This research is aimed at investigating how high glucose affects the proliferation and apoptosis in periodontal ligament stem cells (PDLSCs) in the presence of TNF-α (10 ng/ml) for 2 to 6 days. Cell proliferation and cell cycle were evaluated by CCK-8, EdU incorporation assay, and flow cytometry. Cell apoptosis was assessed by annexin V/PI staining. Protein expression was detected by western blotting. Cellular ROS expression was evaluated by CellROX labeling and flow cytometry. Specific antibodies targeting TNFR1 and TNFR2 were used to block TNF-α signaling. Vitamin C was also used to verify if the blockage of ROS can rescue PDLSCs in the presence of high glucose and TNF-α.  PDLSCs obtained from periodontal healthy permanent teeth were treated under either high-glucose condition  or normal glucose condition  in the presence or absence of TNF-α-induced cell viability inhibition  on day 6. High glucose increased protein expression of TNFR1 compared with the control group on day 2 (1.24-fold) and day 6 (1.26-fold). Blocking TNFR1 totally reversed the proliferative inhibition in G30+TNF-α group. The addition of vitamin C or TNFR1 antibody totally reversed the elevation of intracellular ROS expression caused by high glucose and TNF-α. Vitamin C partially restored cell proliferation in the presence of high glucose and TNF-α.  CCK-8 assay showed that high glucose exacerbated TNF-α-induced proliferative inhibition in human periodontal ligament stem cells through the upregulation and activation of TNF receptor 1. Inhibition of intracellular ROS expression by vitamin C partially rescues PDLSCs in terms of cell proliferation.  High glucose exacerbates TNF- Periodontitis, which is caused by a bacterial infection and exacerbated by host immunological reactions, may result in attachment loss, bone resorption, and eventually loss of teeth. Fortunately, periodontal ligament stem cells (PDLSCs) have shown some promise as seeds for regenerating periodontal tissues . PDLSCs α, one of the most critical inflammatory factors in the progress of periodontitis [α in the gingival crevicular fluid and periodontal inflammatory status [α regulates cell proliferation, differentiation, and apoptosis by binding to its membrane-bound receptors [κB, AP-1, or caspase-8. In contrast, TNFR2 does not have a death domain on its intracellular region and is expressed in several types of cells, including astrocytes, myocytes, thymocytes, endothelial cells, and human mesenchymal stem cells. Despite lacking the death domain, TNFR2 also regulates cell proliferation and differentiation through activation of NF-κB and AP-1. Besides, TNFR2 regulates the rate of combination between TNF-α and TNFR1, possibly by increasing the local concentration of TNF-α at the cell surface through rapid ligand passing mechanism [α treatment in terms of proliferation inhibition when compared with CD146-negative periodontal fibroblasts. We also found that protein expression of both TNFR1 and TNFR2 in CD146-positive PDLSCs was 2-fold higher than that of CD146-negative periodontal ligament cells. However, which type of TNF receptor is mainly responsible for the effects of TNF-α in PDLSCs remains unclear.TNF-dontitis , is mainy status . TNF-α receptors . TNFR1, echanism . In our echanism , CD146-pIt is well established that diabetes mellitus increases the risk and severity of periodontitis, especially in patients with poor metabolic control . Indeed,α in the gingival crevicular fluid (GCF) of periodontitis patients increases up to 9.5 ng/ml [α secretion (4.6-fold increase) of periodontitis patients in the presence of Gram-negative bacterial challenge [α in periodontal tissues is also higher in Goto-Kakizaki rats (type 2 diabetes model) than that of the normoglycemic Wistar rats [α in the periodontium. Hyperglycemia and a high concentration of TNF-α are two common pathological conditions in diabetic periodontitis patients, but how they affect PDLSCs is still unknown. Therefore, the aim of this study was to investigate how high glucose and TNF-α affect cell proliferation and apoptosis in human PDLSCs and to clarify possible mechanisms involved in this process. For this, human periodontal ligament stem cells were incubated with TNF-α under high-glucose conditions, and cell proliferation and apoptosis were detected. The function of two types of TNF receptors was also investigated. The intracellular ROS expression was examined, and the protective effect of vitamin C was also evaluated.It has been reported that the average level of TNF-.5 ng/ml . In addihallenge . In ligatar rats . These d3) followed by digestion with 2 mg/ml type 1 collagenase  for 30 min at 37°C. After being passed through a 70 μm strainer, cells were collected and suspended in α-MEM  containing 10% (v/v) FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin . All cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The culture medium was changed every three days. The cells were subcultured when they reached 70% confluence using 0.25% (w/v) trypsin and 0.02% (w/v) EDTA .The primary culture of periodontal ligament cells (PDLCs) was performed as described in our previous study . Briefly7 cells) were prepared as single-cell suspensions using trypsin/EDTA buffer and resuspended in 60 μl D-PBS buffer containing 0.5% bovine serum albumin and 2 mM EDTA. 20 μl of FcR blocking reagent and 20 μl of CD146 microbeads were added. The mixture was then incubated at 4-8°C for 15 min. Then, the cells were washed with 1 ml D-PBS buffer, centrifuged at 300 × g for 10 min, and resuspended in 500 ml D-PBS buffer. Magnetic separation was performed by applying the cell suspensions to an LS column, which was placed in the magnetic field of a MidiMACS separator. The column was washed with buffer three times so that unlabeled cells were washed away. The CD146 labeled cells were collected by removing the column from the magnetic field and flushing the column with 5 ml D-PBS buffer. CD146-positive cells were centrifuged at 300 × g for 10 min and resuspended in a culture medium.Human periodontal ligament stem cells were isolated using human CD146 microbead kit with LS columns and a MidiMACS separator  according to product instruction. In brief, passage-3 PDLCs  were added into 100 μl cell samples, respectively. Samples were incubated at 4°C for 30 min in the dark and analyzed by flow cytometer .For analysis of mesenchymal stem cell surface markers, cells from the second passage of CD146+PDLCs after magnetic bead isolation were analyzed using a Human MSC Analysis Kit  for CD105, CD90, CD73, CD44, CD34, CD11b, CD19, CD45, and HLA-DR according to the product manual. Briefly, CD146+PDLCs were suspended in a buffer solution at 5 × 10For the colony-forming assay, the first passage of CD146+PDLCs was seeded (150 cells per dish) in 10 cm-diameter culture dishes . After 14 days of culture, cells were fixed in 4% paraformaldehyde  and stained with 0.1% toluidine blue  for 5 min. The number of colonies per dish was determined visually under a light microscope . Colonies containing more than 50 cells were considered one colony.CD146+PDLCs were cultured for 14 days, fixed in 4% paraformaldehyde, and blocked with 10% goat serum . The cells were incubated with mouse anti-human Stro-1 antibody (1 : 100), mouse anti-cytokeratin antibody (1 : 200), or mouse anti-Vimentin antibody   for 2 h at 37°C. The samples were then incubated with rabbit anti-mouse immunoglobulin  for 1 h at 37°C and stained with 3,3′-diaminobenzidine (DAB) for 3 min and hematoxylin for 1 min. Samples were observed under a microscope .4 cells per well) and cultured for 2 days. Then, cells were cultured with osteogenic medium  or adipogenic medium  for 21 days or 14 days, respectively. The osteogenic and adipogenic medium were changed every 3 days. Cells were fixed in 4% paraformaldehyde and stained with 1% alizarin red for osteogenesis assay or 3 mg/ml oil red O for adipogenesis analysis . Photographs were captured using an inverted microscope .CD146+PDLCs were seeded in 6-well dishes (5 × 10Control group (G5.6 group): cells were incubated in a medium containing 5.6 mM D-glucose α treatment group (G5.6+TNF-α group): cells were incubated in a medium containing 5.6 mM D-glucose and TNF-α ;TNF-High-glucose treatment group (G30 group): cells were incubated in a medium containing 30 mM D-glucoseα treatment group (G30+TNF-α group): cells were incubated in a medium containing 30 mM D-glucose and TNF-α (10 ng/ml)High-glucose and TNF-Periodontal ligament stem cells were cultured under several different conditions as follows:The osmotic effect was also evaluated by the addition of mannitol . Cells were incubated in a medium containing 5.6 mM D-glucose and 24.4 mM mannitol.μg/ml, MAB625, R&D Systems, Minneapolis, MN, USA) or mouse anti-human TNFR2 monoclonal antibody  was added into the medium 1 hour before high-glucose and TNF-α treatment. Mouse IgG1 antibody was used as an isotype control . One hour later, the medium was replaced by a medium containing D-gl ucose (30 mM) and TNF-α (10 ng/ml). Then, another freshly prepared antibody was simultaneously added into the medium at a final concentration of 10 μg/ml. The medium was changed every 3 days following the same protocol.For TNFR1 or TNFR2 blockage experiments, mouse anti-human TNFR1 monoclonal antibody  was added into medium free of ascorbic acid  1 hour before high-glucose and TNF-α treatment. One hour later, the medium was replaced by a medium containing D-glucose (30 mM) and TNF-α (10 ng/ml). And another freshly prepared vitamin C solution was simultaneously added into the medium at a final concentration of 200 μM. The medium was changed every 3 days following the same protocol.With regard to vitamin C treatment, ascorbic acid (200 3 cells per well) and cultured for 6 days under different conditions as planned. Cell viability was tested every 24 hours: relative cell viability (%) = (ODαTNF−–ODbackground)/(ODcontrol–ODbackground) × 100%. Growth curves were drawn by SPSS software version 22.0  using the Y = A × eB×time model.Cell viability was evaluated by CCK-8 kit . PDLSCs were seeded in 96-well plates (2 × 10μg) were separated on 10% SDS-PAGE gels  and transferred onto the polyvinylidene difluoride membranes . Membranes were incubated with primary antibodies: rabbit anti-human TNFR1 (1 : 1000), rabbit anti-human TNFR2 , and mouse anti-human β-actin . Secondary antibodies were goat anti-mouse IgG-HRP (1 : 1000) and goat anti-rabbit IgG-HRP . Images were obtained by chemiluminescence using an ECL-Western blot kit  and analyzed using ImageJ software .Briefly, total proteins were extracted from the cells using RIPA lysis buffer  and protease inhibitor mix,  and an ultrasonic cell disruptor . The protein concentrations were determined by BCA assay . Equal protein samples  and cultured for 24 hours. Then, cells were incubated under different conditions as planned for 4 days. Cells were harvested and fixed in 70% ethanol at 4°Cfor 24 h. Cells were washed twice with ice-cold phosphate buffer saline  and incubated with RNase and propidium iodide  for 30 min, and then cell cycle analysis was performed by a flow cytometer . Relative S phase fraction (%) was calculated from dividing the S phase fraction of the test group by that of the control group.PDLSCs were plated in 25 cm2 flask  and cultured for 24 hours. Then, cells were incubated under different conditions as planned for 3 days. EdU reagent was added into the culture medium at a final concentration of 10 μM. 24 hours later, cells were harvested, fixed, and permeabilized in the tube. Cells were incubated with Click-iT™ Plus reaction cocktails for 30 min at room temperature in the dark. Cells were washed once with 3 ml of wash reagent and analyzed by a flow cytometer .Cell proliferation was detected by 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay following the product instruction . In brief, PDLSCs were plated in 25 cm2 flask  and cultured for 24 hours. Then, cells were incubated under different conditions as planned for 6 days. Cells were harvested and resuspended in 1 ml culture medium containing cellROX reagent . Cells were incubated at 37°C for 60 min, and then intracellular ROS expression was detected by a flow cytometer . The relative intracellular ROS level was calculated from dividing the ROS level of the test group by that of the control group.PDLSCs were plated in 25 cm4 cell per well) and cultured for 24 hours. Then, cells were incubated under different conditions as planned for 2 days. Cells were fixed in 4% paraformaldehyde for 1 hour, incubated in 3% H2O2 for 10 min, and permeabilized in the reagent containing 0.1% trisodium citrate and 0.1% Triton X-100 for 2 min on ice. Then, cells were labeled with fluorescein-dUTP and peroxidase-labeled anti-fluorescein antibody. The TUNEL reaction was visualized with diaminobenzidine (DAB). A dark accumulation of DAB in the cells (nuclei and apoptotic bodies) was judged to indicate a positive reaction to TUNEL. The nuclei of cells were stained with 500 ng/ml DAPI . For each well, at least 10 random microscope fields (100x) were examined using an inverted fluorescence microscopy  and analyzed using Image-Pro Plus software.Cell apoptosis on day 2 was detected using TUNEL (TdT-mediated dUTP nick-end labeling) assay kit . In brief, PDLSCs were plated in 48-well plates  and cultured for 24 hours. Then, cells were incubated under different conditions as planned for 6 days. Cells were harvested by trypsin (EDTA free) and washed twice with PBS. Then, cells were incubated with Annexin-V and PI reagent in a binding buffer for 15 min in the dark. Cell apoptosis was analyzed by a flow cytometer .Cell apoptosis on day 6 was detected using Annexin-V/PI kit . PDLSCs were plated in 25 cmt-test. p values under 0.05 were considered statistically significant.All experiments were replicated 3 times using periodontal ligament stem cells obtained from 3 different individuals. A total of 20 healthy individuals were included in this study. Data were analyzed using SPSS software version 22.0 . The data were expressed as mean ± standard deviation and analyzed using one-way ANOVA followed by post hoc LSD After 7–14 days, primitive periodontal ligament cells (PDLCs) attached and started to proliferate. CD146-positive (CD146+) PDLCs were isolated from passage-3 PDLCs using immunomagnetic beads. Second passage CD146+PDLCs were positive for MSC markers, including CD105 (92.16%)  were considered periodontal ligament stem cells (PDLSCs) in the present study.α treatment inhibited PDLSC cell viability  compared with the control group (α group (α) than in the control group. Interestingly, high glucose exacerbated the inhibitory effect of TNF-α. Cell density further decreased in the high-glucose and TNF-α treatment group (G30+TNF-α) compared with the TNF-α group  and day 6 . The protein expression of TNFR2 was also assessed . Cell density in the G30+TNF-α+anti-TNFR1+anti-TNFR2 group was recovered to the level of the control group , even though it was lower than the control group, but there was no statistical significance. Cell density in the G30+TNF-α+anti-TNFR1 group was also recovered to the level of the control group  . Similar to the results of the CCK-8 assay, the relative S phase fraction of the G30+TNF-α group further decreased  compared with the G5.6+TNF-α group. Consistent with results of the CCK-8 assay, blockage of TNFR1 could totally restore the relative S phase fraction in the presence of high glucose and TNF-α .The cell cycle was also detected by flow cytometry on day 4. As shown in α treatment decreased CDK4 expression , and the expression of CDK4 in the G30+TNF-α group further decreased compared with the G5.6+TNF-α group .The protein expression of cyclin-dependent kinase 4 (CDK4) was also evaluated by western blotting on day 6. As shown in α treatment, cell apoptosis was evaluated by both TUNEL/DAPI staining , the increase of apoptosis cells is relatively small (4.6%). In contrast, the change of the relative S phase fraction in the G30+TNF-α group was intense , and high glucose further promoted the elevation of intracellular ROS in the presence of TNF-α   was found to be able to reverse the elevation of ROS expression caused by high glucose and TNF-α    were severely decreased compared with the control group (100.0%) and G5.6+TNF-α group (67.0%) .EdU incorporation assay was performed to verify whether the blockage of ROS can reverse proliferative inhibition caused by high glucose and TNF- (67.0%) . Howeverα  under high-glucose conditions (25 mM) for 7 days [Impaired periodontal wound healing is a common symptom of diabetic periodontitis patients. It is reported that the blood glucose levels of Akita mice  reach 600 mg/dl (33.3 mM) at the age of 3 weeks . Glucoser 7 days . In addir 7 days . Howeverα in gingival crevicular fluids (GCF). The concentration of TNF-α in GCF of periodontally healthy individuals is 2.73 ng/ml [α treatment in a dose-dependent manner. In our previous study [α at different concentrations for 48 hours. TNF-α at 2.5 ng/ml and 5 ng/ml were found to promote cell proliferation, but TNF-α at 10 ng/ml showed the apoptotic effect on PDLSCs, suggesting that severe inflammatory condition is detrimental to PDLSCs. As expected, in the present study, PDLSCs incubated with TNF-α at 10 ng/ml for 6 days showed lower cell viability and higher apoptosis rate compared with the control group. However, the apoptosis rate of the G30+TNF-α group was only 4% higher than that of the control group, suggesting that the decrease in cell viability was mainly caused by proliferative inhibition.According to clinical researches, there is a positive correlation between the severity of periodontal inflammation and the level of TNF-73 ng/ml , and it 73 ng/ml  and chro73 ng/ml . In contα were significantly amplified in the presence of high glucose even though high glucose itself did not do harm to PDLSCs. Esposito and colleagues revealed that TNF-α concentration was acutely increased by hyperglycemia in human subjects through an oxidative mechanism [α is closely related to diabetic complications by inducing endothelial cell apoptosis under high-glucose condition [α is based on its concentration, we speculated whether high glucose exacerbated inhibitory effects of TNF-α by increasing its expression. After culture in a high-glucose medium (30 mM) with or without TNF-α treatment for 2 days, the level of TNF-α in the cell supernatant and cell lysate was measured by enzyme-linked immunosorbent assay , which is consistent with Shu and colleagues' finding that basal expression of TNF-α in periodontal ligament cells is about 10 pg per 106 cells. On the contrary, the concentration of TNF-α in the cell lysate of the G30+TNF-α group was 30% higher than that of the G5.6+TNF-α group. These results indicate that high glucose facilitated the binding between exogenous TNF-α and TNF receptors rather than increased the endogenous expression of TNF-α.In this study, the harmful effects of TNF-echanism , and theondition . Since tκB, and activating protein 1 (AP-1) [κB, and AP-1 are found to be activated in hyperglycemia conditions and are involved in the development of diabetic complications [α, suggesting a positive feedback regulation. However, further research is needed to clarify whether the upregulation of TNFR1 in high-glucose conditions is mediated by C/EBP, NF-κB, or AP-1. The functions of TNFR1 and TNFR2 under high-glucose conditions were also evaluated using specific antibodies. TNFR1, instead of TNFR2, was found to be responsible for proliferative inhibition and apoptosis. Our results are in accordance with other researchers' findings that hepatocyte apoptosis [α and TNFR2 being far lower than that between TNF-α and TNFR1. Therefore, TNFR2 acts as a ligand-passing machine that facilitates the function of TNFR1 [Furthermore, the expression of two kinds of TNF receptors was detected by western blotting. Protein expression of TNFR1 was increased by high glucose, whereas expression of TNFR2 was unchanged under high-glucose conditions. The expression of TNFR1 can be upregulated by several transcription factors, including CCAAT/enhancer binding protein (C/EBP), NF-1 (AP-1) . C/EBP, ications , 33. Thepoptosis  and retipoptosis  caused bof TNFR1 .2 into the electron transport chain. The overloaded electron transport chain generates ROS by passing electrons to molecular oxygen [α. It is reported that human skin fibroblasts release ROS in a time- and dose-dependent manner after stimulation with TNF-α [α-induced ROS elevation in vascular smooth muscle cells is further augmented in high-glucose conditions [α and high glucose. It is reported that the addition of exogenous ROS or agents that trigger an increase in endogenous ROS generation sensitize many types of cells  to TNF-induced apoptosis possibly through inhibition of NF-κB transcription of survival genes [α-induced ROS expression was also further increased in the presence of high glucose. However, PDLSC apoptosis was not alleviated by the treatment of vitamin C, suggesting that other mechanisms are involved in this process. In contrast, the decrease in cell viability caused by high-glucose and TNF-α treatment was markedly reversed by vitamin C treatment, indicating that ROS plays a crucial role in cell cycle arrest. It is well established that DNA damage caused by ROS can activate p53, which in turn upregulates p21cip1. The upregulation of p21cip1 inhibits the activation of CDK2 and induces cell cycle arrest in the G1 phase [INK4a is also upregulated by elevated ROS, which binds to CDK4/6 and arrests cells in the G1 phase [α under high-glucose condition exhibited cell cycle arrest in the G0/G1 phase (73.8%) compared with the control group (52.5%). Blocking ROS production by 200 μM vitamin C markedly reduced the proportion of G0/G1 phase from 75.1% to 64.9%  [μM was used as an antioxidant and exhibited excellent protective effects on PDLSCs in terms of cell proliferation. It is reported by Wei and colleagues [μg/ml (113.6 μM) is capable of enhancing the proliferation capacity and osteogenic differentiation efficiency of PDLSCs through inducing telomerase activity. However, the administration of vitamin C only partially reverse TNF-α- and high glucose-induced cell cycle arrest in PDLSCs, suggesting that there are other signaling pathways other than ROS involved in TNF-α-mediated cell cycle arrest. For example, it is reported that p53 activation, as well as p21 accumulation, is not necessary for TNF-α-induced G1 arrest. Instead, inhibition of Rb phosphorylation by TNF-α without activation of p53 is considered a possible mechanism [α conditions  on cell proliferation . Frei and colleagues' study [Vitamin C (ascorbic acid) is a water-soluble antioxidant that can scavenge several types of oxygen radicals, including singlet oxygen and hydroxyl radicals. The concentration of vitamin C in GCF is 207.3  (72 μM) . In addi (72 μM) . Therefolleagues  that vitechanism . In the s' study  revealedcip1, or p16INK4a are involved in the process of high glucose-induced ROS elevation and proliferation inhibition.This study has potential limitations. First, PDLSCs were treated under a constant high-glucose condition (30 mM) for a relatively short time (6 days). Indeed, blood glucose fluctuation aggravated aorta endothelial cell apoptosis  and acceα-induced proliferation inhibition and cell apoptosis through upregulation and activation TNFR1.High glucose (30 mM) does not cause cell apoptosis or cell viability inhibition but exacerbates TNF-α synergistically elevate intracellular ROS levels in PDLSCs. Vitamin C at 200 μM can partially restore cell viability by inhibiting intracellular ROS expression, increasing S phase entrance and mitosis.High glucose and TNF-


First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Carly York is first author on ‘ Carly YorkWhat is your scientific background and the general focus of your lab?Xenopus laevis) to better understand their ecological impact as an invasive species. While my scientific path has been far from linear, I have gained invaluable skills by working in different animal systems.I am an animal physiologist who has studied a number of different organisms including equines, cephalopods and amphibians. My master's degree research, which I completed at Western Kentucky University under the supervision of Dr. Bruce Schulte, focused on equine social behavior and stress physiology. I then completed my doctorate at Old Dominion University with Dr. Ian Bartol, where I investigated how squid evade predators using an array of sensory modalities and a jet propulsive escape response. I am currently an Assistant Professor of Biology at Lenoir-Rhyne University, where I study the sensory physiology of the African Clawed Frog  imaging system DDPTV . In addition to using new technology, we were working with squid, which are notoriously uncooperative animals. Waiting for a squid to compliantly swim in a water tunnel while a laser beam shines on them requires extreme patience.In terms of our results, we were surprised by the high efficiencies that we calculated in these escape jets throughout paralarval, juvenile and adult life stages. Historically, jet propulsion at high velocities has been considered to have low efficiency compared to caudal fin propulsion typically found in fish. However, our findings indicate that jet propulsion is a high-velocity, propulsively efficient escape mechanism throughout ontogeny in squid. We were also surprised to learn that squid have flexibility in escape responses, which was evident by the observation of two different escape jet modes throughout ontogeny. Escape jet I is more efficient in juveniles and adults and may be the mode used when a threat is not eminent. Escape jet II is less efficient than escape jet I and may be used when a predatory attack is unavoidable, making a rapid escape integral for survival.“As a doctoral student, you often underestimate the amount of luck required for successful data collection.”What, in your opinion, are some of the greatest achievements in your field and how has this influenced your research?The development of PIV was one of the greatest technological advancements for studying squid biomechanics. PIV is an optical measurement technique that allows for the measurement of the velocity and vorticity fields within a region of flow. By evaluating the 3D flow features of a squid's escape jet, we can visualize the vortices produced by this behavior, and calculate the efficiency in a new way.What's next for you?X. laevis) and investigating their sensory physiology. I am particularly interested in focusing on how the lateral line system contributes to prey acquisition throughout ontogeny, and how their sensory physiology plays a role in the ecology of these animals as invasive species.As an assistant professor at a primarily undergraduate institution, my goals are to maintain a research program that allows for students to get hands on experience in the lab. I am currently working with a colony of African Clawed Frogs (


Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (Nc) displaying the hepatitis B virus “a” determinant (aD) were produced in Spodoptera frugiperda (Sf9) insect cells. BALB/c mice immunised with the purified chimeric Nc-aD VLPs elicited a sustained titre of anti-aD antibody, which was significantly higher than that elicited by a commercially available hepatitis B vaccine and Escherichia coli-produced Nc-aD VLPs. Immunophenotyping showed that the Sf9-produced Nc-aD VLPs induced proliferation of cytotoxic T-lymphocytes and NK1.1 natural killer cells. Furthermore, enzyme-linked immunospot (ELISPOT)analysis showed the presence of antibody-secreting memory B cells in the mice splenocytes stimulated with the synthetic aD peptide. The significant humoral, natural killer cell and memory B cell immune responses induced by the Sf9-produced Nc-aD VLPs suggest that they present good prospects for use as a hepatitis B vaccine candidate.Chimeric virus-like particles (VLPs) have been widely exploited for various purposes including their use as vaccine candidates, particularly due to their ability to induce stronger immune responses than VLPs consisting of single viral proteins. In the present study, VLPs of the  Hepadnaviridae  and denatured by heating for 10 min. The sample was then loaded into SDS-polyacrylamide gel [12% (w/v)] and electrophoresed at 16 mA for 80 min. The electrophoresed gel was stained with staining solution  for 15 min and destained with destaining solution  until the protein bands became visible.Proteins samples on an SDS-PAGE gel were transferred onto a nitrocellulose membrane by a semi-dry transblotter . The membrane was then blocked with skimmed milk  in tris-buffered saline (TBS)  at room temperature (RT) for 1 h. The blocked membrane was then washed three times with TBS-tween (TBST) buffer [TBS containing 0.1% (v/v) Tween 20] before the anti-His monoclonal antibody  or, the anti-HBsAg monoclonal antibody ] was added and incubated overnight at 4 °C. The membrane was again washed three times with TBST buffer and incubated with the diluted anti-mouse antibody , or the anti-guinea pig antibody conjugated to alkaline phosphatase  for 1 h at RT. Following another washing step with TBST, the membrane was incubated with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT), with gentle rocking until protein bands became visible. Colour development was stopped by washing the membrane in water. Sf9 and E. coli cells using IMAC were adsorbed to 200-mesh copper grids for 5 min. The grids were then stained with uranyl acetate solution  for 5 min. The grids were dried in air, and micrographs were taken with a transmission electron microscope . The Nc-aD proteins  purified from Sf9 cells were pooled and used for the immunisation. The first and second boosters were administered on days 35 (5th week) and 56 (8th week), respectively. The positive control groups were immunised with Engerix B  and E. coli-produced Nc-aD VLPs , respectively. The negative control groups were immunised with Sf9-produced Nc  and E. coli-produced Nc , respectively. With the exception of Engerix B, the aforementioned positive and negative controls were all mixed with the alum adjuvant (100 µL) before administration.Five to six weeks old female BALB/c mice were randomly assigned to seven immunisation groups (n = 8) and acclimatised for 2 weeks. The mice were then immunised subcutaneously with the vaccine candidate consisting of the Nc-aD VLPs  and the adjuvant . Nc-aD VLPs purified from the culture supernatant and lysate of g for 10 min at RT. The sera were collected and stored at −80 °C.Blood (100 µL) was collected from each mouse via submandibular bleeding on days 14, 35, 56, 77, 98, 119, 140, 161 and 182, respectively. On days 14, 35 and 56, blood collection was performed before immunisation. Blood samples were kept at RT for 30 min until clotting was observed, followed by two rounds of centrifugation at 1000 × p-nitrophenyl phosphate , and incubation in the dark for 20 min. The absorbance measurement was performed at 405 nm using the ELx800 microtiter plate reader .Enzyme-linked immunosorbent assay (ELISA) was performed using the sera collected from immunised mice on days 14, 35, 56, 77, 98, 119, 140, 161 and 182, respectively. A synthetic aD peptide  was coated overnight on a 96-well microtiter plate at 4 °C. The wells were then washed three times using TBST, and blocked with milk-diluent  at RT for 1 h. The wells were washed again using TBST, and loaded with the diluted serum samples , and incubated for 1.5 h at RT. Again, the wells were washed three times using TBST, the alkaline phosphatase-conjugated anti-mouse monoclonal antibody  was added to each well, and followed by incubation for 1.5 h at RT. Finally, the wells were washed with TBST, and colour development was performed by the addition of g for 5 min at 4 °C, and the pellet of splenocytes was resuspended gently in ice-cold erythrocyte lysis buffer , followed by incubation on ice for 10 min. Then, ice-cold PBS (6 mL) was added, and the splenocytes suspension was centrifuged at 2000 × g for 5 min at 4 °C. Erythrocyte lysis was repeated until the pellet of splenocytes obtained after centrifugation was devoid of red blood cells.BALB/c mice (n = 4) from each immunisation group were sacrificed on day 63 (7 days after the administration of the second booster), their spleens were harvested, and kept in ice-cold PBS (pH 7.4). The spleens were homogenized on a cell strainer (70 µm) in the presence of ice-cold PBS (1 mL). The homogenate was separated by centrifugation at 2000 × 6 cells were dispensed into 1.5 mL tubes. In the dark, anti-CD3 monoclonal antibody, fluorescein isothiocyanate , anti-CD4 monoclonal antibody, phycoerythrin , anti-CD8 monoclonal antibody, allophycocyanin  and anti-NK1.1 monoclonal antibody  were added to the splenocytes, and incubated on ice for 1 h. The antibody-stained splenocytes were washed with PBS (1 mL) containing BSA (1%), at 2000 × g for 5 min at 4 °C. Finally, fixation was performed with paraformaldehyde (1% in 1 mL PBS) and the splenocytes were kept on ice for 24 h. Analysis of the splenocytes (1 × 105 splenocytes) was performed using a flow cytometer . Flow cytometry data were analysed using the NovoExpress software .The cell pellet was gently resuspended in ice-cold PBS (1 mL) containing BSA (1%), and aliquots of 5 × 106 splenocytes were treated with the polyclonal activator (1:1000 dilution in CTL medium) and incubated for 4 days at 37 °C with 10% CO2. Then the splenocytes (1.25 × 105) treated with the polyclonal activator were added into the wells of a 96-well ELISPOT plate, which had been coated with a synthetic aD peptide (10 µg). The splenocytes were incubated at 37 °C for 24 h in 10% CO2. The plates were then washed two times with PBS (pH 7.4) and two times with PBST (PBS containing 0.05% Tween-20). Then, the biotin-labelled anti-murine IgG antibody  solution was added per well, followed by overnight incubation in the dark at 4 °C. The wells were again washed three times with PBST, and alkaline phosphatase-conjugated streptavidin solution  was added. Plate incubation was performed for 1 h at RT, away from light. The wells were again, washed twice with PBST and twice with distilled water. Colour development was performed by the addition of the blue developer solution (80 µL), and the plate was incubated for 20 min at RT, away from light. The reaction was stopped by gently rinsing the plate three times with tap water, and the plate was air-dried. Finally, the spots formed by antibody secreting cells were counted with the ELISPOT plate reader . The results were considered as positive based on two criteria: if ASCs were observed in at least 2 out of the triplicate measurements, and if the number of observed ASCs was at least twice the number of ASCs in the negative controls.ELISPOT assay was performed using the Mouse IgG Single-Colour ELISPOT kit , according to the manufacturer’s protocol with some modifications. Briefly, the spleens of mice (n = 4) from each immunisation group were harvested on day 182. Then, 1 × 10p values less than 0.001 are considered as significant, and those less than 0.0001 are considered as extremely significant. The statistical analysis was performed using the IBM SPSS statistics software .Variations in the immunogenicity of the various administered injections tested using ELISA, variations in the number of gated mice splenocytes during immunophenotyping, and variations in the number of spots counted in ELISPOT, were analysed with the one-way analysis of variance (ANOVA). The Duncan’s multiple-range tests were used. Sf9 cells infected with 10% of the P1 baculovirus stock. The purified Nc-aD was eluted from the IMAC column using 500 mM imidazole, dialysed in sodium phosphate buffer, and analysed using SDS-PAGE and western blotting. A protein band of ~52 kDa was detected . The highest antibody titre was recorded from serum samples collected on day 77, which corresponds to 21 days after the administration of the second booster. A decline in the antibody titre was observed in sera from days 98 and 119, after which the antibody titre appeared to reach a plateau. In general, the antibody titre elicited by Sf9-produced Nc-aD VLPs was higher than those of E. coli-produced Nc-aD VLPs and Engerix B.The Nc-aD VLPs were subcutaneously administered into BALB/c mice, and the collected serum samples were analysed with ELISA. As shown in + and CD4+ cells), CTL (CD3+ and CD8+ cells) and NK cells (NK1.1+ cells) in the splenocytes of sacrificed mice , which is an indication of increased CTL activities. Immunophenotyping was performed on day 63 after the administration of the second booster in order to quantitate the helper T lymphocytes . Interestingly, Sf9-produced Nc VLPs, without the aD, induced a significantly higher NK cell response than E. coli-produced Nc-aD VLPs and Engerix B. Also, there was no significant difference between the percentage of NK1.1+ cells in mice inoculated with E. coli-produced Nc-aD VLPs and E. coli-produced Nc VLPs. These observations suggest that the Nc VLPs alone have some effects on NK cell induction.Sf9-produced Nc-aD VLPs, E. coli-produced Nc-aD VLPs and Engerix B had significant higher numbers of ASCs per 1.25 × 105 splenocytes (p < 0.001), as compared to the groups injected with buffer and Nc VLPs produced in Sf9 and E. coli. Interestingly, splenocytes harvested from the mice immunised with Sf9-produced Nc-aD VLPs had the highest memory B cell response.On day 182, the mice from all immunisation groups were sacrificed and their splenocytes were stimulated with a synthetic aD peptide to assay the presence of memory B cells or ASCs. The ASCs were counted as spots on the ELISPOT plate following colour development . The resO- and N-glycosylation, can enhance the immunogenicity and biological activity of recombinant VLPs. Hyakumura et al. [N-glycosylation sites in the S-HBsAg yielded more densely glycosylated VLPs with enhanced immunogenicity, when the particles were expressed in HEK293T mammalian cells. They demonstrated a significant correlation between glycan abundance and immunogenicity of S-HBsAg in BALB/c mice. However, VLP production in mammalian cell lines is often hampered by high production cost and low VLP yield, as compared to yeast cells. These single-celled fungi also have some drawbacks as yeast-produced VLPs are non-secretory, which complicates the downstream purification process due to the presence of cellular debris. HBsAg expressed in Pichia pastoris was shown to be localised within the endoplasmic reticulum, and extraction from detergent-treated cell lysates was only possible after several downstream steps [Saccharomyces cerevisiae involved laborious steps, which include the lysis of cells with detergent and alumina, followed by lyophilisation of the crude lysate, and separation of the particles using sepharyl-S column chromatography and size-exclusion chromatography [Sf9 cell line [N-glycosylated VLPs [E. coli by fusing the epitope to the ice nucleation protein, an outer membrane protein of Pseudomonas syringae.In the absence of any effective treatment for hepatitis B infection, the administration of vaccines remains the most effective control measure . Howevera et al.  demonstram steps . The stutography . Since ttography . Howevertography . HBsAg eell line . The bacted VLPs . Althougted VLPs , concernMrNV VLPs, previous studies by our group suggested that the Sf9 expression system is a more effective system than E. coli, for the production of VLPs for immunologic and structural studies, owing to the fact that the VLPs produced in Sf9 are more stable than those produced in E. coli [Sf9-produced Nc VLPs were stable from pH 2 to 12, and from 5 to 45 °C [Sf9-produced Nc VLPs is 10 nm larger than those produced in E. coli [With regards to the  E. coli ,30. The to 45 °C . In addi E. coli . Fifis e E. coli  demonstrSf9 cells using the baculovirus expression system were purified using IMAC. The VLPs were heterogeneous in size, ranging from ~21 nm to ~55 nm in diameter, as observed under a transmission electron microscope. The heterogeneous population of the VLPs could be attributed to the fusion of aD at the C-terminal end of Nc. Kueh et al. [Sf9 cells assembled into homogenous VLPs with a diameter of ~40 nm. Fusion of a small ubiquitin-like modifier (SUMO) protein at the N- or C-terminus of the Nc of a closely related shrimp nodavirus, Penaeus vannamei nodavirus (PvNV), resulted in heterogeneous VLPs [Sf9-produced enhanced green fluorescent protein (eGFP) fused to the C-terminus of the core protein of hepatitis C virus assembled into heterogeneous VLPs ranging from 30 to 55 nm in diameter. Another study by Luque et al. [Sf9 cells.The Nc-aD VLPs produced in h et al.  demonstrous VLPs . The addous VLPs . Katsaroous VLPs  showed te et al.  revealedSf9-expressed Nc-aD VLPs into the BALB/c mice elicited a significant production of aD-specific antibody, which was maintained to the endpoint of the study (day 182), demonstrating that the high antibody titre was sustained for 126 days after the second booster injection. The Antibody level reached a plateau from days 119 to 182, suggesting a very slow rate of decline in the elicited humoral immune response  was significantly higher than the antibody titres of all other groups even at their peaks (day 77). Ryu et al. [Sf9-produced Nc-aD VLPs to elicit such a significant titre of anti-aD antibody presents good prospects as a new hepatitis B vaccine candidate.When compared to the u et al.  demonstrSf9-produced Nc-aD VLPs into the mice induced CTL proliferation, as evidenced by a higher CD8+/CD4+ ratio in this immunisation group, but not in the negative control groups . Although the increase in CTL ratio induced by E. coli-produced Nc-aD VLPs (0.69) and Engerix B (0.66) is higher than that induced by Sf9-produced Nc-aD VLPs (0.65), the difference is not statistically significant. The observed CTL proliferation could have been induced by the aD component of the immunogens. This is corroborated by a study by Pride et al. [in vivo.Administration of e et al.  who demoe et al. ,40. The e et al. ,42. It ie et al. . A studye et al. . The micSf9-produced Nc-aD VLPs resulted in an increase in the NK cell population in the immunised mice  were administered, at 21 days interval, into BALB/c mice. The chimeric VLPs induced a robust antigen-specific humoral immune response, which was sustained to 126 days after the second booster dose, until the end-point of the animal study. Immunophenotyping revealed that the Sf9-produced Nc-aD VLPs induced CTL and NK cell activities in the immunised mice. ELISPOT assay for memory B cells showed that the Nc-aD VLPs induced the production of ASCs detected in polyclonally activated mice splenocytes stimulated with the synthetic aD peptide on day 182, indicating the induction of a memory B cell immunity. By and large, the Sf9-produced Nc-aD VLPs induced significant humoral, CTL, NK cell and memory B cell immune responses.Nc-aD VLPs were produced in


To develop a paediatric radiology themed escape room session for undergraduate education and secondly, to determine participant satisfaction and improvement in knowledge.A paediatric radiology escape room with accompanying tutorial was developed around key learning objectives set within the RCR and ESR undergraduate curriculum. Students were recruited from two different universities and undertook the escape room themed teaching. An 8-question single best answer (SBA) test was completed before, immediately after and at 2 weeks post-teaching to determine participant improvement and retention of knowledge. The general feedback was also collected.The escape room sessions were held three times, for 19 students (6–7 students per session). All groups completed the escape room in ≤ 20 min. Students enjoyed the experience, assigning an average satisfaction score of 9.4/10 (range 7–10). The majority  preferred this method of teaching to a lecture-based tutorial alone, although all said they found the tutorial component useful. For the SBA test, there was an average increase in 3.6 marks (range 1–6 marks) per participant between before and after the escape room. This improved knowledge was mostly sustained after 2 weeks, with an average increase of 3.4 marks difference (range 1 to 6) per participant compared to before the teaching.A paediatric radiology themed escape room is a feasible teaching method, enjoyed by participants and associated with an increase in radiological knowledge. Further work with larger sample size and direct comparison with other traditional teaching methods is required. A paediatric radiology themed escape room is a feasible teaching method.Students enjoyed the escape room, and most preferred it over didactic lectures.Improvement in paediatric radiology knowledge was maintained after the teaching.The use of interactive teaching and games in healthcare education has a positive impact on the learning process , with moGiven the variability of undergraduate radiology education delivered in universities across the UK  and EuroThe aim of this study was therefore two-fold: firstly, to develop a paediatric radiology themed escape room session for undergraduate education and secondly, to assess participant satisfaction and improvement in paediatric radiological knowledge.Ethical approval was not required for this study, assessing the anonymised student feedback and knowledge from a teaching event. All students provided consent for the use of their anonymised feedback in this study. No external funding was provided for this project.A paediatric radiology themed escape room session and 8-question single best answer (SBA) test was devised by the senior author (S.S.), a paediatric radiologist . Both the SBA test and escape room were constructed with specific learning objectives in mind, mapped to outcomes from the Royal College of Radiologists (RCR)  and EuroThe escape room consisted of four radiological ‘puzzles’ (relating to the objectives above), that could be solved in any order Figs. , 3 and 4Prior to running the undergraduate teaching session, a ‘practice run’ was piloted on two paediatric radiology fellows active in undergraduate teaching  to identify errors in the design, feedback on level of difficulty and to inform estimated time required to complete the puzzles. Both radiology fellows were blinded to the escape room design prior to the practice run.Undergraduate medical students from two different universities  were recruited. The escape room teaching sessions were held on two separate dates, both in October 2019.A single escape room based teaching session was held for the UCL students, which took place during working hours as part of their weekly compulsory undergraduate medical teaching programme at the senior author’s institution, organised by the in-house postgraduate medical education department . Two sessions of escape room based teaching were held for SGUL students, both taking place in the early evening as part of an undergraduate radiology society event, attended by students on a voluntary basis. In this second setting, the students attending were recruited by event posters and email, Facebook and social media alerts.Each teaching session lasted 90 min. In the first 10 min of the sessions, students completed the SBA test to gauge their baseline radiological knowledge. The rules of the escape room were then explained by the senior author . Once in the room, a timer was started to add a competitive element. A time limit of 30 min was set. The senior author (SS) was present in the room for all sessions to provide general supervision and observation, but the students were not allowed to ask for any hints. Following the completion of the escape room, a 45-min radiology tutorial was delivered by the senior author (S.S.), covering a ‘walk-through’ of the key findings in the escape room puzzles and the aforementioned learning objectives.At the end of the teaching, students engaged in a team photo, completed a feedback form of their experience  and descriptive analyses using the total, mean and range of feedback scores and test scores were performed.Devising the escape room took place over a period of 2 weeks. This included sourcing the individual equipment for the room, preparing the puzzles and testing the design. The total estimated cost for the materials was £93.83  completed the escape room across the three escape room teaching sessions. There were 7/19 (36.8%) male participants. Most of the participants were in their final undergraduate year (i.e. year 6) of training (8/19 (42.1%)), with others from year 5 , year 4 , year 3 , year 2 , year 1  and one recently qualified foundation year 1 doctor .None of the participants had previously received paediatric radiology teaching, although most  had prior general radiology teaching. Only three students  reported no prior radiology teaching. They were in years 1 and 2 of undergraduate study. Of the 16 students who had previous radiology teaching, the most common method for teaching were small group tutorials , with 6/16 (37.5%) receiving large group lectures and 5/16 (31.3%) having completed placements in a radiology department. Approximately, a third of students  had previously completed an escape room before .Session 1: 14 min 57 s; six final years (year 6) studentsSession 2: 20 min; 1 year 1, one year 2, one year 3, two year 4 and one year 5 studentSession 3: 16 min 28 s; one year 1, one year 2, one year 3, one year 4, two year 6 students and one recently qualified junior doctor (foundation year 1).The escape room teaching session was run three separate times for different teams of participants. A 6-person team participated in the first two sessions, and 7-person team in the third session. The escape room was solved by all three teams within the 30 min time-limit, with the following times and participant seniority levels:Feedback regarding overall enjoyment and the running of the escape room is provided in Table All students completed the pre-escape room and immediate post-escape room SBA tests at the time of the teaching. The online SBA test was completed by 13/19 (68.4%) students 2 weeks after the teaching session.The mean test score (out of 8) prior to the teaching was 3.7 (range 2–6), improving to 7.3 (range 4–8) immediately after the teaching session, and 7.3 (range 5–8) 2 weeks later. When assessing the difference in scores on a ‘per participant’ level, this corresponded to an average increase of 3.6 marks (range 2–6) between pre- and post-teaching sessions; but a slight reduction of 0.5 marks (range − 2 to + 1) between scores at post-teaching and 2 weeks later. The average difference between the pre-teaching (i.e. baseline) and 2 weeks test was an increase in 3.4 marks (range 1–6) per participant. Only 5/13 (38.5%) students performed worse at 2 weeks compared to their immediate post-session test. All participants scored higher immediately after, and at 2 weeks after the teaching session compared with their baseline marks.The number of correct answers on a per-question level amongst the students is outlined in Table A paediatric radiology themed escape room is a feasible, enjoyable and educational method for delivering undergraduate radiology education. All students improved their knowledge of paediatric radiology immediately after the teaching session, with sustained improvement after a 2-week period compared to baseline.p < 0.01). Whilst our results are similar in finding an increase in knowledge immediately after the escape room, the authors did not test the students’ knowledge retention at a later date. Our results showed that whilst a third of students scored lower on their ‘2 weeks post-session test’ than immediately after the teaching, all students maintained a higher test score than before the teaching, indicating a level of knowledge improvement and retention.When comparing prior studies relating to healthcare education-themed escape rooms   [Figure A2: The Single Best Answer (SBA) Test provided to all participants before, immediately after and at two weeks post escape room themed radiology teaching. The answers to the quiz are as follows: 1b, 2d, 3b, 4d, 5d, 6b, 7b, 8a. Question 6 was based on the systematic review by Kemp et al3, demonstrating highest association with rib fractures with suspected physical abuse, but insufficient evidence to quantify probability of association with corner metaphyseal fractures. Figure A3: Escape room rules and regulations. These instructions were read to students prior to entering the escape room and a copy of the rules were also left on the central table within the escape room itself as a reminder of good behaviour. Figure A4: Escape room backstory. This fictional story was read to the participants prior to entering the escape room in order to ‘set the mood’ and create and fun and engaging atmosphere. Figure A5: Teaching feedback form completed by students after the escape room themed teaching session to assess levels of enjoyment, difficulty and design of the escape room. Figure A6: A list of inventory and equipment purchased in order to develop and set up the escape room themed teaching. The location for sourcing the material and the costs incurred (including postage costs where bought online) are provided. Figure A7: Individualised free text comments provided by participants on the feedback forms after the teaching sessions.diology) . Figure


The recent introduction of biological agents has revolutionized the treatment of chronic immune-inflammatory diseases; however, this new therapy did not come without significant side effects.Through large controlled studies indicating decrease in the number of uveitis flares, the role of TNF inhibitors therapy for non-infectious uveitis gained more ground. Paradoxically to its therapeutic effect, there are reports associating these drugs with the onset or recurrence of inflammatory eye disease.A number of studies have suggested possible roles for anti-TNF-α agents in precipitating or worsening an underlying inflammatory process, including the hypothesis of a disequilibrium in cytokine balance, but to date the mechanisms responsible for these adverse events are not fully understood.A PubMed literature search was performed using the following terms: ophthalmic complication, uveitis, inflammatory eye disease, optic neuritis, neuropathy, adverse events, anti-TNF, TNF alpha inhibitor, infliximab, etanercept, adalimumab, golimumab, certolizumab, and biologics. The data presented in this study was mainly derived from the use of TNF inhibitors in rheumatology, essentially because these drugs have been used for a longer period in this medical field.Many of the ocular adverse events reported on this review may be considered a paradoxical effect of anti-TNF therapy. We found a variety of data associating new onset of uveitis with anti-TNF therapy for rheumatic conditions, predominantly under etanercept.In conclusion, although there is increasing data on ocular adverse events, it remains to be seen whether the suggested link between TNF inhibitors and the onset of ocular inflammation is substantiated by more quality data. Nevertheless, the awareness of potential treatment side effects with anti-TNF should be highlighted. Autoimmune inflammatory diseases affect approximately 7.6–9.4% of the world population, especially among the young and middle-aged women. These diseases are on the leading causes of mortality all around the world and are frequently accompanied by severe and chronic morbidity .For the last two decades, the introduction of biological agents has revolutionized the treatment of chronic immune-inflammatory diseases, such as chronic inflammatory rheumatic diseases, and, more recently, uveitis . AlthougAnti-TNF drugs are usually well tolerated; however, with the increase of biological therapies, a variety of adverse effects has emerged through mechanisms that are not fully understood. The main adverse event is infusion or injection site reactions, but they are typically manageable. Less common, although potentially more severe, complications include reactivation of tuberculosis and increased incidence of other infections; cytopenias, or, rarely, aplastic anemia; increased risk of malignancy; worsening of congestive cardiac failure; hepatotoxicity; and exacerbation or development of demyelinating disease . ParadoxIn this review, we are going to focus on a less documented aspect of this therapy: ocular adverse events. These range from a common conjunctivitis to endophthalmitis and can potentially be sight-threatening, highlightening the relevance of closely monitoring ocular manifestations in patients treated with anti-TNF-α agents.It is important to emphasize that when assessing safety profile, the results obtained differ depending on the underlying disease, which may play a crucial role in the rate, type, and severity of adverse events .The aim of this study was to review the current evidence on ocular adverse events of anti-TNF agents.A literature search was performed in PubMed. The search was focused on the most recent literature but included all articles published after 1990. The keywords ophthalmic complication, uveitis, inflammatory eye disease, optic neuritis, neuropathy, adverse events, anti-TNF, TNF alpha inhibitor, infliximab, etanercept, adalimumab, golimumab, certolizumab, and biologics were used for this search. For this review, mainly studies conducted in medical fields outside ophthalmology were included. Because TNF inhibitors were first implemented in other specialties , large series of patients have been treated already for a very long time, and a higher quality of evidence can be found in these fields’ literature. References from the selected manuscripts were searched for additional relevant studies.TNF-α is a 26 kDa homotrimeric transmembrane protein that is expressed on the surface of macrophages, T-lymphocytes, natural killer cells, smooth muscle cells, and fibroblasts . In the This type of therapy includes medications structurally composed of monoclonal antibodies, their Fab portions, or even antibody fusion proteins, which may target cytokines and membrane receptors , 13. TheUveitis is a common, sight-threatening ocular disease, broadly characterized as the inflammation of the uvea. It can have both infectious and noninfectious origin and leads to 5–10% of visual impairment worldwide , being cThe current standard of treatment for centers not specialized in uveitis relies on corticosteroid therapy to eliminate inflammation . While bTNF inhibitors began to emerge as a possible therapy for uveitis when a number of large controlled studies indicated that patients with immune-mediated conditions treated with anti-TNF-α agents had a significant decrease in the number of uveitis flares . The resThe VISUAL I and II trials evidenced the ability of adalimumab to reduce the number of uveitis flares compared with placebo  , 19 and A recent systematic review found that adalimumab reduced the risk of treatment failure by 43–75% and might be a promising choice in reducing inflammatory activity, gaining VA, and sparing corticosteroid use in non-infectious uveitis .Although the remaining anti-TNF therapies are considered off-label for uveitis, they are still used to control ocular inflammation associated with systemic diseases , 22 and Because biological TNF-α antagonists have become prominent agents in the management of patients with chronic immune-mediated diseases and their use has increased over the past decade, several adverse events related to its use have been reported, including ocular ones .Paradoxically to its therapeutic effect, there are reports associating TNF inhibitors with the onset or recurrence of inflammatory eye disease, particularly with etanercept . Most caThe role of anti-TNF agents in the pathogenesis of ocular adverse events when used for treatment of diseases in which ocular involvement is recognized as part of the clinical spectrum is controversial. New onset uveitis and development of uveitis in the absence of articular activity after initiation of treatment , 42, 43 It has been hypothesized  that uveHowever, the increasing evidence associating these therapies to a paradoxical adverse event in ocular inflammation strongly points toward a true cause-effect relation. Furthermore, in many cases, uveitis resolved after treatment withdrawal , and re-The mechanisms whereby TNF-α inhibitors produce paradoxical inflammatory effects are unclear. One hypothesis involves the inverse and interdependent relationship between TNF-α and interferon levels (both α and γ) and the role of increased interferon in the development of autoimmune diseases . Others P < 0.01), and this was again the case after exclusion of patients whose underlying disease was associated with uveitis. In a French study [In the database of spontaneously reported uveitis under anti-TNF therapy in the USA , TNF-α sch study  gatherinThere are several hypothesis trying to explain this difference. Regarding pharmacokinetics , etanercEtanercept  was the first anti-TNF-a approved by the FDA in 1998. It is a humanized, recombinant dimeric fusion of a human Fc molecule and two p75 TNF receptors. Etanercept prevents binding of TNF-α and TNF-β to cell surface TNF receptors by binding these circulating factors. Unlike other agents within its class, etanercept does not induce lysis of TNF-producing cells.A registry-based study in the USA found 43 cases of uveitis associated with etanercept . Other sThe possible reasons for this phenomenon have been previously discussed in this review. One possible explanation is that inhibition with infliximab shuts down both TNFRp55- and TNFRp75-mediated events, whereas etanercept leaves TNFRp75-mediated signaling at least partially intact . AnotherScleritis is strongly associated with RA, with an overall prevalence of scleritis in patients with RA reported to range from 0.67 to 6.3% , 61.Le Garrec at al . reporteLater, another study  describeVarious demyelinating disorders have been reported in association with anti-tumor necrosis factor α (TNF-α) agents. Many papers showed the association between optic neuritis and etanercept , 63–66. Ocular myositis is a rare disorder characterized by inflammation of single or multiple extra-ocular eye muscles presenting with painful diplopia and/or ophthalmoplegia. Two reports of ocular myositis occurring under etanercept treatment for rheumatoid arthritis were found in the literature , 68.Infliximab  is a chimeric mouse-human monoclonal antibody that binds and neutralizes both circulating and membrane-bound TNF-α and was the second anti-TNF-α agent to receive FDA approval in 1999.The same registry-based study mentioned with etanercept found 12 cases of uveitis associated with infliximab . A retroThe exact number of cases that present ON as an effect of infliximab or other TNF inhibitors is unknown but seems to be much higher than described in the literature . Many reImmunosuppressive disease and therapy are a known risk factor for endophthalmitis . DiffereAdalimumab  is a fully humanized monoclonal antibody against TNF-α. Since its approval by the FDA in 2002, it has become one of the most prescribed biologics in the USA , 85. It p > 0.5, OR = 1.6). A report by Seve et al. [Although in the registry based study by Lim et al. , there we et al.  describeWe found a case report  of a womOnce more in the study by Lim et al. , the autA study evaluating the ocular toxicity of escalating doses of intravitreous adalimumab (Humira) in the rabbit eye  found thP. acnes, since it may induce pathologic reactions in compromised patients and cause endophthalmitis [Only one case report of endophthalmitis associated with adalimumab treatment was found . This rehalmitis .In the literature, there is one case report of internuclear ophthalmoplegia in a woman with history of Crohn’s disease who developed facial numbness, blurred vision, and diplopia on right gaze .Golimumab  is a fully human monoclonal antibody and was approved by the FDA in 2009.Certolizumab pegol  is approved for the treatment of moderate to severe rheumatoid arthritis, psoriatic arthritis, and Crohn’s disease. Unlike fellow anti-TNF monoclonal antibodies, this agent is a polyethylene glycolated Fab fragment.n = 169), with fewer reported with infliximab (n = 122) and adalimumab (n = 55).As these are newer types of anti-TNF-α, there is still very little evidence on ocular side effects for both. In the SABER study , when reThe spectrum of TNF blockers is constantly expanding. Since the expiration of the patent for infliximab in 2015, a number of biosimilars have obtained EMA and FDA approval and are used in clinical practice. In 2017, biosimilars for adalimumab also became available.Real-world clinical practice experience and data on these novel drugs are still limited; however, a systematic review showed that biosimilar has a comparable safety and efficacy spectrum to the originating drugs . To thisAlthough anti-TNF biopharmaceuticals have emerged as a major evolution in the treatment of immune-mediated diseases, the decision to start a patient on anti-TNF therapy has to take into account potentially serious side effects.Anti-TNF-α is being increasingly used to treat ocular inflammation, and adalimumab and infliximab have the greatest body of data supporting their use in non-infectious uveitis (NIU). These agents are considered appropriate therapy for most forms of NIU but may be considered first-line therapy for uveitis associated with Behçet’s disease and juvenile idiopathic arthritis . NeverthIn addition, a wide range of paradoxical adverse events (PAEs) have been reported . A numbeUnder these circumstances, many of the ocular adverse events reported on this review may be regarded as a paradoxical effect of anti-TNF therapy, particularly uveitis. In this review, we found ample data associating new onset of uveitis with anti-TNF therapy for rheumatic conditions, predominantly in patients with spondyloarthritis and under etanercept , 29. WheAlthough awareness of potential treatment side effects should be highlighted, it remains to be seen whether the suggested link between TNF inhibitors and the onset of uveitis is substantiated by more quality data. We gathered mainly case reports and retrospective studies which, considering the broad spectrum of uveitis causes and its potential link to the underlying auto-immune diseases treated with anti-TNF-α, can lead to confounding errors. Future large, prospective, post-marketing studies including patients treated with conventional therapies as controls may be useful in order to evaluate the real risk of patients receiving biological therapies developing uveitis.Another ocular adverse effect that was found to be consistently associated with anti-TNF-α therapy is optic neuritis. The relationship between anti-TNF-α and induction of demyelinating diseases remains unclear, but in a number of cases the chronology of clinical events is suggestive. A randomized, placebo-controlled study of 168 patients with MS (excluding patients with rapidly progressive MS) treated with lenercept found an increase in the frequency of exacerbations and a trend toward more severe exacerbations when compared with the placebo .Optic neuritis is a serious complication that may lead to irreversible vision loss; therefore, patients being treated with a TNF-α antagonist should be closely monitored for the development of ophthalmological or neurological signs and symptoms. Clinical presentation, serological and radiographic findings, and quick responses to discontinuation of potentially offending drugs suggest ON associated with an anti-TNF agent , 65, 71.If diagnosed, treatment for ON, including pulse steroids followed by oral steroids, should be started. In a literature search, nine out of fifteen patients experienced complete resolution after treatment . AdditioIn a French national survey , the autTreatment strategies for ocular adverse events dictate discontinuation of the offending drug. Substitution for steroids is a good option –67, 71; Although there is increasing data on ocular adverse events associated with anti-TNF-α therapy, most available information comes from uncontrolled studies, and therefore solid conclusions are not possible. In spite of the limited quality of the available data, this review is important to gather the current information and shed light on the possibility of potential adverse and paradoxical effects of anti-TNF agents that rheumatologists and ophthalmologists should be aware.Diagnosis and treatment must be co-managed by rheumatologists and ophthalmologists. In some cases, cessation of the drug is mandatory due to persisting and/or recurrent eye disease.Finally, our findings do not alter the current indications for the use of biologics but rather recommend a close surveillance of ocular status in patients using anti-TNF and open the discussion about the need of better quality evidence on this matter.


The heart failure (HF) epidemic is growing and approximately half of the HF patients have heart failure with preserved ejection fraction (HFpEF). HFpEF is a heterogeneous syndrome, characterized by a preserved left ventricular ejection fraction (LVEF ≥ 50%) with diastolic dysfunction, and is associated with high morbidity and mortality. Underlying comorbidities of HFpEF, i.e., hypertension, type 2 diabetes mellitus, obesity, and renal failure, lead to a systemic pro-inflammatory state, thereby affecting normal cardiac function. Increased inflammatory biomarkers predict incident HFpEF and are higher in patients with HFpEF as compared with heart failure with reduced ejection fraction (HFrEF). Randomized trials in HFpEF patients using traditional HF medication failed to demonstrate a clear benefit on hard endpoints . Therefore, therapies targeting underlying comorbidities and systemic inflammation in early HFpEF may provide better opportunities. Here, we provide an overview of the current state and future perspectives of immunomodulatory therapies for HFpEF.The online version of this article (10.1007/s12265-020-10026-3) contains supplementary material, which is available to authorized users. Heart failure (HF) with preserved ejection fraction (HFpEF) accounts for ≈ 50% of all heart failure (HF) patients (≈ 6.2 million American adults with HF from 2013 to 2016) with increased prevalence in the elderly population (> 70 years) and women  . HFpEF iThus far, randomized trials in HFpEF patients using traditional HF medication failed to demonstrate a benefit on mortality and hospitalization for HF (HHF) . DespiteTrials targeting systemic inflammation in HFpEF are currently lacking. Therefore, we summarize preclinical research and ongoing trials focusing on hypertension and T2DM, which have a known inflammatory phenotype.Hypertension is the most common comorbidity (> 65%) in HFpEF and associated with inflammation . AngioteApproximately 45% of all HFpEF patients have T2DM, and T2DM patients are twice as likely to develop HF . T2DM inMetformin decreases hepatic glucose production, increases tissue insulin sensitivity, and is associated with improved cardiac outcome based on experimental models. Metformin attenuated LV remodeling in a hypertensive rat model by inhibiting aldosterone-induced fibroblast activation . A studyThiazolidinediones (glitazones) stimulate fatty acid storage in adipocytes, thereby increasing glucose utilization and lowering serum glucose level. However, they may cause sodium and water retention leading to congestion . DipeptiThis novel class of drugs showed benefits beyond glycemic control in preventing HF in T2DM patients. SGLT2i reduces renal glucose reabsorption and increases urinary glucose excretion, next to restoration of sodium delivery, thereby increasing diuresis –29. The PPAR-γ agonists (glitazones) reduce inflammation, oxidative stress, and hypertrophy in adipocytes and cardiomyocytes . In a HFObesity and hyperlipidemia are important comorbidities in HFpEF and linked to systemic inflammation . Weight Although inflammation is a major driver in the pathophysiology of HFpEF, trials primarily focusing on systemic inflammatory pathways in HFpEF are currently lacking. Targeting specific components of the inflammatory process might decrease cardiac remodeling and benefit cardiac function. Based on experimental data, important novel therapeutic options are discussed below and summarized in Fig. Normally, NO is produced by cardiac microvascular endothelial cells resulting in activation of soluble guanylate cyclase (sGC) in cardiomyocytes and vascular smooth muscle cells. sGC in turn produces cyclic guanosine monophosphate (cGMP), which regulates protein kinase G (PKG) . HoweverPDE5i inhibit breakdown of cGMP levels, thus activating protein kinase G, which resulted in decreased LV/cardiomyocyte stiffness and LV relaxation time and prevention of HFpEF in Zucker diabetic fatty rats . HoweverNitrate and nitrite increase NO bioavailability, contributing to vascular integrity and function. In the NEAT-HFpEF trial, nitrate isosorbide mononitrate caused HFpEF patients to be less active and no QoL improvement was observed , 47. In The sGCs riociguat has proven anti-fibrotic, anti-proliferative, and anti-inflammatory effects . HoweverPro-inflammatory cytokines, including TNF-α, IL-1, and IL-6, can lead to progressive LV dysfunction and remodeling, pulmonary edema, and cardiomyopathy . TNF-α pThe PDEi pentoxifylline improved LVEF and reduction of TNF-α and Fas/Apo-1 in HFpEF. However, TNF-α was still increased compared with healthy individuals and CRP was not measured . ThalidoStudies in HF patients with the aldosteric antagonists for the TNF receptor etanercept showed improved QoL, increased 6-min walk distance, and improved LVEF . HoweverIL-1β is primarily produced by inflammatory cells and a product of cytoplasmic innate immune complexes, the inflammasomes –64. CrohUric acid is known to predict mortality and the need for heart transplantation in HF patients. Uric acid is a by-product of purine metabolism via the xanthine oxidase pathway. Uric acid in its crystallized form can trigger IL-1β-mediated inflammation via activation of the NLRP3 inflammasome. The OPTIME-HF trial evaluated the xanthine oxidase inhibitor oxypurinol, which decreases production of uric acid, in HFrEF patients (LVEF ≤ 40%), but no improvement of morbidity, mortality, or QoL was seen .In the CANTOS trial, patients with prior MI and hs-CRP levels of > 2 mg/mL were treated with the IL-1β neutralizing antibody canakinumab once every 3 months and had a median follow-up of 3.7 years, leading to less HHF . In a suRecently, accumulation of unfolded proteins was discovered in both humans and animal models with HFpEF , and unfMethotrexate is a disease-modifying antirheumatic drug and is primarily used in rheumatoid arthritis. However, it was associated with reduced cardiovascular events, including HHF . In a smHigh-density lipoprotein (HDL) and its main protein component apolipoprotein A-I (ApoA-I) have immunomodulatory properties, which pose an interesting target in patients with HFpEF . ExperimB cell responses are crucial in host defense and tightly regulated by our immune system. However, in HFpEF, systemic inflammation combined with exposure of cardiac proteins by damaged or dead cardiomyocytes may break immune tolerance and result in the activation of autoreactive T and B cells . B cell A key feature of B cells is the production of immunoglobulins, and autoreactive B cells can produce antibodies targeting myocardial proteins . BindingProgenitor cells, including mesenchymal stromal cells (MSC) and cardiac progenitor cells, are able to improve cardiac function, despite poor cardiac engraftment , 100. BeT cells are found in cardiac tissue of HF patients , 111 andPrevalence of HFpEF differs between sexes and races , and mosMany questions can be raised on patient selection for HFpEF trials. Given the very heterogeneous HFpEF population, it might not be sensible to aim for a generalized therapy. We should rather consider the sex differences, differences in LVEF, presence of comorbidities, degree of LVDD, and levels of NT-proBNP, troponin, and CRP . As manyThe translational challenge to bring promising therapies from experimental models to HFpEF patients as seen with methotrexate use highlights the need for suitable preclinical models, which implements several aspects of the syndrome. Until now, development of new therapies based on underlying disease mechanisms is progressing slowly, as animal models of HFpEF are not yet well established and mimic either one or a selection of risk factors, such as LVH, hypertension, ageing, obesity, or diabetes , 119. BeImmunomodulation in HFpEF holds encouraging results, but new (immunomodulatory) therapies based on disease mechanisms call for better experimental models for HFpEF. With this review, we aimed to provide an overview of the current state and future perspectives of immunomodulatory therapies for HFpEF based on preclinical experimental models and early clinical data. Promising future therapies include IL-1β antagonists , which show improvement of exercise capacity and QoL and reduce inflammation upon HFpEF , 67. AddESM 1(DOCX 24 kb)


Tumour necrosis factor (TNF) inhibitors are used in the treatment of certain autoimmune diseases but given the role of TNF in tumour biology and atherosclerosis, such therapies may influence the risk of cancer and cardiovascular disease. We conducted a Mendelian randomization study to explore whether TNF levels are causally related to cardiovascular disease and cancer.Single-nucleotide polymorphisms associated with TNF levels at genome-wide significance were identified from a genome-wide association study of 30 912 European-ancestry individuals. Three TNF-associated single-nucleotide polymorphisms associated with higher risk of autoimmune diseases were used as instrumental variables. Summary-level data for 14 cardiovascular diseases, overall cancer and 14 site-specific cancers were obtained from UK Biobank and consortia.Genetically-predicted TNF levels were positively associated with coronary artery disease (odds ratio (OR) 2.25; 95% confidence interval (CI) 1.50, 3.37) and ischaemic stroke , and inversely associated with overall cancer , breast cancer , and colorectal cancer . There were suggestive associations of TNF with venous thromboembolism , endometrial cancer , and lung cancer .This study found evidence of causal associations of increased TNF levels with higher risk of common cardiovascular diseases and lower risk of overall and certain cancers. Tumour necrosis factor (TNF) is a pro-inflammatory cytokine secreted primarily by immune cells. It is involved in a broad range of both homoeostatic and pathophysiological processes, such as immunity, inflammation, cell proliferation, apoptosis and lipid metabolism. As such, anti-TNF agents have become cornerstone in the treatment of autoimmune inflammatory conditions such as rheumatoid arthritis and inflammatory bowel disease. However, the potential therapeutic, or even deleterious, effects of targeting TNF in other inflammatory conditions, such as cardiovascular disease and cancer remains equivocal. Mendelian randomization (MR) is an epidemiological approach using genetic variants as instrumental variables for an exposure to strengthen the causal inference in an exposure-outcome association by reducing residual confounding and reverse causality.In the present MR study, we provided the first causal evidence of positive associations of TNF levels with atherothrombotic disease (coronary artery disease and ischaemic stroke) and venous thromboembolism. Furthermore, we revealed inverse associations of TNF levels with risk of overall cancer and several site-specific cancers . We confirmed that higher TNF levels were strongly associated with established TNF-driven diseases (rheumatoid arthritis and inflammatory bowel disease) which added strong support to the validity of the genetic instrument used and the reliability of our findings.This study reveals evidence of causal associations of increased TNF levels with higher risk of common cardiovascular diseases and lower risk of overall and certain cancers. These results may inform decisions concerning potential benefits and risks of TNF inhibitor therapy. In detail, clinicians need to assess potential increased cancer risk derived from anti-TNF therapy usage especially amongst individuals with inherited or acquired high risk of cancer, and in addition, may use anti-TNF medicine as a potential prevention approach for people with excessive cardiovascular risk and a potential treatment strategy for patients with impaired cardiovascular condition. The study also indicates that randomized controlled trials are warranted to verify our findings and comprehensively evaluate the benefits and risks of anti-TNF therapy in populations with different health conditions.Alt-text: Unlabelled box1Tumour necrosis factor (TNF) is a pro-inflammatory cytokine secreted primarily by immune cells. It is involved in a broad range of both homoeostatic and pathophysiological processes, such as immunity, inflammation, cell proliferation, apoptosis and lipid metabolism Atherosclerosis is a chronic inflammatory disease of the arterial wall, driven by immune cells and cytokines at all stages, and, TNF-deficient mice have reduced plaque size Cancer is characterized by uncontrolled cell proliferation and survival. As a pro-inflammatory cytokine, TNF can promote all stages of carcinogenesis including survival, angiogenesis, and metastasis. TNF levels are raised in multiple cancer types, are reduced by chemotherapy and the reduction is associated with patient outcomes Utilizing genetic variants as instrumental variables for an exposure , Mendelian randomization (MR) can improve the causal inference of an exposure-outcome association Here, we aimed to evaluate the CVDs and cancers that are causally associated with TNF levels and which could be targeted with TNF-modifying therapies. We conducted a two-sample Mendelian randomization study to explore the associations of genetically predicted TNF levels with risk of 14 CVDs, overall cancer, and 14 site-specific cancers. To validate the instrumental variables, we assessed whether genetically predicted TNF levels were associated with higher risk of rheumatoid arthritis and inflammatory bowel disease.22.1This is a two-sample MR study design based on summary-level data. An MR analysis depends on the assumptions that the genetic variants: 2.2P<5 × 10−8) (Supplementary Table 1). A genetic instrument containing rs10744774, rs3184504 and rs7182229 was associated with an expected increased odds of rheumatoid arthritis and inflammatory bowel disease. The TNF-raising allele of rs2857602 was associated with lower odds of these autoimmune diseases and was regarded as an unreliable instrumental variable for TNF. We therefore used three SNPs  as instrumental variables for TNF levels in the primary analysis; all four SNPs were used in a supplementary analysis. The two SNPs on chromosome 12 (rs10744774 and rs3184504) were in modest linkage disequilibrium (r2 = 0.18) based on 1000 G reference panel. The genotype associations with TNF levels were adjusted for age2, sex, body mass index, and study-specific variables such as genetic principal components and relatedness A meta-analysis of genome-wide association studies (GWASs) of 25 cohorts encompassing 30 912 European-descent individuals identified four single-nucleotide polymorphisms (SNPs) associated with TNF levels at genome-wide significance ( × 10−8)  [12]. Tor2=1). From UK Biobank, we included CVDs and cancers with at least 1000 cases to ensure sufficient statistical power to detect moderate to strong associations. The SNP-outcome associations in UK Biobank and most consortia were adjusted for age, sex, and genetic principal components. Detailed information of included outcomes is displayed in Fourteen CVDs, overall cancer, and 14 site-specific cancers were included as outcomes in this MR study . Summary2.3http://www.phenoscanner.medschl.cam.ac.uk/). One or more of the SNPs related to TNF were associated with autoimmune diseases , various immune and blood cells, haemoglobin levels, hypothyroidism, diastolic blood pressure, total and low-density lipoprotein cholesterol, and height (Supplementary Table 2).To evaluate whether the exclusion restriction assumption is likely to hold, possible pleiotropic associations of the instrumental variables with other phenotypes were assessed by searching a database of human genotype-phenotype associations (PhenoScanner V2) ) as strong evidence of causal associations. Associations with p values below 0.05 but above 1.7 × 10−3 were treated as suggestive evidence of associations. All analyses were two-sided and performed using TwoSampleMR and MendelianRandomization packages in R 3.6.0.The inverse-variance weighted method with adjustment for correlations amongst the SNPs 2.5The funders had no role in study design, data collection, interpretation, or the decision to submit the work for publication.3Supplementary figure 1).The associations of TNF levels instrumented by three SNPs with the CVD and cancer outcomes are displayed in 4In the present MR study, we provided the first causal evidence of positive associations of TNF levels with atherothrombotic disease (coronary artery disease and ischaemic stroke) and venous thromboembolism. Furthermore, we revealed inverse associations of TNF levels with risk of overall cancer and several site-specific cancers . We confirmed that higher TNF levels were strongly associated with established TNF-driven diseases (rheumatoid arthritis and inflammatory bowel disease) which added strong support to the validity of the genetic instrument used and the reliability of our findings.4.1A correlation between a TNF-related SNP and CVD was found in a study with 587 patients, which showed an association between the TNFA rs1800629 gene variant and cardiovascular complications in patients with rheumatoid arthritis albeit confined within individuals carrying the rheumatoid shared epitope Atherothrombotic disease is a chronic inflammatory disease of the arterial wall and has been shown to be TNF-driven. A possible positive association of TNF with ischaemic stroke 4.2With regard to overall cancer risk, randomized controlled trials and observational studies assessing the effect of TNF inhibitor treatment, primarily in rheumatoid arthritis and inflammatory bowel disease patients, have yielded inconclusive results ,36. ThisTNFA−308 A allele was associated with a lower risk of breast cancer amongst European populations Previous studies of TNF levels in relation to risk of colorectal cancer are inconsistent. Carcinogenic effects have been suggested by a clinical study of 30 colorectal cancer patients in which the TNF gene was significantly overexpressed in cancerous tissue compared with adjacent normal colorectal tissue 4.3Increased risk of cardiovascular disease with genetically predicted high TNF level sheds light on the usage of anti-TNF medicine as a potential prevention approach for people with excessive risk of CVD and a potential treatment strategy for patients with impaired cardiovascular condition. In addition, clinicians need to assess the potential increased CVD risk derived from TNF therapy especially amongst individuals with inherited or acquired high risk of CVD. With regard to the observed protective effect of TNF on cancer, our study reveals two important clinical considerations. Firstly, it suggests recombinant TNF therapy as a potential therapy in such cancers, in particular colorectal and breast cancers. Phase 2 trials of recombinant TNF across a range of cancer types have so far not proven successful in causing tumour responses 4.4The MR design, which diminishes confounding and reverse causality, was the major strength of this study. Additionally, we comprehensively assessed the causal associations of TNF levels with a broad range of CVD and cancer outcomes. Data were mainly extracted from individuals of European ancestry, except for a few outcomes with a small portion of individuals of non-European ancestry. Moreover, the SNP-exposure and SNP-outcome estimates were adjusted for principal components for ancestry. Thus, population stratification bias is unlikely to have had an essential effect on our results. On the other hand, this population confinement, on a certain degree, compromised the generalizability of the study results to other populations, such as Asians, African Americans, etc. A major limitation is that the number of cases was few for some CVDs and site-specific cancers, which resulted in low precision of the estimates. Thus, we may have missed weak associations.The results of this MR study should be interpreted in light of the pleiotropic effects of TNF, which plays a role in a wide range of biological processes, such as immunity, inflammation, apoptosis, lipid metabolism, and coagulation This MR study found evidence of causal associations of increased TNF levels with higher risk of coronary artery disease, ischaemic stroke, and venous thromboembolism and decreased risk of overall, colorectal, breast, endometrial, and lung cancer. Along with previous observational studies ,10, the Study design: S.Y., P.C., M.B., M.V., S.K., A.M.M., A.L., S.B., S.C.L.; data acquisition and analysis: S.Y., A.M.M., S.B., S.C.L.; figures and writing S.Y.; reviewing and editing: S.Y., P.C., M.B., M.V., S.K., A.M.M., A.L., A.L., S.B., S.C.L.SCL reports grants from Swedish Research Council , grants from Swedish Research Council for Health, Working Life and Welfare , grants from Swedish Heart-Lung Foundation , during the conduct of the study. AMM reports grants from EC-Innovative Medicines Initiative (BigData@Heart), during the conduct of the study; .All other authors declare no conflicts of interest.