposed patients. However, as the experience of ustekinumab in anti-TNF-naïve patients is still quite limited, this conclusion should be interpreted with caution.The information from the pivotal studies on the efficacy of ustekinumab, specifically in patients with primary anti-TNF failure, is quite limited, and is summarised in Schmitt et al.  characteIn summary, as it was the case with vedolizumab, despite primary non-response to an anti-TNF drug, IBD patients may respond to ustekinumab in a limited but considerable number of cases.In a recent meta-analysis, treatment response to a second biologic, particularly with ustekinumab, was shown to be poorer in anti-TNF therapy primary non-responders, in comparison with secondary non-responders .In another meta-analysis, where only two randomised controlled trials (CERTIFI and UNITI-1) were included, clinical response was significantly higher for patients who received ustekinumab compared with those who received placebo in secondary non-responders, but in the case of primary non-responders this benefit could not be confirmed . The resMore recently, Singh et al.  analysedIn summary, anti-TNF primary non-response seems to be a predictor of ustekinumab failure, unlike loss of response.Studies comparing second-line anti-TNF agents and ustekinumab specifically in patients with primary failure to anti-TNF treatment are lacking. However, several studies have compared anti-TNF and ustekinumab treatment in anti-TNF experienced patients without considering the type of failure to the anti-TNF treatment . These studies are summarised below.Ahmed et al.  providedMore recently, Cerpa Arencibia et al.  evaluateIn summary, studies comparing second-line anti-TNF agents and ustekinumab specifically in patients with primary failure to anti-TNF treatment are lacking. The very few studies that have compared anti-TNF and ustekinumab treatment in anti-TNF experienced patients in general (without considering the type of anti-TNF failure) suggest better results with ustekinumab than with a second anti-TNF drug.Randomised controlled trials directly comparing vedolizumab and ustekinumab are currently not available. Most studies indirectly comparing these two drugs in anti-TNF experienced patients do not provide effectiveness results according to the type of failure to the anti-TNF treatment . These studies are summarised below.Biemans et al.  comparedAlric et al.  comparedTownsend et al.  comparedManlay et al.  comparedFinally, several meta-analyses have compared the efficacy of different biologic agents for the treatment of IBD. Their general results, and their data in patients with anti-TNF failure, are summarised in Pagnini et al.  analysedKawalec et al.  performe(a)The number of patients included in each study is relatively small.(b)The studies are mostly non-randomised, and many of them even retrospective.(c)The results of the studies are difficult to compare due to differences in patient populations, methodology and statistical analysis. For example, initial trials of infliximab comprised anti-TNF naïve patients, while significant proportions of patients included in trials for recent anti-TNF (or other biologics) had prior anti-TNF exposure.(d)The results of some studies are difficult to interpret, since only responders to induction therapy were enrolled in the maintenance studies, which were supplemented with responders from open-label cohorts of which no outcomes are presented.(e)Even though most of the observational studies were adjusted for potential confounding factors performing a propensity score matching, the validity of this analysis relies on the untestable assumption that all confounders were accounted for.(f)Most of the studies do not include endoscopic follow-up and therefore data on mucosal healing is not available; thus, only clinical effectiveness is generally assessed.(g)The duration of follow-up is limited; ideally, studies with longer follow-up duration are required to confirm outcomes over a prolonged period.(h)Finally, most studies indirectly comparing vedolizumab and ustekinumab in anti-TNF experienced patients do not provide effectiveness results according to the type of failure to the anti-TNF treatment  .In summary, some data, mainly from real-life, indirectly comparing vedolizumab and ustekinumab in CD patients with prior failure to anti-TNF agents, suggest that ustekinumab could be more effective than vedolizumab. However, the following potential limitations , should be taken into consideration when interpreting these indirect comparisons:Approximately one-third of IBD patients do not respond to anti-TNF therapy, which represents a challenging scenario. In the last few years, novel molecules have become available with mechanisms of action different from anti-TNF blockade, and many more are tested in the therapeutic pipeline. Consequently, therapeutic options for anti-TNF-experienced IBD patients are constantly expanding, creating new opportunities for the management of difficult-to-treat patients. Identifying patients who are most likely to benefit from specific agents is of paramount importance to help best positioning IBD therapies. In the present study, we have reviewed, for the first time, current data on the two main strategies to treat a patient with primary non-response to an anti-TNF agent: switching to a second anti-TNF or swapping for a drug with another mechanisms of action (mainly vedolizumab and ustekinumab). The conclusions of the present review are summarized in The efficacy of a second anti-TNF seems to depend on the cause for switching, being lower when the reason to withdraw the first anti-TNF is primary failure. Nevertheless, switching to another anti-TNF even after primary failure may still be a valid therapeutic option in some patients, suggesting that primary non-response to anti-TNF therapy may not be necessarily a class-effect phenomenon. In any case, patients who have failed treatment with anti-TNF agents are probably a more refractory group, and this seems to be valid not only for second-line anti-TNF therapy but also for any other rescue biologic agent. Thus, both vedolizumab and ustekinumab have generally been shown to be less effective in anti-TNF exposed patients, although the experience of these biologics in anti-TNF-naïve patients  is still quite limited. Nevertheless, despite primary non-response to an anti-TNF drug, patients may respond to vedolizumab or ustekinumab in a limited but considerable number of cases. The cause for swapping (primary vs. secondary anti-TNF failure) seems to have limited effect on the efficacy of vedolizumab treatment, although a lower efficacy of this biologic in patients with primary anti-TNF failure cannot be ruled out. In the case of ustekinumab, anti-TNF primary non-response seems to be a clearer predictor of treatment failure (compared with those patients with loss of response to anti-TNF therapy).Unfortunately, the two main strategies to treat specifically a patient with primary non-response to an anti-TNF agent—switching to a second anti-TNF or swapping for vedolizumab or ustekinumab—which constitutes the main issue raised in the present article, have not been properly compared . Therefore, no conclusion can be drawn about the best strategy (switch or swap) in the specific scenario of anti-TNF primary non-responders.Finally, limited data, mainly coming from real-life, indirectly comparing vedolizumab and ustekinumab in CD patients with prior failure to anti-TNF, suggest that ustekinumab could be more effective than vedolizumab. However, these studies  suffer from relevant methodological limitations, and do not provide enough information according to the type of failure to the anti-TNF treatment .In summary, the data reviewed in the present study clearly emphasise the imperative need to carry out head-to-head randomised trials in patients exposed to anti-TNF agents in general, and specifically in those with primary non-response to these agents. These warranted future randomised studies should directly compare the two main strategies to specifically treat a patient with primary non-response to an anti-TNF agent: switching to a second anti-TNF or swapping for a drug with another mechanisms of action. Additionally, these desirable studies should also compare vedolizumab vs. ustekinumab (and vs. tofacitinib) in the setting of anti-TNF primary failure. Only in this way we can obtain reliable information to inform clinicians regarding the optimal positioning of biologic therapies in IBD.


Enfortumab vedotin is a first-in-class Nectin-4-directed antibody-drug conjugate approved by the US Food and Drug Administration for the treatment of patients with locally advanced or metastatic urothelial cancer (la/mUC) previously treated with a platinum-based chemotherapy and a programmed death receptor-1/programmed death-ligand 1 (PD-1/L1) inhibitor, or patients with la/mUC who are ineligible for cisplatin-based chemotherapy and have previously received one or more prior lines of therapy. Enfortumab vedotin is the only drug to have demonstrated survival benefit versus chemotherapy in a randomized controlled trial in patients with la/mUC previously treated with platinum-based chemotherapy and a PD-1/L1 inhibitor. The development of dermatologic events following the administration of enfortumab vedotin is anticipated given the expression of Nectin-4 in epidermal keratinocytes and skin appendages . There is the potential for rare but severe and possibly fatal cutaneous adverse reactions, including Stevens-Johnson syndrome and toxic epidermal necrosis, as described in the boxed warning of the US prescribing information for enfortumab vedotin. This manuscript describes the presumed pathophysiology and manifestations of dermatologic reactions related to enfortumab vedotin, and presents recommendations for prevention and treatment, to provide oncologists and other healthcare providers with an awareness of these potential adverse events to best anticipate and manage them. Enfortumab vedotin is a Nectin-4-directed antibody-drug conjugate approved by the US FDA for the treatment of patients with locally advanced or metastatic urothelial cancer. This article describes dermatologic reactions related to enfortumab vedotin and presents prevention and treatment recommendations. There is a need for efficacious and tolerable treatments for patients with locally advanced/metastatic urothelial carcinoma (la/mUC). Enfortumab vedotin is a first-in-class Nectin-4-directed antibody-drug conjugate for the treatment of la/mUC that has shown an overall survival benefit over standard-of-care chemotherapy. As Nectin-4 is expressed in epidermal keratinocytes and skin appendages, dermatologic events are common, anticipated treatment-related adverse events. Understanding the features of treatment-related dermatologic events and the prevention and management protocols should equip the healthcare team to educate, monitor, and manage patients who develop these reactions to minimize toxicity, maximize clinical benefit, and maintain quality of life.1 demonstrating an unmet need for efficacious and tolerable treatments for patients with la/mUC. Only recently have developments such as immunotherapies and targeted therapies expanded treatment options and improved outcomes in patients with la/mUC. Dermatologic events are frequent, anticipated adverse events (AEs) associated with immunotherapies and targeted therapies, and require timely and appropriate management to maintain quality of life and continuation of therapy.2-6Locally advanced or metastatic urothelial carcinoma (la/mUC) is an aggressive disease associated with a poor prognosis. The historical 5-year survival rate for metastatic disease in the US is 6.4%,7-10 Nectins are thought to mediate cell-cell adhesions at adherens junctions, and to regulate several cellular activities, including cell motility, differentiation, polarization, proliferation, and survival.12 Nectin-4 is also highly expressed in urothelial as well as breast, ovarian, gastric, pancreatic, head/neck, esophageal, and lung carcinomas,13-17 and overexpression is associated with disease progression or poor survival in multiple tumor types,15-22 thus making Nectin-4 a molecular target with therapeutic potential.One molecular target expressed in la/mUC is Nectin-4, a member of the family of immunoglobulin (Ig)-like adhesion molecules normally expressed in human epidermal keratinocytes and skin appendages , as well as in the transitional epithelium of the bladder, salivary gland ducts, esophagus, breast, and stomach .7-10 Nec23 In the US, enfortumab vedotin is indicated for the treatment of patients with la/mUC previously treated with a platinum-based chemotherapy and a programmed death receptor-1/programmed death-ligand 1 (PD-1/L1) inhibitor, or patients with la/mUC ineligible for cisplatin-based chemotherapy who have previously received one or more prior lines of therapy.24 These indications are based on 2 global clinical trials evaluating enfortumab vedotin in patients with la/mUC: EV-201 (NCT03219333), a phase II, single-arm in patients previously treated with a PD-1/L1 inhibitor, and a platinum-based chemotherapy (Cohort 1) or were ineligible for cisplatin (Cohort 2),13 and EV-301 (NCT03474107), a randomized, open-label, phase III trial in patients with la/mUC previously treated with platinum-based chemotherapy and a PD-1/L1 inhibitor.25 In EV-301, enfortumab vedotin monotherapy versus chemotherapy (single-agent taxane [docetaxel or paclitaxel] or vinflunine) significantly improved overall survival .25 The incidence of all-grade and grade 3 or higher treatment-related AEs was similar in the 2 treatment groups. The most common grade 3 or higher treatment-related AEs in the enfortumab vedotin arm included maculopapular rash, fatigue, and decreased neutrophil count compared with decreased neutrophil/white-cell count, anemia, and febrile neutropenia in the chemotherapy arm. Additionally, prespecified analyses of AEs of special interest (AESI) using composite categories of related AEs included any skin reaction (representing a set of dermatologic AEs), peripheral neuropathy, and hyperglycemia. In EV-301, all-grade treatment-related skin reactions, inclusive of all dermatologic AEs, occurred in 47% of patients who received enfortumab vedotin versus 15.8% of patients in the chemotherapy group. Skin reaction was the most common treatment-related AESI of grade 3 or higher in the enfortumab vedotin group, occurring in 14.5% of patients as compared to 0.7% in the chemotherapy group.25 Across enfortumab vedotin clinical trials, rare events of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) occurred and are included in the boxed warning of the US prescribing information for enfortumab vedotin; these events are described in detail later.Enfortumab vedotin is a first-in-class antibody-drug conjugate (ADC) consisting of a Nectin-4-directed, fully human IgG1 monoclonal antibody conjugated to monomethyl auristatin E (MMAE), a microtubule-disrupting agent, via a protease-cleavable linker.In this manuscript, we discuss the putative pathophysiology of enfortumab vedotin-related dermatologic events, varying clinical presentation, and potential predisposing risk factors. In addition, we describe recommendations for the prevention, monitoring, diagnosis, and management of these events, including when referral for specialized care is indicated.24 Preclinical data suggest that in addition to direct cytotoxicity, enfortumab vedotin may have additional mechanisms of action including the bystander effect, which occurs when intracellular MMAE released from enfortumab vedotin diffuses across cell membranes and causes apoptosis in adjacent tumor cells.26 The mechanism by which enfortumab vedotin causes dermatologic events is presumed to be through the delivery of MMAE into Nectin-4-expressing normal tissue, such as the epidermis and epithelium of sweat glands and hair follicles , morphology, symptomatology, and severity, with onset generally within the first cycle of treatment .13,24,2533 Such therapies, including enfortumab vedotin, may cause rare but potentially life-threatening skin reactions collectively known as severe cutaneous adverse reactions. Severe cutaneous adverse reactions are a subset of skin reactions that are predominantly caused by drugs and include SJS, TEN, symmetrical drug-related intertriginous and flexural exanthema (SDRIFE), drug reaction with eosinophilia and systemic symptoms (DRESS)/drug-induced hypersensitivity syndrome, and acute generalized exanthematous pustulosis (AGEP). These reactions can present with both dermatologic and non-dermatologic symptoms . The different types of severe cutaneous adverse reactions vary in terms of mortality rate, systemic involvement, and sequelae.34 For example, SDRIFE is distinct from other severe cutaneous adverse reactions due to its absence of systemic involvement. Although complications with SDRIFE are extremely rare given its self-limited course,34 more serious severe cutaneous adverse reactions must be ruled out to ensure proper management.35 SJS/TEN is associated with the highest mortality rate of all severe cutaneous adverse reactions.32 While the pathogenesis of severe cutaneous adverse reactions is not fully understood, they are thought to be Type IV delayed hypersensitivity reactions involving activation of CD8+ cytotoxic T cells and other immune cells, as well as secretion of cytokines and chemokines.36Dermatologic events commonly seen with enfortumab vedotin also occur with other targeted anticancer therapies as well as with immunotherapies.24In the integrated safety population of 680 patients across enfortumab vedotin monotherapy trials, 55% of patients who received treatment at the approved dose of 1.25 mg/kg experienced skin reactions (as an AESI composite term encompassing the broadest capture of cutaneous events). The most common manifestations of skin reactions were maculopapular rash (23%) and pruritus (33%). Grade ≥3 skin reactions occurred in 13% of patients and included maculopapular rash, rash erythematous, rash or drug eruption, SDRIFE, dermatitis bullous, dermatitis exfoliative, and palmar-plantar erythrodysesthesia syndrome.37 In these studies, severe cutaneous adverse reactions were reported as a composite term of dermatologic and non-dermatologic events because of potential ocular and mucosal involvement and included the terms “stomatitis,” “drug eruption,” “dermatitis bullous” or “blister,” “skin exfoliation,” “exfoliative rash,” “erythema multiforme,” “fixed eruption,” “pemphigus,” “toxic skin eruption,” “conjunctivitis,” and “mouth ulceration”. These events occurred in 20% of patients in EV-301.25 The incidence of enfortumab vedotin-related dermatologic events reported in the EV-301 clinical trial is summarized in In the enfortumab vedotin clinical trials, the median time to onset of the first observed skin reaction was within the first cycle.25 Skin reactions led to discontinuation of treatment in 2.6% of patients in the integrated safety population (n = 680). Among the 59 patients in the integrated safety population who experienced a skin reaction necessitating dose interruption and who then restarted enfortumab vedotin treatment, 24% who restarted at the same dose and 16% who restarted at a reduced dose experienced recurrent severe skin reactions. Treatment-emergent skin reactions that resulted in dose reductions in EV-301 occurred in 8% of patients , serious AEs of severe cutaneous adverse reaction were reported in 11 patients (1.5%) and included “dermatitis bullous” (0.4%), “drug eruption” (0.4%), “blister” (0.1%), “conjunctivitis” (0.1%), “SJS” (0.1%), “stomatitis” (0.1%), and “toxic skin eruption” (0.1%) (data on file).Severe cutaneous adverse reactions have also been reported in the post-marketing setting. From the US Food and Drug Administration (FDA) approval of enfortumab vedotin in December 2019 through October 2020, 15 patients receiving enfortumab vedotin experienced severe cutaneous adverse reactions, some of whom had more than one event and some of whom died (data on file). Given the complexity and incomplete information associated with voluntary post-marketing reporting, the number of fatal outcomes caused by a severe cutaneous adverse reaction cannot be accurately determined. While post-marketing safety data cannot accurately estimate the frequency of these events or establish a causal relationship to drug exposure, it can provide insight into the nature of enfortumab vedotin-related dermatologic events.38There are currently no established risk factors for developing dermatologic events while on enfortumab vedotin treatment. General characteristics that may predispose patients receiving targeted anticancer treatment or immunotherapies to experience dermatologic events include prior cutaneous reactions to previous lines of anticancer therapies, including immunotherapies that can cause delayed immune-related AEs. Other predisposing factors include prior personal or family history of a dermatologic condition, allergic reactions, or xerosis; skin damage due to solar or therapeutic radiation; advanced age; and renal and/or hepatic impairment.Routine skin assessments starting with the initiation of enfortumab vedotin are important since dermatologic events typically occur within the first cycle, and frequent and thorough follow-up is key as the appearance of dermatologic events often evolves over time. Enfortumab vedotin-related dermatologic events tend to spare the face and are more typically seen in the intertriginous, flexural, acral, and truncal areas. The common progression observed clinically is erythema that then fades or darkens, often accompanied by desquamation as the reaction resolves. The patient and their caretakers should be informed that rashes and severe skin reactions, as well as ocular and mucosal symptoms, have occurred after administration of enfortumab vedotin, and that they should notify the treating clinician immediately of any new or worsening dermatologic events.32 Careful examination for mucosal involvement is important, as >90% of patients with SJS/TEN experience involvement of the oral, ocular, or genital mucosa.39 The appearance of these symptoms should be an indication for immediate evaluation and intervention, as early recognition and drug discontinuation are associated with better prognosis.32 Evaluation may include biopsy(ies) and close follow-up to assess the progression and evolution of the dermatologic event over time. A histological analysis demonstrating widespread full-thickness epidermal necrosis and detachment is suggestive of SJS/TEN and should prompt urgent referral for specialized care to confirm the diagnosis. It is critical that histologic findings are interpreted in conjunction with the full clinical picture and a thorough history. Major complications from secondary infections such as sepsis and cellulitis, long-term effects involving the skin and eyes, and the significant acute morbidity and mortality associated with SJS/TEN are further reasons why early diagnosis of these diseases is crucial.40Severe cutaneous adverse reactions are frequently preceded by symptoms that can assist in early diagnosis, including malaise, fever ≥100.4°F, ocular and mucosal symptoms, and dermatodynia.24The recommended dose of enfortumab vedotin is 1.25 mg/kg (up to a maximum dose of 125 mg) administered as an intravenous infusion over 30 minutes on Days 1, 8, and 15 of a 28-day cycle until disease progression or unacceptable toxicity. Starting with the first cycle and throughout treatment, patients should be closely and frequently monitored for dermatologic events.42 Clinical experience supports the prophylactic use of barrier-protecting agents such as zinc-containing moisturizers , especially in intertriginous areas like the axillae and groin, as well as ultraviolet radiation protection with sunscreen for photo-exposed areas  . Topical28 likely due to the relatively short half-life of the ADC and MMAE .24 Treatment can then be resumed at the same dose level, or with a dose reduction by one dose level at the discretion of the treating physician.24 Based on the clinical experience, oral corticosteroids  are advisable for grade 3 events. For cases of suspected SJS/TEN, treatment with enfortumab vedotin should be withheld immediately and management with dermatologic consultation should be considered.24 For grade 4 or recurrent  grade 3 dermatologic events or confirmed SJS/TEN, treatment should be permanently discontinued,24 and admission to intensive care or burn unit may be warranted. The management of the ocular and mucosal symptoms that may be present in patients with SJS/TEN is beyond the scope of this manuscript; specialist consultation should be considered to manage these symptoms.For severe (grade 3) dermatologic events, referral for specialized dermatologic care to assist in differential diagnosis and management should be considered. Enfortumab vedotin should be withheld until improvement to grade ≤1 . Clinica33Recommendations for the management of AEs apart from the dermatologic events discussed here and also seen with other targeted therapies and immunotherapies have been detailed elsewhere.A 69-year-old man with metastatic urothelial cancer was commenced on enfortumab vedotin after progression of disease on prior immunotherapy. On Day 15 of Cycle 1 of enfortumab vedotin treatment, the patient developed pruritic, erythematous macules located in the folds of both elbows and the back of the neck. Topical diphenhydramine was effective in relieving the pruritus. Over the course of Cycles 2 and 3, the rash continued to progress despite the use of topical clobetasol, emollient and barrier treatment, and topical and systemic antihistamines, and the decision was made to withhold enfortumab vedotin for 3 weeks and re-evaluate.At the next follow-up, the skin reaction had improved to grade 1, and Cycles 4 and 5 were administered at the full dose. Treatment was complicated by lower extremity cellulitis with blistering that responded to cephalexin. However, due to the worsening rash and the effect on the patient’s quality of life, several dose delays and treatment holds ranging from 1 week to 2 months were undertaken. The rash resolved completely after the prolonged 2-month treatment hold, and Cycle 6 of enfortumab vedotin was resumed at a reduced dose of 1 mg/kg.Within 3 weeks of restarting enfortumab vedotin, a grade 2 pruritic maculopapular rash had again developed on the arms, abdomen, and legs of the patient, accompanied by sloughing of the skin on his legs, although the patient did not report any significant effect of the rash on his activities of daily living. He was using white petrolatum and clobetasol, both as needed. Additional cycles of enfortumab vedotin were marked by dose delays due to recurring rash and unrelated toxicities, including peripheral neuropathy. The patient achieved a complete response after 7 cycles of treatment before the patient ultimately discontinued treatment due to disease progression.Enfortumab vedotin monotherapy has demonstrated an overall survival benefit compared with chemotherapy in patients with la/mUC previously treated with platinum-based chemotherapy and a PD-1/L1 inhibitor. Enfortumab vedotin has also shown encouraging anti-tumor activity in patients with la/mUC ineligible for cisplatin therapy who have previously received one or more prior therapies. Enfortumab vedotin induces cell death through the preferential delivery of MMAE to Nectin-4-expressing cells. As Nectin-4 is normally expressed in epidermal keratinocytes and skin appendages, dermatologic events are common treatment-related AEs and should be anticipated by treating oncologists. Clinicians should monitor the patient frequently throughout treatment for the development of dermatologic events, starting early in the first cycle of treatment when the onset of these events typically occurs. Patients and caregivers should be educated on the signs and symptoms of dermatologic events and advised to report new or changing skin reactions to their treating clinician. Preventative strategies, including topical barrier-protecting agents, especially in intertriginous locations, and sunscreen in photo-exposed areas, are recommended. Mild-to-moderate dermatologic events can be managed with supportive care . For grade 3 dermatologic events, treatment with enfortumab vedotin should be withheld immediately, and referral for dermatologic consultation should be considered. Enfortumab vedotin should also be withheld for cases of suspected SJS/TEN and specialized care with dermatologic consultation should be considered. Treatment should be discontinued in any patients with confirmed SJS/TEN, any grade 4 skin reactions, or recurrent grade 3 dermatologic events. Monitoring for early signs of severe cutaneous adverse reactions is critical, as rare, fatal cases have been reported in clinical trials and in the post-marketing setting. By having an awareness of the pathophysiology of enfortumab vedotin-related dermatologic events as well as protocols for prevention and management, oncologists should be better equipped to educate, appropriately monitor, and manage patients who develop these reactions to minimize toxicity and maximize clinical benefit. The evaluation of the clinical activity and safety of enfortumab vedotin in earlier disease settings in urothelial cancer and in other solid tumors is currently ongoing.


Tumour necrosis factor (TNF) is an inflammatory cytokine produced in response to viral infections that promotes the recruitment and activation of leukocytes to sites of infection. This TNF-based host response is essential to limit virus spreading, thus poxviruses have evolutionarily adopted diverse molecular mechanisms to counteract TNF antiviral action. These include the expression of poxvirus-encoded soluble receptors or proteins able to bind and neutralize TNF and other members of the TNF ligand superfamily, acting as decoy receptors. This article reviews in detail the various TNF decoy receptors identified to date in the genomes from different poxvirus species, with a special focus on their impact on poxvirus pathogenesis and their potential use as therapeutic molecules. TNF is a potent pro-inflammatory cytokine with a broad range of biological effects, ranging from the activation of inflammatory gene programs to cell differentiation or apoptosis induction while also playing an essential role in the host control of many viral infections ,2. It waTNF is first produced as a 26 kDa class II transmembrane protein (tmTNF) on the surface of cells. Then, tmTNF is cleaved by the metalloprotease TNF-converting enzyme (TACE) to be released as a soluble homotrimeric cytokine (sTNF) of about 51 kDa that can execute its function far away from the site where it is synthesized [Far from just being a precursor, tmTNF is a key regulator for many immune and inflammatory processes, as both forms of TNF (tmTNF and sTNF) can bind to two different transmembrane cellular receptors with different affinities . WhereasSoluble versions of TNFR1 and TNFR2 may also exist as a result of alternative splicing or proteolytic cleavage ,11. To rIn addition, tmTNF can induce reverse signalling, a process by which this membrane-bound cytokine can also act as a receptor and trigger intracellular signalling in tmTNF-bearing cells. This tmTNF-elicited reverse signalling is key for the activation and regulation of T cells, macrophages and natural killer (NK) cells ,16.Another TNFLSF member is lymphotoxin-α (LTα), the closest homolog to TNF with 51% sequence similarity, which mainly exists as a soluble homotrimer secreted from T and B lymphocytes. LTα differs from TNF in the amino terminal part, which resembles a typical signal peptide, making LTα secretion extremely efficient. Indeed, unlike most members of TNFLSF, LTα can only anchor to the cell surface after association with membrane-bound lymphotoxin-β (LTβ) to form a heterotrimer . LTα binOne of the main physiological roles of TNF is the mediation of acute inflammation during virus infection. TNF is locally induced early after virus infection and enhances its own expression together with other pro-inflammatory cytokines and also the secretion of chemokines that promote the recruitment and activation of leukocytes. In animal models of infection, TNF, synergistically with interferon (IFN) γ, has been shown to interfere with the replication of diverse viruses, such as hepatitis B virus  or murinThe specific relevance of TNF and TNF signalling in the protection against poxvirus infections is also largely known and supported by experimental data from in vivo studies. For instance, mouse strains naturally resistant to a lethal poxvirus infection, such as ectromelia virus (ECTV), became more susceptible after TNFR deletion, since 60% of the knockout animals succumbed to infection . Indeed,Poxviruses are known for their broad repertoire of immunomodulatory proteins dedicated to evading the host response during infection. One of their most distinctive immune evasion mechanisms is the secretion of viral proteins from infected cells with the ability to bind key host cytokines before engagement with cellular receptors, thus acting as decoy receptors . These pvTNFRs are soluble proteins that mimicking the extracellular domains of TNFRSF and bind to members of TNFLSF in solution, preventing their interaction with cognate receptors at the cell surface . To dateAlthough the structure of just one vTNFR has been experimentally determined , it is wAs stated before, vTNFRs represent soluble viral versions of the membrane-anchored cellular receptors. However, VACV CrmE can additionally associate to the cell surface, maintaining its TNF binding properties intact. Since CrmE lacks a transmembrane domain, this association might occur through interaction with cellular proteins, remaining attached to the cell surface before its cleavage to act as a soluble inhibitor. Alternatively, CrmE might be retained through its N-terminal region at the cell surface, allowing CRDs exposure to bind soluble TNF .Diversity also extends to the number of gene copies encoding vTNFRs among different poxviruses. For example, while one copy of the CrmC gene is found in CPXV, CrmB and CrmD are encoded by two gene copies in CPXV and ECTV, respectively. The number of gene copies encoding the same vTNFR can even vary across poxvirus species to influence its expression levels. Remarkably, although CrmD is expressed at lower levels than CrmC by CPXV, two gene copies in ECTV provide higher expression levels of CrmD during infection .One of the most fascinating aspects of vTNFRs is their degree of specificity to bind diverse members of the TNFLSF together with their specialization to discriminate them from different animal species. CrmC  and CrmEIt is worth noting that vTNFRs CrmB, C, D and E are differentially distributed among poxvirus species. It seems that those poxviruses with a strict host range, like variola virus (VARV) and ECTV, usually express just one of them. For instance, a strictly human poxvirus like VARV only expresses CrmB as functional vTNFR and the mouse-specific ECTV expresses only CrmD ,62,63. OThis specific disposal of vTNFRs among viral species seems to be the result of their co-evolution with their hosts and would indicate that poxviruses have adapted their anti-TNF strategies to the specific TNF response they need to counteract. For example, although CrmB orthologues encoded by CPXV, MPXV, and VARV show high sequence similarity, they exhibit very different ligand binding properties. CrmB from VARV is the most effective at binding and inhibiting human TNF . SimilarIt is also remarkable that CrmC and CrmE can bind both human and mouse TNF with high affinity, but they are only able to inhibit the biological activity of mouse TNF and human TNF, respectively. This may be explained by specific differences in how vTNFRs recognize their ligands, as binding nonessential residues may increase affinity but may not interfere with the cellular recognition, thus being irrelevant for neutralizing activity ,52. RegavTNFRs are considered more similar to TNFR2, as they share more sequence similarity with the extracellular domain of TNFR2 (32%) than to the one of TNFR1 (22%). In addition, the structure of CrmE , the uniMost of the tmTNF activity overlaps with sTNF, however tmTNF can additionally trigger reverse signalling after interaction with TNFR at the cell surface and dampen inflammatory processes ,38,40,72The particular, the behaviour of CrmE deserves a special mention, as it binds both murine sTNF and tmTNF with high affinity but can only inhibit murine tmTNF activity. These data suggest that the molecular structures from mouse tmTNF and sTNF are different and raise two possibilities to explain this surprising behaviour: either tmTNF and sTNF bind to CrmE through different residues, or these interacting residues are the same, but the biological activity of these cytokines relies on different sites. In any case, the contribution of tmTNF signalling to the defence against poxvirus infections in mice becomes evident due to the fact that the expression of recombinant CrmE surprisingly enhanced the virulence of a VACV strain lacking other vTNFRs . Besides their CRDs, CrmB and CrmD contain an additional C-terminal domain, termed Smallpox virus-Encoded Chemokine Receptor (SECRET), with the ability to bind with high affinity and block the activity of a particular set of chemokines involved in mucosal and skin inflammation. This domain shares no sequence similarity to cellular TNFRs or any other known cellular protein and represents an independent folding domain that can exert its function either independently or fused to a TNFR . The modAlthough the SECRET domain is not related to any cellular gene, sequence analysis of poxviral genomes revealed three additional genes encoding separate SECRET domain-containing proteins (SCPs) . DespiteA fine characterization of the functional boundaries between CRDs and the SECRET domain from CrmD has recently confirmed that CRD4, although initially assigned to the TNF binding region, contributes to the SECRET domain-mediated chemokine binding. This incertitude was already hinted at when Asp167 and Glu169 residues in CRD4 from CrmD were identified as key chemokine-binding determinants in the structure of the SECRET domain–CX3CL1 complex . In lineT2 protein was the first vTNFR identified in poxvirus  and to dGiven its dual activity as a TNF and apoptosis inhibitor, it is not surprising that M-T2 was addressed as an obvious virulence factor. Indeed, the infection of susceptible European rabbits with MYXV lacking both gene copies encoding M-T2 resulted in disease attenuation and diminished mortality, indicating a critical role for this vTNFR in the development of myxomatosis .vCD30 was identified as a soluble viral version of the cellular receptor CD30, which is also known as TNFRSF8 and expressed by a small subset of activated T and B cells . vCD30 iIn addition to vTNFRs, some poxvirus species have developed their own vTNFBPs to impede TNF signalling. The 2L protein was the first vTNFBP, with no sequence homology to any known cellular TNFR, identified from Tanapox virus (TPXV)-infected cell supernatants. Later, two homologs were found in the other two existing yatapoxvirus: Yaba-like disease virus (YLDV) and Yaba monkey tumor virus (YMTV). Similar to some vTNFRs, 2L binds human TNF with very high affinity to prevent cytokine signalling through TNFR1 and TNFR2 , howeverThis structural analysis also showed that the 2L interaction with TNF resembles the TNF interaction with the cellular TNFR, a central TNF trimmer bound by three symmetrically arranged 2L molecules. These data raise the hypothesis that 2L might have originated from a fragmented gene encoding an MHC-I molecule that was subsequently incorporated and modified by poxviruses to bind and block TNF.Besides the 2L protein from yatapoxviruses, proteins SPV003 and DPV008 from swinepox virus and deerpox virus, respectively, are also considered as vTNFBPs ,92. In cAlthough we have previously mentioned the relative importance of CD30 and T2, the contribution of vTNFRs to poxvirus pathogenesis was initially unclear, as most reports generating viruses lacking expression of vTNFRs showed very limited effects or were not conclusive. For example, deletion of CrmB resulted in an attenuated phenotype compared to parental virus in an intracranial model of CPXV , a routeIn contrast, CrmD is the only functional TNF inhibitor in ECTV, a strict mouse pathogen causing mousepox disease, making ECTV infection of susceptible mice a suitable model to study the contribution of this vTNFR to poxvirus pathogenesis. In contrast with the observations described above, the infection of susceptible BALB/c mice with an ECTV lacking CrmD expression (ECTV∆CrmD), and therefore in the absence of any TNF blocking activity, resulted in a dramatic virus attenuation and a difference of six orders of magnitude in LD50 compared to wild type virus . In the TNF modulation by ECTV represents an excellent example illustrating how virus evasion strategies can increase the understanding of the host immune regulation mechanisms. In this sense, the analysis of the specific contribution to mousepox pathogenesis of CRDs and SECRET domains from CrmD revealed that chemokines and TNF cooperate during virus infection to develop the inflammatory response and provide anti-viral immunity. Thus, the reinsertion of either the anti-TNF (CRDs domain) or the anti-chemokine (SECRET domain) activities into ECTV∆CrmD did not restore full virulence, indicating that both functions need to operate together to evade the potent antiviral response. In line with this, a recent study evidenced the deleterious consequences of an excessive TNF based response during poxvirus infections. The intranasal infection of mouse strains genetically resistant to mousepox with ECTV∆CrmD caused uniform mortality due to excessive TNF action and a dysregulated inflammatory response . In thisDysregulated TNF production has also been postulated as being responsible for the pathology in many inflammatory disorders, such as rheumatoid arthritis, psoriasis, vasculitis or Crohn's disease . In thesSome vTNFRs show broader ligand specificity or are more potent inhibitors of TNF signalling than etanercept , supportChemokines are also considered an important target for the development of anti-inflammatory therapies, as they mediate leukocyte migration . As descCrmD has also inspired an additional modification related to ligand specificity intended to prevent some of the adverse effects from actual anti-TNF therapies . EtanercvTNFRs could have an additional application in poxvirus vaccination. Although current vaccines against virulent poxviruses have proven their efficacy in the past, there is still uncertainty about the duration of their immunity and their adverse effects, raising considerable interest to develop new and safer vaccines. In this sense, immunization with CrmD protects mice from otherwise lethal mousepox, most likely due to antibody-mediated blockade of CrmD . A similIn conclusion, these soluble decoy receptors constitute a highly efficient strategy to prevent TNF effects, as deduced from the recent evidence demonstrating the relevant contribution of vTNFRs to poxvirus virulence and pathogenesis. Thus, further research on this repertoire of TNF decoy receptors might inspire additional modifications to improve current therapies for inflammatory diseases, or even the generation of new anti-inflammatory molecules with a viral origin.