In this study, we designed a series of DNVs, based on an insulin–phospholipid complex (IPC-DNVs), to investigate the influence of drug dose, buccal administration methods, and key quality characteristics of IPC-DNVs for buccal delivery. IPC-DNVs showed a non-linear dose–response relationship between 8 and 12 IU. There was no significant effect of drug delivery site  or ligation time (15 or 30 min) on buccal absorption of IPC-DNVs. However, the area above the curve of reduction in blood glucose level overtime (AAC0–6h) for oral mucosa administration was significantly higher than that for buccal mucosa administration. Increasing the drug concentration in IPC-DNVs led to a decrease in AAC0–6h. This might be due to local leakage of DNVs, while squeezing through biological barriers with high concentration of insulin, thus hindering the subsequent delivery of DNVs. IPC-DNVs, measuring 80–220 nm in size, did not significantly affect AAC0–6h. However, when the size was increased to approximately 400 nm, AAC0–6h decreased, thus suggesting that IPC-DNVs with reasonable size were more effective. Additionally, increased deformability of IPC-DNVs might cause drugs to leak easily, thus reducing the promoting effect of buccal absorption. Our results clarified the effect of characteristics of IPC-DNVs on buccal delivery in vivo and provided meaningful support for the design of dosage form of DNVs.Deformable nanovesicles (DNVs) have been used in the buccal delivery of biomacromolecules due to their ability to enhance drug penetration. However, no breakthroughs have been made until now due to limited understanding of the factors affecting  However, most biological macromolecules, such as proteins and peptides, must be administered by injection due to physio-chemical and biological restrictions, often leading to poor patient compliance. Hence, the need to realize alternative noninvasive routes to administer biomacromolecules has always been an important issue in the field of drug research.in vivo. It is crucial to bridge this gap to optimize the formulation of DNVs and decrease the risk of failure in clinical trials.Among the different alternative noninvasive routes of drug delivery, such as buccal delivery, transdermal delivery , and the key quality characteristics of IPC-DNVs. Normal rabbit esophageal ligation was used as the animal model to assess the hypoglycemic effect after buccal administration of IPC-DNVs. The ability of buccal delivery of IPC-DNVs was evaluated by calculating the area above the curve of reduction in blood glucose level over time (AAC0–6h) and the relative pharmacological bioavailability (Fp).In our previous study, we developed a novel DNVs loaded with IPC (IPC-DNVs) for buccal absorption. These IPC-DNVs had a high drug entrapment efficiency and high relative pharmacological bioavailability (15.53%) . Lecithin  was obtained from Shanghai Toshisun Biology & Technology Co. Ltd. . Sodium deoxycholate (SDC), D-(+)-Trehalose and sodium pentobarbital were obtained from Sigma-Aldrich . α-Lactose monohydrate was purchased from Aladdin . Other chemicals and solvents used were of analytical or chromatographic grade.2.2.Male big-eared Japanese rabbits were purchased from Beijing HFK Bioscience Co. Ltd. . All rabbits were raised in the Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College . The experiments were performed with the approval of the Laboratory Animal Care and Use Committee of the Peking Union Medical College.2.3.2.3.1.IPC-DNVs were prepared using the thin-film hydration method, as described in our previous study .2.3.3.To obtain IPC-DNVs with different drug concentrations and sizes, lyophilized IPC-DNVs were prepared using the freeze-drying technology, as described in our previous study  were prepared by dissolving lyophilized IPC-DNVs in different volumes of distilled water. IPC-DNVs with different sizes  were prepared by changing the amount and proportion of the cryoprotectant  in lyophilized IPC-DNVs.2.4.2.4.1.The average particle size and size distribution were determined by dynamic light scattering technique, using a Zetasizer Nano ZSP , at an angle of 173° at 25 °C. Prior to analysis, the samples were diluted ten times with distilled water.2.4.2.The entrapment efficiency of deformable nanovesicles based on insulin–phospholipid complex (IPC-DNVs) was determined by a fast ultrafiltration method, as described previously  of IPC-DNVs. Briefly, 1 mL of each prepared IPC-DNVs suspension was placed in a centrifugal filter tube  and centrifuged at 4000 rpm for 40 min to separate free and entrapped insulin in the IPC-DNVs. After ultrafiltration, the amount of insulin in the ultrafiltrates was measured using RP-HPLC. Subsequently, the centrifugal filter tube was washed thrice with PBS at 2500 rpm for 10 min and the ultrafiltrate was collected and analyzed using the RP-HPLC to determine the drug absorbed by the centrifugal filter tube. EE% was calculated according to the following equation:2.4.3.2/s) was calculated according to the following formula .The deformability of IPC-DNVs was determined using a stainless-steel pressure filter device. A constant pressure of 0.45 MPa was applied for 5 min to extrude the IPC-DNVs through the 50 nm polycarbonate membranes. The size of IPC-DNVs was measured using the size analyzer, as mentioned previously. The deformability index of the IPC-DNVs  from Spectrum Labs (USA), and introduced into 15 mL of release media at 37 °C, with a rotation speed of 80 rpm. For the release studies, 15 mL of sample was taken out and replaced with 15 mL fresh medium at, 1, 3, 8 and 12 h. Each study was carried out in triplicate and the concentrations of insulin was determined using reverse phase high-performance liquid chromatography (RP-HPLC) with an Agilent Technologies 1200 series HPLC system  and a 300SB-C18 column . The drug release profile was compared with that of insulin solution.The drug release of IPC-DNVs 2.6.The stability of insulin solution and IPC-DNVs, against degradation by isolated aminopeptidase N was evaluated by incubating them with artificial saliva, containing isolated aminopeptidase N. Briefly, 100 µL of IPC-DNVs suspension was diluted to 10 mL with artificial saliva, containing aminopeptidase N (0.625 μg/mL), and incubated in a shaking water bath (100 rpm) at 37 °C. Aliquots (500 μL) were withdrawn at fixed intervals and put in an ice bath for 2 min to stop the reaction. Thereafter, the samples were analyzed using the RP-HPLC assay. Each experiment was performed in triplicate.2.7.The relative pharmacological bioactivity of insulin delivered via the transmucosal route by IPC-DNVs in different drug doses, delivery sites, ligation times, drug concentrations, sizes, and deformabilities was evaluated in normal rabbits by assessing the hypoglycemic effects.Insulin solution subcutaneous administration group (INS-subcutaneous): 3 mg/mL, 1 IU/kg.Insulin solution buccal administration control group (control): 3 mg/mL, 10 IU/kg.IPC-DNVs group : drug dose ; delivery site , 10 IU/kg; ligation time , 10 IU/kg; buccal administration.IPC-DNVs with different quality characteristics group: drug concentration  ; size ; deformability ; 10 IU/kg; buccal administration.Before the experiment, the rabbits were randomly divided into groups of three as follows:After fasting for 2 h, the rabbits were weighed and anesthetized with pentobarbital  through the ear margin vein. During anesthesia, the animals were restrained in a supine position to a board. The esophagus was surgically ligated to prevent swallowing. The drug solution for buccal administration  was divided into four parts; two parts for administration via sublingual mucosa, and two parts for administration via left and right buccal mucosa. The sublingual mucosa and the buccal mucosa groups were administered only to the sublingual mucosa or buccal mucosa. After administration of the drug solution for 30 min (except the group with ligation time of 15 min), the esophagus was untied, and the response of the animals was observed. Blood samples were collected from the ear veins at predetermined time intervals  after DNVs administration and blood glucose levels were measured using a Blood Analyzer . Only the animals with initial blood glucose value between 7 and 10 mmol/L and those which maintained a constant body temperature throughout the experiment were selected for buccal administration. At the end of the experiment, the animals were euthanized by administering an intravenous overdose of pentobarbital (2%).buccal and Doses.c. are the insulin doses administered through the buccal or subcutaneous route, respectively, and AAC is the area above the curve of the reduction in blood glucose level over time.The relative pharmacological bioavailability (Fp) of insulin, after buccal administration, was calculated using the following equation . The drug release profiles  indicate4.4.0–5h and Fp values of all the doses. A significant difference was observed in the respective AAC0–5h and Fp values of doses 8 IU/kg and 10 IU/kg or 12 IU/kg, suggesting a non-liner dose response relationship in the range of 8–12 IU/kg. Interestingly, a relative lower Fp was observed at a dose of 8 IU/kg, which may be related to the mechanism of self-regulating glucose balance in normal animals , after buccal administration are shown in s Benzo, . When th4.5.0–6h and Fp values are shown in 0–6h and Fp values. Sublingual and buccal mucosa have become the key routes for drug delivery due to their non-keratinized nature. However, different oral sites with the same pattern of epithelial differentiation (non-keratinization) do not necessarily exhibit the same permeability properties  remains the ideal mode for buccal delivery.The hypoglycemic effects after sublingual mucosal, buccal mucosal, and oral mucosal administration of IPC-DNVs are shown in 4.6.0–6h and Fp values are shown in in vivo pharmacokinetics or pharmacodynamics studies.Drugs administered through buccal delivery cannot stay at the administration site for a long time due to mechanical stress and the continuous secretion of saliva , and the key quality characteristics of IPC-DNVs  on buccal delivery. We found that IPC-DNVs can be fully absorbed in 15 min and that they showed a non-linear dose–response relationship between insulin dose of 8 IU/kg and 12 IU/kg. The delivery site and ligation time had no influence on buccal absorption. However, the AAC0–6h of oral mucosal administration was significantly higher than that of the buccal mucosal administration, suggesting that oral mucosal administration is more suitable for the buccal delivery of DNVs. Moreover, increased drug concentration led to decrease in AAC0–6h and Fp. This may be due to the local leakage and accumulation of IPC-DNVs, with high concentration of insulin squeezing through the biological barrier hindering the delivery of subsequent IPC-DNVs. In addition, in terms of two key characteristics of IPC-DNVs, increasing size beyond a certain range (80 nm–220 nm) had no significant effect on the oral absorption. However, when the size was increased to 400 nm, the AAC0–6h decreased significantly, indicating that in the prescription design of DNVs, the size should be controlled within a reasonable. Further, increasing deformability above the existing deformability range was found to be ineffective in promoting absorption. It is also necessary to optimize the deformability, combined with in vivo efficacy for better drug delivery through the buccal route. Our results not only clarified the characteristics of IPC-DNVs on buccal delivery, but also provide meaningful evidence for dosage form design and administration methods for buccal delivery system of IPC-DNVs.


J. Appl. Cryst. (2021), 54, 1029–1033]Jill Trewhella Jill Trewhella [ are corrected.Errors in the article by  Journal of Physics – Conference Series, Vol. 247, 2010. The correct numbers of papers and pages for SAS2002 should be 106 papers, 495 pages; for SAS-99 should be 105 papers, 445 pages; and for SAS-96 should be 63 papers, 327 pages.Table 1 in the article by Trewhella 2021 containsIn regard to the origins of canSAS, it was originally an initiative of staff at the Institut Laue–Langevin, European Synchrotron Radiation Facility (ESRF) and ESRF Collaborative Research Groups that came to be taken up by the broader SAS community with support at the SAS conferences.


Tumour necrosis factor‐α (TNF‐α), a crucial cytokine, has various homeostatic and pathogenic bioactivities. The aim of this study was to assess the neuroprotective effect of ketamine against TNF‐α‐induced motor dysfunction and neuronal necroptosis in male C57BL/6J mice in vivo and HT‐22 cell lines in vitro. The behavioural testing results of the present study indicate that ketamine ameliorated TNF‐α‐induced neurological dysfunction. Moreover, immunohistochemical staining results showed that TNF‐α‐induced brain dysfunction was caused by necroptosis and microglial activation, which could be attenuated by ketamine pre‐treatment inhibiting reactive oxygen species production and mixed lineage kinase domain‐like phosphorylation in hippocampal neurons. Therefore, we concluded that ketamine may have neuroprotective effects as a potent inhibitor of necroptosis, which provides a new theoretical and experimental basis for the application of ketamine in TNF‐α‐induced necroptosis‐associated diseases. We also evaluated the effects of ketamine on the long‐term physical functions of post‐TNF‐α mice, hippocampal damage, neuronal loss, and oxidative stress using the open field test, Nissl staining, immunofluorescence, flow cytometry and western blotting. Furthermore, the present study addressed the possible contribution of the clinical transformation of ketamine to the pathological mechanism in the SIRS model.22.1Adult male C57BL/6J wild‐type mice  were bred in specific pathogen‐free conditions, and housed in air‐conditioned, temperature‐controlled rooms with a 12 hours/12h light/dark cycle , 22‐25°C ambient temperature, and ad libitum access to food and water in the Laboratory Animal Center of Xiamen University. Prior to experimentation, animals were allowed to habituate to the new housing environment for 7 days. All procedures and animal use were approved by the Animal Ethics Committee of Xiamen University . Every effort was made to minimize stress to the animals.2.2The mice were randomly assigned into three groups treated as follows—TNF‐α group: mice intravenously received murine TNF‐α (eBioscience) (10 μg/mouse) in 200 μL of endotoxin‐free phosphate‐buffered saline (PBS) (pH 6.8); ketamine pre‐treated group: mice were intraperitoneally injected with ketamine  (20 mg/kg) 20 minutes prior to TNF‐α injection; and control group: mice intravenously received an equal volume of Dulbecco's PBS (vehicle) 20 minutes before the challenge with TNF‐α  and 50 cm high walls made of grey polyvinyl chloride plastic. On the day of the test, mice were transported to the testing room and left in their home cages for 1 hour prior to testing. At the initiation of each session, a mouse was placed in a particular corner of the arena and allowed to explore for 5 minutes. The apparatus was cleaned with 70% ethanol prior to testing each animal. Time was subsequently recorded, and each mouse was allowed to explore the testing area for 10 minutes. The statistical data were recorded and analysed using the Noldus EthoVision XT system (Ugo Basile SLE).2.4The mice were sacrificed under isoflurane anaesthesia 48 hours after tail‐vein injection of TNF‐α, and then perfused through the ascending aorta with 100 mL of normal saline followed by 100 mL of 4% (w/v) paraformaldehyde in 0.1 M PBS (pH 7.4). The brain of each mouse was dissected and removed from the skull, and resected‐brain tissue was fixed in 4% paraformaldehyde for 24 hours at 4°C. Tissues were subsequently stored overnight in 30% sucrose phosphate buffer until the tissue sank to the bottom of the solution. Tissue sections (8 μm) were cut in the coronal plane using a freezing microtome  and mounted on gelatine‐coated slides. The sections were then stained with 0.1% cresyl violet solution  at 37°C for 30 minutes. The sections were subsequently rinsed in distilled water, rehydrated using a descending alcohol series, and checked microscopically for optimal results. Tissues were dehydrated in 100% ethanol, washed in xylene and finally scanned using an Olympus BX53 Scanner (Olympus Corporation) at a magnification of ×20/×4.2.5Brain tissue containing the hippocampus was embedded in optimal cutting temperature compound and then cut into thick coronal sections (30 μm) using a freezing microtome. Sections were collected in dishes containing 0.01 M PBS. Free‐floating sections were permeabilized in permeabilization buffer containing 0.25% (v/v) Triton X‐100 (Beyotime Institute of Biotechnology) for 15 minutes and subsequently blocked for 30 minutes with 3% bovine serum albumin (Beyotime Institute of Biotechnology) in 0.01 M PBS with gentle agitation. Tissues were subsequently incubated with the following primary antibodies: mouse anti‐RNA binding Fox‐1 homolog 3 (NeuN) , rabbit anti‐MLKL (phospho S345) , rabbit anti‐ionized calcium binding adaptor molecule 1 (Iba1)  and rat anti‐CD68  in primary antibody dilution medium (Beyotime Institute of Biotechnology) overnight at 4°C. The sections were then incubated with a mixture of the following secondary antibodies: Alexa 488‐donkey anti‐mouse IgG ; Alexa 594‐donkey anti‐rabbit IgG ; Alexa 488‐donkey anti‐rabbit IgG , and Alexa 594‐donkey anti‐rat IgG  in secondary antibody dilution medium (Beyotime Institute of Biotechnology) for 2 hours at room temperature. Thereafter, sections were mounted onto gelatine‐coated glass slides, air‐dried and cover‐slipped with anti‐fade mounting medium with dihydrochloride (Beyotime Institute of Biotechnology). Samples were gently washed thrice with 0.01 M PBS for 10 minutes before each step. Sections were observed using a fluorescence microscope .2.62. Recombinant TNF‐α was purified as described previously.Mouse hippocampal HT‐22 cells were cultured in DMEM  supplemented with 10% foetal bovine serum  and 1% antibiotic/antimycotic  at 37°C in 5% CO2.7HT‐22 cells were treated with the indicated drug for ~12 hours. Cell survival was determined using the Cell Titer‐Glo Luminescent Cell Viability Assay kit (Promega Corporation) according to the manufacturer's protocols. Luminescence was recorded using the Omega POLAR Star (BMG Labtech GmbH).2.8Reactive oxygen species levels were measured using a fluorometric intracellular ROS kit , according to the manufacturer's protocols. Stained cells were viewed using a fluorescence microscope. Cells were treated with TNF‐α/z‐VAD or TNF‐α/z‐VAD/ketamine for 3 hours, and fluorescence was measured using a CytoFLEXS flow cytometer .2.9HT‐22 cells were collected and centrifuged in 0.01 M PBS solution, and the supernatant was removed. The pellets were dissolved in PRO‐PREP solution by vortexing and ultrasonication for western blot analysis. Briefly, proteins were separated using 10% SDS‐PAGE gels along with the marker  and then transferred onto PVDF membranes . The membranes were blocked using 5% skim milk for 40 minutes to reduce non‐specific binding and then incubated with primary antibodies  at 4°C overnight. Subsequent to being incubated with mice /rabbit  horseradish peroxidase–conjugated secondary antibodies  for 2 hours at room temperature, protein bands were visualized using an enhanced chemiluminescence detection kit . ImageJ software  was used to determine the band intensities to reflect the expression of each protein.2.10P <.05 was considered to indicate a statistically significant difference.Independent experiments were performed in duplicate or triplicate, and data were analysed using GraphPad Prism 7.0 . Data are presented as mean ± standard error of the mean unless indicated otherwise. One‐way analysis of variance and the Bonferroni multiple comparison post hoc test were used. 33.1Tumour necrosis factor‐α is a crucial mediator of neuroinflammation, and elevated levels of TNF‐α are associated with various neurodegenerative conditions and contribute to neurotoxicity.3.2To further explore whether TNF‐α‐induced motor dysfunction was associated with brain injury, Nissl staining of neurons was performed to evaluate neurological dysfunction and TNF‐α‐induced neuronal damage. It was observed that administration of TNF‐α caused a reduction in neuronal density in the hippocampus, particularly in the carbonic anhydrase 3 region. At the same time, pre‐treatment with ketamine prevented the TNF‐α‐induced loss of hippocampal neuron number and density in vivo Figure . We also3.3The effect of a single systemic challenge with TNF‐α on C57BL/6J mice was examined using the microglial activation markers Iba‐1 and CD68. Confocal microscopy results showed an increase in Iba‐1‐CD68 double‐positive cells after TNF‐α administration, which was attenuated by ketamine pre‐treatment Figure . Overall3.4The subsequent experiments were designed to elucidate the modulatory effects of ketamine on the necroptosis signalling cascade. HT‐22 hippocampal cells have been reported to be sensitive to TNF‐α only upon caspase blockage and subsequently undergo necroptosis.3.5In vitro ROS assays revealed that after 4 hours, there was enhanced expression of ROS in TNF‐α/z‐VAD‐treated HT‐22 cells compared with that in the control group Figure . In addi4The present study revealed that surviving mice with TNF‐α‐induced SIRS had motor function decline, and the open field test data indicated that these mice had problems related to anxiety and exploration. However, based solely on the aforementioned observations, it could not be discerned whether this phenomenon was a result of decreased activity levels caused by dyskinesia. Furthermore, TNF‐α‐induced motor dysfunction was attenuated by pre‐treatment with ketamine. We also demonstrated that TNF‐α‐induced brain injury led to neuronal necroptosis and microglial activation in C57BL/6J mice. Collectively, these data indicate that the neuroprotective effect of ketamine against TNF‐α‐induced necroptosis was mediated via inhibition of ROS production and MLKL phosphorylation in hippocampal neurons.Systemic inflammatory response syndrome, an over‐reactive immuno‐inflammatory response, represents a significant disease burden and is associated with long‐term physical, cognitive and psychosocial morbidity.Classically, necroptotic cell death is known to be characterized by disrupted plasma membrane; however, the downstream events leading to membrane collapse are far from being clarified. ROS production and accumulation have been suggested to be required for necroptosis in cells such as L929, the human 5‐8F NPC cell line and HT‐29 human colon cancer cells.Ketamine is a traditional narcotic analgesic and psychotomimetic drug with abuse potential in medical practice. It was considered a notable and attractive “drug of the year” when a rapid and sustained anti‐depressant profile, with selective rescue of eliminated spines and restoration of coordinated activity in multicellular ensembles, was revealed.Microglia, the resident macrophages of the CNS, are activated by a limited number of stimuli, including lipopolysaccharide, which can mediate neuroinflammation.Overall, this study provided experimental evidence that necroptosis of hippocampal neurons may be induced by TNF‐α both in vivo and in vitro, and could be attenuated by ketamine via inhibition of ROS production and MLKL phosphorylation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Lu Wang: Data curation (lead); Formal analysis ; Investigation (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Bin Deng: Conceptualization ; Funding acquisition ; Project administration (lead); Writing‐review & editing (lead). Panpan Yan: Formal analysis ; Methodology ; Writing‐original draft . Huanghui Wu: Methodology ; Validation ; Writing‐review & editing (supporting). Chunhui Li: Data curation ; Methodology . Hongrui Zhu: Investigation ; Methodology . Jiwei Du: Conceptualization (lead); Project administration ; Validation . Lichao Hou: Conceptualization (lead); Funding acquisition (lead); Project administration ; Resources ; Writing‐review & editing (supporting).Fig S1‐S2Click here for additional data file.


Recombinant MrNV capsid protein has been shown to effectively deliver plasmid DNA and dsRNA into Sf9 insect cells and shrimp tissues. To extend its application to cancer cell-targeting drug delivery, we created three different types of chimeric MrNV virus-like particles (VLPs)  that have specificity toward the epidermal growth factor receptor (EGFR), a cancer cell biomarker, by incorporating the EGFR-specific GE11 peptide at 3 different locations within the host cell recognition site of the capsid. All three chimeric MrNV-VLPs preserved the ability to form a mulberry-like VLP structure and to encapsulate EGFP DNA plasmid with an efficiency comparable to that previously reported for normal MrNV (N-MrNV). Compared to N-MrNV, the chimeric R-MrNV and E-MrNV carrying the exposed GE-11 peptide showed a significantly enhanced binding and internalization abilities that were specific towards EGFR expression in colorectal cancer cells (SW480). Specific targeting of chimeric MrNV to EGFR was proven by both EGFR silencing with siRNA vector and a competition with excess GE-11 peptide as well as the use of EGFR-negative colorectal cells (SW620) and breast cancer cells (MCF7). We demonstrated here that both chimeric R-MrNV and E-MrNV could be used to encapsulate cargo such as exogenous DNA and deliver it specifically to EGFR-positive cells. Our study presents the potential use of surface-modified VLPs of shrimp virus origin as nanocontainers for targeted cancer drug delivery. This is simply due to their excellent physical properties such as self-assembling to form nano-sized symmetrical particles, controllable disassembly/reassembly, and practical surface functionality modifications through either chemical modification or genetic reengineering2. These surface modified VLPs, so-called “chimeric VLPs”, are intended for multiple research purposes. Interior modification mostly aims at improving both capabilities of VLPs to encapsulate their cargoes more effectively as well as traceability of these particles inside the targeted cells. On the other hand, exterior modification is aimed for either triggering immune response of the host cells or enhancing specific recognition of VLPs towards the desirable targeted cells or tissues3. The latter has been extensively reported with the objective to specifically deliver therapeutic compounds into the targeted cancer cells4. To date, several functional ligands or molecules that are known for their specific binding to cancer cells, e.g., antibody fragments5, short peptides6, DNA/RNA aptamers7 have been used to generate chimeric VLPs. Likewise, several kinds of VLPs have been used in the clinical trial as the hosted VLPs with their own intrinsic physical benefits including HBVc10, MS211, Qβ13, and CPMV14.Among several delivery systems at the nanoscale, virus-like particles (VLPs) designed from capsid proteins of many non-enveloped viruses are considered the most outstanding biomaterials that that surpass other biological containersMacrobrachium rosenbergii nodavirus (MrNV) is a shrimp infectious, non-enveloped virus with an icosahedral symmetry (T = 3) and a size of 26–27 nm in diameter20. Interestingly, only the RNA2 gene  is responsible for encoding capsid protein which makes it rather straightforward to produce recombinant protein to generate the self-assembling VLP21. Apart from this simplicity, its ability to withstand harsh conditions, particularly, strong digestive enzyme digestion as well as its controllable particle assembly has brought it an excellent deliverable nanocontainer. Principally, MrNV-VLPs could22. The controllability of MrNV-VLPs to encapsulate nucleotide-based agents (DNA vector and dsRNA) against the severely infectious virus through the uses of EGTA + DTT  is also well established both in vitro and in vivo23.24  to the MrNV-VLPs significantly increases the levels of natural killer and cytotoxic T cells as well as enhances interferon-gamma (IFN-γ) secretion in mice26. Notably, most of the placement of foreign epitopes into the VLP exterior is at any individual 4 loops of the protruding (P) domain  in several human cancer cells1. The resulting chimeric EGFR-targeting MrNV VLPs that we created exhibited great potential as nano-containers for targeted delivery of therapeutic compounds to cancer cells.MrNV capsid consists of shell (S) domain and protruding (P) domain, the latter of which is the host cell recognition site24 Fig. . It has ain Fig.  located 27. The GE11 peptide was placed at 3 different sites in the MrNV protruding (P) domain, creating 3 forms of chimeric VLPs  protein folding prediction server28. The MrNV template (PDB#6H2B) was chosen based on the scores obtained from fold recognition and threading algorithms in Phyre2. The icosahedral models of the chimeric MrNV-GE11-VLPs were created based on the icosahedral structure of N-MrNV as a referencing atomic structure displaying the highest secondary structure agreement with T = 3 quasi-equivalent29. All visualization was performed using the UCSF Chimera software (http://www.cgl.ucsf.edu).We designed chimeric GE11-MrNV-VLPs using the sequence information from normal MrNV (N-MrNV) capsid protein (Genbank accession No. EU150129) and the 12 amino-acid sequences of GE11 peptide, YHWYGYTPQNVIEscherichia coli (BL21) was performed by the heat shock method  and immediately followed by ice incubation (5 min). Transformed E. coli were inoculated in SOC  medium , selected on the LB agar plates containing ampicillin (50 g/ml). The positive clones, verified by restriction endonuclease digestion and DNA sequencing, were transformed into E. coli and further inoculated in LB broth containing 50 µg/ml of ampicillin overnight at 37 °C until the absorbance of 0.6–0.8 at 600 nm (A600) was reached. Protein expression was induced by adding 1 mM IPTG to the culture followed by an overnight incubation . After induction, the cells were pelleted by centrifugation  and resuspended in PBS containing 500 mM NaCl and 2 mM phenylmethylsulfonyl fluoride (PMSF). The cells were ruptured by sonication  and centrifuged . The collected supernatant was loaded onto a Nickel column chromatography. After several washes with a washing buffer , the bound proteins were eluted with elution buffer . The proteins were dialyzed against PBS overnight at 4 °C and further subjected to a sucrose gradient (10–40%) ultracentrifugation . Each fraction was collected and analyzed by SDS-PAGE and immunoblotting. The concentration of purified GE11-MrNV-VLP was measured by the NanoDrop2000 Spectrophotometer .All of the chimeric MrNV-GE11 capsid sequences were synthesized and ligated into the pET16b expression vector . Transformation of the recombinant vector into competent Recombinant chimeric GE11-MrNV-VLPs obtained from all purification steps were resolved in 12.5% SDS-PAGE and Coomassie blue staining. The proteins were either transferred to a polyvinylidene fluoride (PVDF) membrane  or directly blotted onto the membrane using a slot blot device They were .submerged in 2% BSA and 5% skim milk to block non-specific antibody staining and washed with PBS containing 0.05% Tween (PBST). Thereafter, transferred proteins were probed with either monoclonal anti-MrNV or anti-His or anti-EGFR or anti-GAPDH antibody at the dilution of 1:2000  followed by a goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) at a dilution of 1:5000. After extensive washing with 0.05% PBST, the reactivity of antibody-antigen was detected by an enhanced chemiluminescence method using an ECL kit .For VLP structure verification by transmission electron microscopy (TEM), 10 µl of purified chimeric MrNV-VLP was pipetted onto the carbon-coated EM grids  and allowed to stand for 30 s. Non-adhering VLPs were washed away by droplets of Milli-Q water and blotted away by a filtered paper. The adhered VLPs were stained with 2% uranyl acetate for 30 s and immediately blotted away by a filtered paper. Thereafter, the samples were viewed under a JEOL1230 transmission electron microscope operated at 120 kV .Colorectal cancer cell lines, SW480 (EGFR-positive) and SW620 (EGFR-negative) were maintained in RPMI 1640 + Glutamax I medium supplemented with 10% fetal bovine serum (FBS). The breast cancer cell line, MCF-7 (EGFR-negative) were maintained in DMEM medium supplemented with 10% FBS.5 cells/well) and cultured for 24 h before the binding and internalization assays. For binding experiments, the cells were pre-chilled at 4 °C and then incubated either with N-MrNV-VLP (control) or with chimeric GE11-MrNV-VLPs at the final concentration of 50 μg/ml or approximately 4.6 × 105 VLP particles/ml at 4 °C for 2 h with gentle shaking. The excess or loosely bound VLPs were then washed from the cells by ice-cold culture media.Cells at ~ 90% confluence were detached and seeded in 6-well plates (2 × 10To test the specificity of EGFR-mediated VLP binding by siRNA silencing of EGFR SW480 cells were transfected with either EGFR siRNA (5′-AAGCTCACGCAGTTGGGCACT-3′) or control siRNA   using a lipofectamine RNAiMAX reagent  according to the manufacturer’s protocol for reverse transfection. Two days after siRNA transfection, cells were subjected to the binding assay as described above.To verify the EGFR-specific binding of the GE11-MrNV VLPs by GE11 peptide competition assay, an excess amount of GE11 peptide or a scrambled peptide  was included in the cell culture medium (100 μg/ml) before the incubation of the cells with chimeric VLPs. Following the preincubation with free competing peptide, the cells were subjected to the VLP binding assay as described above. Subsequently, the cells were fixed with 4% paraformaldehyde, washed in PBS, and further processed for analyses by indirect immunofluorescence (IIF) or flow cytometry.For VLP synchronized internalization test, following the VLP binding at 4 °C (without or with peptide competition) as described above, the VLP-containing medium was replaced with a new culture medium without VLPs at 4 °C. The cells were then incubated at 37 °C for 30 min in an incubator and further processed for IIF or flow cytometry as above.22. Briefly, purified chimeric VLPs were treated with 1 mM ethylene glycol tetraacetic acid (EGTA) in the presence of 20 mM dithiothreitol (DTT) and further subjected to ultracentrifugation . Approximately 5 µg of plasmid DNA was added into the disassembled VLPs overnight and CaCl2 was slowly added to the mixture to reach a final concentration of 5 mM. The mixture was then subjected to ultracentrifugation as the condition above.The encapsulation of pEGFP plasmid DNA into chimeric VLPs was performed based on the VLP disassembly/reassembly method described previously® SA, NY) served as positive control. The delivery of the plasmid by the VLPs to SW480 (EGFR-positive) or SW620 (EGFR-negative) cells was assessed based on the expression of EGFP protein in the cell cytoplasm which was visualized by an Olympus FV10i confocal microscope as well as quantified by flow cytometric analysis.Loaded MrNV-VLPs containing 1 µg pEGFP plasmid (as determined by A260) in culture medium were added to the SW480 and SW620 cell layers and allowed to incubate for 48 h. Cells incubated with unloaded VLPs or purified plasmids served as negative control and cells transfected with 1 µg pEGFP using Jetprime transfection reagent , aldehyde-fixed cells were treated with 30 mM glycine in PBS and washed twice with PBST. Non-specific antibody staining was blocked with 4% BSA in PBST prior to incubation with a monoclonal mouse anti-MrNV antibody (1:500). The cells were washed and incubated with Alexa 488-conjugated goat anti-mouse antibody at a dilution of 1:1000 (Invitrogen). After washing, cells were counterstained with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) for nuclear staining and mounted with 50% glycerine in PBS . The stained cells were observed under an Olympus FV1000 confocal microscope  using the krypton and argon laser excitation lines and either 350 nm (blue) or 520 nm (green) band-pass emission filter. All images were acquired under a Kalman line-by-line scanning mode to prevent any cross talk of the fluorescence emission.4 cells were collected with a BD FACSCanto™ flow cytometer . Along with the green fluorescent signal of Alexa 488, the forward and side scatter parameters (FSC and SSC) were also recorded for differentiating the single cells from the surrounding debris and doublets. The data were further analyzed by FACSDiva™ Software . Plots represented mean ± SEM of normalized data. When the binding and internalization ability between GE11-MrNV VLPs vs. N-MrNV-VLPs were compared, the data were presented as the percentage of GE11-MrNV+ cells normalized with respect to the percentage of N-MrNV+ cells (%GE11-MrNV+/%N-MrNV+) × 100%. For the EGFR siRNA treatment to inhibit the EGFR-specific binding of each type of MrNV VLPs, the data were presented as the median fluorescence intensity (MFI) of EGFR-siRNA-treated cells normalized with respect to the MFI of Luc-siRNA-treated cells (control), (MFI siEGFR/MFI siLUC) × 100%.For flow cytometry, SW480, SW620 and MCF7 cells treated with N-MrNV-VLP and chimeric GE11-MrNV-VLPs at different time points were trypsinized and fixed in 4% prechilled paraformaldehyde in PBS. Cells were washed with PBS and permeabilized  in 0.3% saponin in PBS. After blocking, cell were incubated with anti-MrNV and the corresponding Alexa 488 conjugated secondary antibodies under similar conditions described above. Data of approximately 1 × 10Figure E. coli yielded the amount of purified proteins of about 2 mg/g of total proteins with the purity of about 90–95% in all samples. Protein profilings by a Coomassie blue staining of recombinant GE11-MrNV capsid proteins revealed a single major protein band with a molecular mass of about 42.5 kDa (for N-MrNV and R-MrNV), 43.0 kDa (for I-MrNV), and 43.5 kDa (for E-MrNV) . Following the incubation of the cells with different chimeric VLPs at 4 °C, the binding of R-MrNV and E-MrNV particles could be noted in the cells as an intense, punctate fluorescent staining by anti-MrNV antibody  and E-MrNV (p < 0.05). It should be noted that the binding of R-MrNV was more greatly affected (about 70%) than that of E-MrNV (about 45%)  the binding of the chimeric GE11-MrNV VLPs to the cell surface  were noted when compared to the percentages of EGFP-positive SW480 cells , with 60% less EGFP- expressing cells than the siLuc control counterpart  on SW480 cells in order to determine the dependence of the cargo delivery by the VLPs on EGFR expression. Generally, cells treated with control siRNA (siLuc) always showed a higher plasmid delivery and thus higher levels of cells expressing EGFP compared to those treated with siEGFR. Notably, for the EGFR-knockdown cells treated with EGFP-loaded R-MrNV and E-MrNV, the levels EGFP-positive cells reduced markedly -VLP by inserting p18 peptide (derived from the V3 loop of HIV-1 gp120) into the P-domain sequence of HEV capsid protein which alters the surface property of chimeric VLPs and allows them to escape from anti-HEV reactivity35. Similarly, engineering of Qβ-VLP by inserting human epidermal growth factor (EGF) into its exterior improves the interacting property of this chimeric VLP in inducing autophosphorylation of EGFR and enhancing apoptosis of A431 cells13. In this study, placement of GE11 peptide into the pillar structure of MrNV-VLP apparently altered VLP’s interaction property towards the human cancer cell line, which otherwise its interaction with human cells would be negligible  virus particle at the AB loop of the viral fiber knob  which significantly decreases binding of Ad5 towards the recipient cells while reversely enhances the internalization of the chimeric Ad5 into EGFR positive cell lines36. The other example of chemically modified nodavirus  with tumour homing peptide has shown recently to successfully deliver hydrophobic drugs into EGFR-positive breast cancer cells, a closely-resembled example to MrNV reported herein37.Application of VLPs becomes a well-established platform that provides the solution for specifically targeted drug delivery because of their abilities to encapsulate several types of cargoes after systemic functionalization of VLP for enhancing receptor recognition. In addition, through genetic engineering of the available capsid sequences, several modes of capsid alterations either via insertion, deletion or substitution of ligands on the specific site(s) of the capsid proteins are rather practical. Such modifications apparently improve biological and physiological properties of VLPsle Figs. . Higher 38. In case of MrNV, previous studies have shown that the P domain localized at the C-terminus of MrNV capsid plays an important role in binding and internalization in the host cells39. The atomic structure of MrNV resolved from cryo-EM indicated that the exposing surface of the P domain exhibits 4 loop- or pillar-structure located at amino acids 268–355 that represent potential insertions for targeting peptide24. In this study, placement of GE11 peptide at the 4th pillar (positioned 352–363 or R-MrNV) showed a high efficiency of chimeric MrNV-VLPs to interact with EGFR receptor on the cancer cell surface  also offered an efficient binding and internalization of chimeric VLPs into the cancer cells. This may be explained by the non-disturbance of the P-domain’s pillar structure by the addition of GE11 peptide, while the overhanging of the short peptide with the linker would allow more flexibility to freely display ligand towards the exposing EGFR receptor. This is in agreement with the previous studies of MrNV where they were engineered with the extended M2e (matrix 2 protein) or hepatitis B surface antigen (HBsAg) on the C-terminus of MrNV capsid and exert the specific property in inducing immunity in vivo26.Apart from GE11 specificity, our results in this study clearly showed the differential binding ability of chimeric GE11 + MrNV-VLP variants towards EGFR positive SW480 cells. Notably, R-MrNV-VLP and E-MrNV-VLP have a far superior binding ability to that of I-MrNV and N-MrNV-VLPs  is enriched with Arg and Lys amino acids and plays a role in binding to negatively charged RNA. This N-ARM domain regulates both length and conformation of ssRNA while interacting with viral capsid interior24. Although this inherent property of MrNV-VLP has already been advantageous for encapsulating nucleotide-based materials into the VLP’s cavity, however, its cargo loading capability can still be maximized to improve the efficiency of the therapeutic compound on the site of the target. This is particular for the case of cancer therapy or DNA vaccination where the local concentration of an active compound that is delivered into target cells is of utmost importance. In this perspective, the future avenue is to modify the interior of chimeric MrNV-VLP with the aim to maximize the encapsulation of active cancer therapeutic compounds. Conclusively, this study demonstrated the utility of chimeric MrNV nanocontainers rationally designed for specific delivery of cargoes, such as DNA and anticancer drugs, to targeted cancer cells. Further development of nanocontainers based on this study benefits the quest for improved targeted therapies.Here, we also launched our effort to encapsulate EGFP plasmid into the cavity of chimeric VLPs with similar encapsulating efficiency as reported earlier in normal MrNVSupplementary Figures.


Tumor necrosis factor (TNF) is one of the main cytokines regulating a pro-inflammatory environment. It has been related to several cell functions, for instance, phagocytosis, apoptosis, proliferation, mitochondrial dynamic. Moreover, during mycobacterial infections, TNF plays an essential role to maintain granuloma formation. Several effector mechanisms have been implicated according to the interactions of the two active forms, soluble TNF (solTNF) and transmembrane TNF (tmTNF), with their receptors TNFR1 and TNFR2. We review the impact of these interactions in the context of mycobacterial infections. TNF is tightly regulated by binding to receptors, however, during mycobacterial infections, upstream activation signalling pathways may be influenced by key regulatory factors either at the membrane or cytosol level. Detailing the structure and activation pathways used by TNF and its receptors, such as its interaction with solTNF/TNFRs versus tmTNF/TNFRs, may bring a better understanding of the molecular mechanisms involved in activation pathways which can be helpful for the development of new therapies aimed at being more efficient against mycobacterial infections. TNF was described for the first time in the middle of 1970; it is a polypeptide considered a potent pro-inflammatory cytokine and encoded in the major histocompatibility complex in human and mice, and it is produced by immune system cells both myeloid and lymphoid origin ,3.TNF is synthesized as a monomeric protein and stored in vesicles, which use the route of rough endoplasmic reticulum (ER) to cross the cytoplasm. TNF monomers form a compact trimer through non-covalent interactions; the TNF-trimeric has high thermodynamic stability, the molecular mass of the human TNF is 50.4-kDa and murine TNF 50-kDa .TACE or also called A Disintegrin and Metalloproteinase (ADAM) domain 17 (ADAM17), cleaves tmTNF between residues Ala76 and Val77 to obtain solTNF . TACE isIt is currently well established that TACE is necessary to deliver solTNF; however, how its catalytic activity is activated or regulated is still limited. Probably, the presence of a functional TACE is dependent on the action of multiple molecules. For instance, in TGF-β-mediated TACE activation, when TGF-β binds to its receptors, the sarcoma kinase (Src) molecule is phosphorylated, mediating NOX 1 activation and producing reactive oxygen species (ROS), which is finally activating TACE . The TNFThree mechanisms proposed to explain how the TACE’ catalytic activity is regulated are discussed below.(1) Nardilysin (NRD)-dependent pathway: NRD is a metalloendopeptidase of the M16 family. NRD binds to TACE (at cytosol or extracellular level) and potentiates its catalytic activity, a process promoted by phorbol esters. The NRD gene’s overexpression increases TNF and induces ADAM10 activation, suggesting that the high TNF shedding could be due to the function of both TACE and ADAM10 ,16  Oxygen deprivation-dependent pathway. Reports suggested that oxygen deprivation induces increased TACE expression and, consequently, high solTNF levels . By thisReports also suggested that solTNF upregulates the transcription factor AP-2α. However, the TACE promoter contains an AP-2α binding sequence, and it can bind in a TNF-dependent manner, indicating that solTNF downregulates TACE expression and function because AP-2α is enhanced   P2 and iRHOM2 pathways. The P2 purinergic receptors have bi-functional effects on TNF release. On the one hand, P2X receptor activation attenuates TNF release and simultaneously, on the other hand, P2Y induces TNF release . ATP indammation   or RHBDF2 is a member of the rhomboid protein family found in the ER. iRHOM2 is considered an essential regulator for the crosstalk TNF/TACE as it helps TACE to get the plasma membrane . Data inTwo receptors have been identified to mediate interactions with TNF, tumor necrosis factor receptor 1 (TNFR1), also called CD120a and p55 (its molecular weight is 55 kDa), and tumor necrosis factor receptor 2 (TNFR2), also called CD120b and p75 (its molecular weight is 75 kDa) . TNFR1 aTNFR1 and TNFR2 are on the cellular membrane or in a soluble form following TACE activation; their cytoplasmic domains are unrelated, and intracellular signalling pathways are independent. TNFR1 is involved in cytotoxicity, whereas TNFR2 plays a role in cytotoxicity and proliferation ,32. As dU) is ubiquitinated to recruit Ikappa B kinase (IKK), and then there is a binding between RIPK1U/NEMO (regulatory subunit IKKγ) to finally activate NF-κB . T. T70]. TIt has been reported that MCF-7 human tumor cells with a high expression level of tmTNF are resistant to cell death associated with solTNF and constitutive NF-κB activation. tmTNF contains a leader sequence (LS) in the cytoplasmic segment (76 amino acid residues) through which tmTNF is anchored into the membrane. LS appears to affect both forward and reverse signalling, and it seems that LS induces directly the constitutive activation of NF-κB ,73 FiguC.Using a pleural cell model, we have previously shown that tmTNF but not solTNF controls the expression of TNFR2 on myeloid cells expressing tmTNF, TNFR1 also contributes but in a minor way. Besides, the inflammatory process of BCG-induced pleurisy was downregulated mainly by tmTNF and TNFR2 . Recent The actin cytoskeleton plays an essential role in different functions of the cell, for instance, intracellular trafficking and cellular contractility, but also it has been recognized that the actin’s dynamic structure is involved in apoptosis and necrosis . The actActivation-induced cell death (AICD) plays a role in regulating peripheral immune tolerance by deleting overactivated or autoreactive T cells. It has been described that NF-κB is required to mediate the expression of the pro-apoptotic molecule called Fas ligand (FasL or CD95L) inducing AICD . It was Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium bovis (M. bovis), and also, there are unwanted effects such as sepsis autoimmunity and neurodegeneration.Diverse proposals of anti-TNF therapies targeting the cytokine or the receptors are underway ,86. AlthAmong the most used inhibitors are Etanercept, a TNFR2 dimeric fusion protein, followed by monoclonal antibodies Adalimumab and Infliximab. Their therapeutic efficacy is dependent on the interaction of the Fc region of the anti-TNF IgG and the FcγR expressed on the cell surface. Moreover, these antibodies have been related to a specific risk of extrapulmonary and disseminated infections .M. tuberculosis reactivation. For instance, the new design of a hypo-fucosylated form of Adalimumab, which was found to have better healing properties due to high affinity for FcγRIII and induction of CD206+ macrophages without apparent adverse effects. However, validation in large cohorts of patients to verify the clinical response is required [Staphylococcus aureus-induced septic arthritis; authors showed that the levels of pro and anti-inflammatory cytokines were modulated via the NF-kB and JNK signalling, which favored an increase of iNOS and RANKL and reduced recruitment of phagocytes at the site of inflammation, and subsequently decreased the generation of ROS and septic arthritis [New therapeutic strategies are proposed to target the TNF axis with a minimum risk of required . Sultanarthritis .Additional studies suggest that the combination of conformational analysis and molecular coupling studies for a cyclic peptide inhibitor of human TNF and TNFR1 could result in a rational design of a new moderate inhibitor of TNF-TNFR1 interaction. The combination of these cyclic peptides was shown to improve severity in rat colitis models, depending on the use of TNF-binding cyclic peptide (TBCP) or TNFR1-binding (TRBCP) . MoleculDominant-negative TNF molecules selectively blocking solTNF but not tmTNF molecules were designed and reported to prevent inflammatory process mainly mediated by solTNF . These sSpohn et al.  reportedA summary of TNFR1 and TNFR2 modulators is presented in The use of new biological is currently limited, and it is due mainly to the high cost of clinical studies and potential risk of side effects in human. Animal models for preclinical studies have been developed, for example mice expressing human TNF molecules to overcome the limitation of analyzing a human TNF inhibitor in mouse . HoweverM. tuberculosis; the current vaccine used against TB is the Bacillus Calmette-Guérin (BCG), which is derived from another mycobacterium called M. bovis. BCG vaccine provides adequate protection against childhood TB, but the level of protection against adult pulmonary TB can be variable [Tuberculosis (TB) is an infectious disease, which is the leading cause of death worldwide from a single infectious agent; the World Health Organization has estimated that in 2019 worldwide, 10 million people fell ill with TB  and 1.2 million deaths . The cauvariable .TNF plays a critical role in host defense mechanisms against mycobacterial infections as shown by many research groups using different models of TNF and TNFRs deficient mice . In vitrM. tuberculosis DNA (MtbDNA) is recognized by murine macrophages, which leads to autophagy induction, TLR-9 expression, and considerable TNF production. Interestingly, only M1 macrophages were fully responsive to MtbDNA [M. tuberculosis infection where TNF, TLRs and autophagy are involved [It has been recently reported that o MtbDNA . Severalinvolved ,120.Most TNF activities have been attributed to solTNF form. At present, the contribution of tmTNF in protective immunity against mycobacterial infections has been also well analyzed by using mutant mice that only express tmTNF and not solTNF. However, as previously discussed, these tmTNF are modified molecules that are retained at the cell membrane representing a model system but not the native tmTNF molecule.M. tuberculosis. tmTNF was sufficient to sustain the cellular activation and to reduce bacterial BCG load by inducing the granuloma formation, and IFN-γ expression although the amounts were lower than those induced when solTNF was expressed [M. tuberculosis infection but not to chronic M. tuberculosis infection as these mice developed an exacerbated inflammation [In 2002, Olleros et al.  reportedxpressed . Using tammation .M. tuberculosis infection around 170 days post-infection contrary to WT, which survive to 300 days, confirming that tmTNF is sufficient to control acute, but not chronic infection.In a different mouse model of tmTNF knocking (KI) mice, Saunders et al.  showed tM. tuberculosis reactivation, it has been reported that tmTNF is not sufficient to support an efficient immunity, although confers protection against acute M. tuberculosis infection. However, the long-term protection requires solTNF in order to decrease inflammatory response and to contain M. tuberculosis reactivation [M. tuberculosis infection, but was not associated with a reduction in bacterial load [Using the same tmTNF KI mice in a mouse model of tivation ,124. Morial load   have been described as natural suppressor cells inhibiting T-helper lymphocytes’ proliferative response. A study has reported a new mechanism to explain the role played by tmTNF in the control of BCG infection. BCG-infection induces MDSC accumulation, and tmTNF expression on MDSC is crucial to activate their suppressive function. Chavez-Galan et al.  suggesteM. tuberculosis infected macrophages could be initiated by TNF-TNFR1 interaction with TRADD (TNFR-associated death domain) and FADD (Fas-associated death domain) association and subsequent aggregation of death effector domains (DEDs), procaspase-8 activation and the DISC  assembly. Finally, effector caspases are activated. TNF-induced apoptosis through molecules receptor-associated that impact caspase activation, resistance to cell death may be influenced by NF-kB [The apoptotic process in by NF-kB .M. tuberculosis induce a miR-30A overexpression in infected macrophages, inhibiting the autophagy and negatively impacting the immune control [M. tuberculosis independently of IFNγ and nitric oxide, and it displays an inverse correlation with macrophages apoptosis, but this apoptosis is not observed under BCG infection, suggesting that regulation of apoptosis and mycobacterial replication by TNFR2 is a virulence dependent pathway [Virulence of mycobacteria is strongly associated with cell death evasion during infection. Virulent strains like  control . A parti control . In this pathway . However pathway ,133.M. tuberculosis H37Rv in U937 and THP-1 cell lines and in human monocyte-derived macrophages, apoptosis inhibition involves the extrinsic apoptosis pathway, but it does not interfere with the mitochondrial apoptosis pathway [M. tuberculosis into the cytoplasm were identified, as responsible for apoptosis suppression. Rv3654c cleaves PFS (polypyrimidine tract binding Protein-associated Splicing Factor), which decreases the expression of caspase-8 [M. tuberculosis infection is not clear. It has been suggested that mitochondrial apoptosis pathway may be a strategy to induce necrosis in infected macrophages during a well-defined stage of the infection [In an infection model with  pathway . Two proaspase-8 . In anotaspase-8 . Howevernfection ,136.M. smegmatis, involving TNF and FasL activation, which upregulates TNFR1, TNFR2 and Fas. In this process, TLR2 was involved as well as in the activation of caspase-8, caspase-9 and caspase-3 [A different mechanism has been proposed by Miller et al. NuoG a subunit of NDH-1 (type I NADH dehydrogenase) is involved in neutralization of NOX2-derived reactive oxygen species (ROS), promoting inhibition of TNF-induced apoptosis . If ROS aspase-3 . In summTNF is a key regulatory cytokine that plays an important role in the innate and adaptive immune responses during mycobacterial infections. However, several mechanisms can influence the course of the infection. The first interactions of TNF and TNF receptors after mycobacterial phagocytosis may play a crucial role inside a specific environment. It is also of interest to consider the balance between the two TNF forms, tmTNF, and solTNF and cells responding to these TNF forms that can influence the outcome of mycobacterial infections.Recent evidence has pointed out the relevance of tmTNF-mediated reverse signalling during mycobacterial infections. It is of interest that this pathway not only favors proinflammatory responses, as discussed in this review, but tmTNF attenuates Th1 cell-mediated inflammatory responses and mediates cell recruitment. These are new insights elucidating tmTNF/TNFRs axis that can be considered as target to control mycobacterial infections, mainly focusing on TB, which still as a major public health worldwide.Moreover, the selective inhibition of solTNF is another important target because it improves survival in an acute infection, nevertheless diverse signalling mechanisms for both forms of TNF, TNFR1 and TNFR2 receptors remain to be elucidated. More research on the biology of TNF is required to design improved therapies that alleviate inflammatory diseases while maintaining protection against mycobacteria. Furthermore, this will lead to personalized treatment as a promise to improve the clinical outcome of patients suffering from mycobacterial infections.


All treatments showed an improvement of structural changes at the axial and peripheral joints on micro-CT imaging, with a significant decrease for roughness (p < 0.05), which reflects both erosion and new bone formation, at the level of the caudal spine. The effect of dual blockade therapy on new bone formation was more prominent at the axial than the peripheral level. Collectively, our study showed that dual blockade therapy significantly reduces inflammation and structural changes, including new bone formation. However, we could not confirm a more pronounced effect of dual inhibition compared to single inhibition.The tumor necrosis factor (TNF) and IL-23/IL-17 axes are the main therapeutic targets in spondyloarthritis. Despite the clinical efficacy of blocking either pathway, monotherapy does not induce remission in all patients and its effect on new bone formation remains unclear. We aimed to study the effect of TNF and IL-17A dual inhibition on clinical disease and structural damage using the HLA-B27/human β2-microglobulin transgenic rat model of SpA. Immunized rats were randomized according to arthritis severity, 1 week after arthritis incidence reached 50%, to be treated twice weekly for a period of 5 weeks with either a dual blockade therapy of an anti-TNF antibody and an anti-IL-17A antibody, a single therapy of either antibody, or PBS as vehicle control. Treatment-blinded observers assessed inflammation and structural damage clinically, histologically and by micro-CT imaging. Both single therapies as well as TNF and IL-17A dual blockade therapy reduced clinical spondylitis and peripheral arthritis effectively and similarly. Clinical improvement was confirmed for all treatments by a reduction of histological inflammation and pannus formation ( Spondyloarthritis (SpA) is the second most common form of chronic inflammatory arthritis . SpA canPrevious pre-clinical and clinical studies imply that the TNF and IL-23/IL-17 pathways operate independently from one another ,25 and mMycobacterium tuberculosis (M. tub.) induced HLA-B27/Human βeta 2 microglobulin (Huβ2m) rat model of SpA [We hypothesized that the dual blockade of TNF and IL-17A effectively targets both inflammation and new bone formation in SpA. To test this hypothesis we performed dual blockade of TNF and IL-17A in the l of SpA .M. tub.-induced HLA-B27/huB2m tg rats (p = 0.031) and anti-TNF monotherapy groups (p = 0.031), while a numerical decrease was observed in the anti-IL17A monotherapy group without statistical significance (p = 0.164) (p = 0.006), anti-IL-17A monotherapy (p = 0.011) and the dual blockade therapy groups (p = 0.008)  b. The cl= 0.008) c. These p = 0.036), and anti-TNF (p = 0.039) therapies compared with the vehicle control (p = 0.037), anti-TNF (p = 0.016) and anti-IL17A (p = 0.021) therapies compared with the vehicle control. Compared with vehicle control, a numerical decrease of bone proliferation in the spine was seen for all treatment modalities.To substantiate the clinical data, we evaluated caudal spine and ankle sections histologically stained for hematoxylin/eosin and Safranin O/Fast Green a,c. At t control b. Similap = 0.047) compared to the vehicle control, while dual blockade and anti-TNF therapies only showed a trend of reduced inflammation , and slightly less for anti-TNF  and anti-IL-17A  monotherapy, respectively (p = 0.013) monotherapy compared to vehicle-treated, diseased rats , and a less pronounced reduction for anti-TNF (−34%) and anti-IL-17A (−14%) monotherapy, respectively, although none reached significance a,b. Simiectively a,b. To esed rats b. A simised rats b. Moreovsed rats a,b. Thessed rats A,B. TogeM. tub. induced HLA-B27/Huβ2m tg rat SpA disease model which offers a great opportunity to study the effects of existing and new treatment regimens on both inflammation and bone remodeling. In this experimental disease model, we tested the effects of single or dual blockade of IL-17A and TNF on axial and peripheral disease in combination with an in-depth assessment of inflammation and structural damage at different disease sites.The TNF and IL-23/IL-17 axes are major contributes to pathology in SpA . Both ofIn this study, TNF blockade resulted in a significant reduction in the severity of spondylitis associated with a significant reduction of axial inflammation and bone roughness at the level of the caudal spine. These results confirm the findings of the studies on the transmembrane TNF (tmTNF) transgenic mouse model of SpA, in which it has been shown that the overexpression of tmTNF drives osteoproliferative joint inflammation seen in SpA. TNF receptor I signaling is essential for inflammation and TNF receptor II signaling contributes to pathological new bone formation ,38. MoreWe have also demonstrated that treatment with anti-IL-17A monotherapy resulted in the significant reduction in severity of arthritis and axial inflammation. IL-17A blockade also resulted in a significant reduction of bone roughness at the level of the caudal spine and bone remodeling at the level of the tarsals. These results confirm previous studies in the HLA-B27/Huβ2m tg rat model where both preventive and therapeutic inhibition of IL-17A reduced inflammation and new bone formation . MoreoveNevertheless, despite these positive observations neither TNF nor IL-17A inhibitors can normalize both inflammation and bone remodeling in all patients and complete remission with either therapeutic remains low. The latter further confirms the need for novel treatment strategies. In our study dual anti-TNF and anti-IL-17A therapy resulted in significant reduction of spondylitis and swelling at the level of the peripheral joints. This was also associated with a significant reduction of axial inflammation, bone roughness at the level of the caudal spine and bone loss at the level of the tarsals. Our results indicate that dual blockade of anti-TNF and anti-IL-17A is effective in experimental SpA and may have added clinical value. In agreement with our results, the dual blockade therapy of anti-IL17A and anti-TNF (infliximab) in the human TNF mouse model, resulted in a significant reduction of synovitis, synovial hyperplasia, and cartilage damage with a similar efficacy as high dose TNF inhibition. Dual inhibition also showed a superior effect on bone remodeling with a significant reduction of the bone remodeling markers RANKL and osteoprotegerin . In clinM. tub. induced HLA-B27/Huβ2m tg rat SpA disease model. In light of this data, we hypothesize that early, profound and sustained suppression of inflammation is essential to arrest or prevent structural damage. This hypothesis is consistent with previous observations in the non-immunized HLA-B27/Huβ2m tg rats where no new bone formation is observed in the absence of inflammation [Dual blockade of TNF and IL-17A demonstrates efficacy in controlling inflammation and structural damage of the caudal spine with a smaller effect on the peripheral joints in the ammation ,52,53, aammation . Follow-M. tub. induced HLA-B27/Huβ2m tg rats.The main limitation of our study was the small group sizes, which probably accounted for the fact that we observed only numerical differences for several outcomes and could not perform formal statistical analysis comparing various treatment arms. Moreover, molecular data could have helped to elucidate the individual and synergistic effects of TNF and IL-17A inhibition, but this was outside the scope of this proof-of-concept study. Despite these limitations, our study is the first to demonstrate the positive effect of TNF and IL-17A dual blockade therapy in the In conclusion, our data indicate that treatment with a dual blockade with TNF inhibitor and IL-17A inhibitor impacts both axial and peripheral inflammation as well as structural damage. Further preclinical and clinical research is needed to assess whether deep and sustained control of inflammation is needed to fully abrogate structural damage in  SpA.The transgenic (tg) 21-3 (HLA-B27/Huβ2m) and 283-2 (Huβ2m) rat lines  of the LMycobacterium tuberculosis  in 100 μL Freund’s incomplete adjuvant (IFA)  [At four weeks of age, orchiectomy was performed using a standard protocol  to prevWA, USA)  by an in3) was measured using plethysmometry and normalized to the day of therapeutic treatment initiation. Spondylitis, characterized by the presence of swelling and bumps in the tail, was identified macroscopically and scored 0–3 per tail. Clinical scoring and plethysmography were performed by an observer, blinded for the treatment. Humane endpoints were 20% body weight loss or the complete swelling of two paws.The presence of arthritis in the paws was identified macroscopically, the severity was scored 0–3 per paw , and a cumulative arthritis severity score for all four paws was calculated. Digital swelling , 15 mg/kg a rat-specific anti-TNF antibody , a combination of both, or PBS as a vehicle control. The dosages of both antibodies were determined by AbbVie through titration in a rat collagen-induced arthritis model for efficacy (data not shown). We chose to use the total dosage of both antibodies for the dual blockade treatment to see a clear effect.The rats of two cages started treatment earlier as arthritis was developing more rapidly than in the other cages. Two rats, one from the anti-IL-17A and one from the dual blockade therapy group, developed only very mild arthritis which was not progressive during the experiment. Therefore, they were excluded and left out of further analyses.3 to include low density  bone and exclude cortical bone; endosteal surface was masked. Moreover, in the analysis of distal calcaneus, 200 slices (3.6 mm) were utilized compromising the distal end of calcaneus with a segmentation threshold of 220–320 mg of HA/cm3 to estimate low-density new periosteal bone formation; endosteal surface was also masked. In the analysis of rear paw tarsals, 200 slices (3.6 mm) comprising the navicular bone and tarsals were used with a segmentation threshold 350 mg of HA/cm3 to include remaining high density cortical bone.The lumbar spine, sacroiliac pelvis region, knees, left hind paw and tails of all animals were isolated, fixed with 4% formalin and transferred to 70% ethanol to be imaged by micro-CT. First, the vehicle-treated rats were scanned to detect areas with bone changes—including new bone formation, roughness and bone loss—subsequently, the affected areas were scanned for the rats of the single and dual blockade therapy groups. Approximately 1000 slices were included (18 mm) in the analysis of caudal spine, compromising 2 caudal vertebra (Ca3-Ca4) and the gap between them. The segmentation threshold used was 200 to 320 mg of HA/cmAfter micro-CT analysis, the hind paws and tails of the rats were decalcified with OsteoSoft decalcifier solution  and embedded in paraffin. Five micrometer sections were stained for hematoxylin and eosin (H&E) or Safranin O Fast Green. For H&E staining, sections were incubated in Mayer’s hematoxylin (no. MHS16) and Eosin Y solution (no. HT110116) . For Safranin O Fast Green staining, sections were incubated in Weigert’s iron hematoxylin (no. HT1079), 0.1% fast green FCF (no. F7252), and 0.1% Safranin O solution (no. HT90432) . The sections were subsequently dehydrated and embedded in Entellan . Separate experimentally blinded observers scored the caudal spine, calcaneus, and tarsal regions semi-quantitatively (0–4) for inflammation, bone proliferation and pannus formation.The data were analyzed using GraphPad Prism 6 and IBM SPSS Statistics 23 software. One-way analysis of variance (for multiple comparisons) was performed on all metric data; for clinical scores and hind paw swelling, the area under the curve was calculated first. All categorical data (semi-quantitative scores) were analyzed by a Mann–Whitney U test.


Inflammatory bowel disease (IBD) is chronic inflammation of the gastrointestinal tract, although its etiology has largely been unclear. Tumor necrosis factor inhibitors (TNF-I) are effective for the treatment. Recently, biosimilars of TNF-I, such as CT-P13, have been developed and are thought to possess equal efficacy and safety to the original TNF-I. Sarcoidosis is also a systemic granulomatous disease of unknown etiology. In steroid-resistant cases of sarcoidosis, TNF-I have been reported effective for achieving resolution. However, the progression of sarcoidosis due to the TNF-I also has been reported. We herein report a case of pulmonary sarcoidosis with a Crohn’s disease (CD) patient developed after a long period administration (15 years) of TNF-I.A 37-year-old woman with CD who had been diagnosed at 22 years old had been treated with the TNF-I . Fifteen years after starting the TNF-I, she developed a fever and right chest pain. Chest computed tomography (CT) revealed clustered small nodules in both lungs and multiple enlarged hilar lymph nodes. Infectious diseases including tuberculosis were negative. Bronchoscopic examination was performed and the biopsy specimens were obtained. A pathological examination demonstrated noncaseating granulomatous lesions and no malignant findings. TNF-I were discontinued because of the possibility of TNF-I-related sarcoidosis. After having discontinued for four months, her symptoms and the lesions had disappeared completely. Fortunately, despite the discontinuation of TNF-I, she has maintained remission.To our knowledge, this is the first case in which sarcoidosis developed after switching from O-IFX to IFX-BS. To clarify the characteristics of the cases with development of sarcoidosis during administration of TNF-I, we searched PubMed and identified 106 cases. When developing an unexplained fever, asthenia, uveitis and skin lesions in patients with TNF-I treatment, sarcoidosis should be suspected. Once the diagnosis of sarcoidosis due to TNF-I was made, the discontinuation of TNF-I and administration of steroid therapy should be executed promptly. When re-starting TNF-I, another TNF-I should be used for disease control. Clinicians should be aware of the possibility of sarcoidosis in patients under anti-TNF therapy.The online version contains supplementary material available at 10.1186/s12876-021-01948-6. Inflammatory bowel disease (IBD) is chronic inflammation of the entire gastrointestinal tract, although its etiology has largely been unclear. Tumor necrosis factor (TNF) inhibitors are known to be effective treatment for treating IBD patients with moderate to severe activity. The cost-effectiveness and efficacy of TNF inhibitors (TNF-I) has been demonstrated through their reduction in the rates of hospitalization and surgery . RecentlSimilar to IBD, sarcoidosis is a systemic granulomatous disease of unknown etiology, affecting various organs, including the lung, heart, lymphatic system and skin. In many cases of sarcoidosis, steroids are effective for treatment, and in case of steroid resistance, TNF-I are reported to be effective. While some studies have reported that the administration of TNF-I caused the progression of sarcoidosis, no reports regarding the relationship between sarcoidosis and infliximab biosimilar (IFX-BS) have been published.We herein report a case of pulmonary sarcoidosis in a Crohn’s disease (CD) patient during fifteen years administration of IFX-BS after switching from original infliximab (O-IFX). To our knowledge, this is the first case of sarcoidosis developing after switching from O-IFX to IFX-BS in a CD patient.A 37-year-old Japanese woman was diagnosed with CD at 22 years of age. She had no relevant family history. At the onset of CD, she had symptoms of fever, abdominal pain, and frequent diarrhea. On total colonoscopy, she was found to have multiple longitudinal ulcers in the terminal ileum with stricture. Her symptoms were severe; thus, we administered O-IFX first, without steroid therapy. Clinical remission was obtained after 3 months of O-IFX treatment. She had maintained clinical remission without any adverse events for twelve years after the administration of O-IFX, and then O-IFX was switched to IFX-BS (CT-P13) after obtaining informed consent, because IFX-BS demonstrated equivalent efficacy and safety in the treatment of CD and the drug price was approximately half that of O-IFX in Japan. After switching to IFX-BS, clinical remission was still maintained for three years.Fifteen years after starting the TNF-I (O-IFX and CT-P13), she developed a fever and right chest pain but had no respiratory symptoms, such as cough or sputum. Laboratory findings showed total bilirubin, 1.5 mg/dL; alanine aminotransferase, 248 U/L; aspartate aminotransferase, 105 U/L; gamma glutamyl aminotransferase, 192 U/L; alkaline phosphatase, 489 U/L; C-reactive protein (CRP), 0.44 mg/dL; anti-nuclear antibody, 1:160. Hepatitis B and C were negative. Chest X-ray and computed tomography (CT) revealed clustered small nodules in both lungs and multiple enlarged hilar lymph nodes  years, and the male:female ratio was 5:3 when including our case. The causative TNF inhibitors were adalimumab (ADA)  and IFX . The symptoms depended on the organs involved. Our case showed an unexplained fever and chest pain because of pleuritis. Asthenia, uveitis, and skin lesions were also reported. There were no specific symptoms.In this case, the serum ACE level was normal. It was reported that serum ACE shows low sensitivity and specificity in the diagnosis of sarcoidosis . In addiWe also investigated the difference in the duration until the development of sarcoidosis under IFX and ADA therapy in the six CD cases without switching cases. The median duration to the development of sarcoidosis in CD patients under IFX (n = 3) and ADA (n = 3) therapy was 12 and 24 months, respectively. Patients receiving ADA therapy showed a longer duration until development than those receiving IFX, which might have influenced the difference in antibody type, such as chimeric human-mouse IgG monoclonal antibody and fully human monoclonal antibody. However, the number of such cases has been limited so far, so the accumulation of more cases will be required to verify whether or not differences in antibodies affects the development of sarcoidosis.Regarding the therapy delivered after the development of sarcoidosis, six of eight cases discontinued TNF-I, two without steroid and four with additional steroid, and two cases continued TNF-I therapy while adding steroid therapy or topical therapy. All six cases who discontinued TNF-I with/without steroids showed the resolution of sarcoidosis. In one case, in which the patient continued TNF-I with steroid treatment, improvement of the lung lesion was observed. In one case in which the patient continued TNF-I with topical steroid treatment, improvement of the lung lesion, but not the skin lesion, was observed. In CD patients who develop sarcoidosis, discontinuation of TNF-I seems to be a feasible treatment. Among 107 cases with various diseases, 47 discontinued TNF-I without the addition of steroid therapy, and sarcoidosis was improved or resolved in 43 (91.5%), while 49 cases discontinued TNF-I with the addition of steroid therapy, and 46 (93.9%) showed improvement or resolution of sarcoidosis. These data support the notion that discontinuing TNF-I is effective for treating patients with chronic inflammatory disease, but the efficacy of additional steroid therapy is unclear.Our case has maintained remission despite having discontinued TNF-I; however, TNF-I may be key drugs for CD patients. Thus, whether or not TNF-I can be re-initiated for these patients is important to clarify. Only one CD patient re-initiated the same TNF inhibitor (O-IFX) with no recurrence of sarcoidosis observed . When exIn conclusion, should be aware that TNF-I can cause several drug related complications, including sarcoidosis. Sarcoidosis due to TNF-I can be caused by IFX-BS and develop even after long-term administration. Clinicians should be aware of the possibility of sarcoidosis in patients under anti-TNF therapy. The efficacy of additional steroid therapy remains unclear; however, TNF-I should be discontinued as soon as possible, even in CD patients who maintain long-term remission with TNF-I. When re-starting TNF-I, another TNF-I should be used for disease control, as relapse of sarcoidosis is frequent when patients are treated with the same TNF-I Table .Table 1TAdditional file 1. To confirm the involvement of these causes and clarify the characteristics of patients likely to develop sarcoidosis under TNF inhibitor therapy, we searched for case reports in PubMed concerning sarcoidosis due to TNF inhibitor using the combination of the heading terms ‘infliximab’, ‘adalimumab’, ‘etanercept’, ‘certolizumab’, ‘golimumab’, ‘infliximab biosimilar’, without ‘Crohn’s disease’, combined with ‘sarcoidosis’.


Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation and pannus formation, with subsequent joint and cartilage degradation. Treatment commonly targets inflammatory cytokines, including tumor necrosis factor (TNF) alpha, which is a potent inflammatory cytokine required for cell signaling, regulation, and apoptosis, as well as for other cellular functions including immune response. TNF alpha inhibitors have demonstrated benefits in improving RA patient outcomes in terms of immune function and symptomatology. While TNF alpha inhibitors are generally beneficial, some studies have demonstrated that TNF alpha inhibitors may increase the risk of adverse cardiovascular events. While this continues to be debated, our study investigates the role of Tumor Necrosis Factor Receptor 1 (TNFR1) and Tumor Necrosis Factor Receptor 2 (TNFR2) in cardiac tissue. TNFR1 is an apoptotic receptor and its inhibition by TNF alpha inhibitors is subsequently cardioprotective. However, TNF alpha inhibitors may be inhibiting TNFR2 receptors even more so than TNFR1 receptors. TNFR2 is primarily a cardioprotective receptor and its greater inhibition results in the cardiovascular morbidity associated with TNF alpha inhibitors. Globally, rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that affects one percent of the population . In the Heart failure and rheumatoid arthritisCongestive heart failure (CHF) is a disease characterized by cardiac pump dysfunction . A varieDiastolic dysfunction is defined as a preserved ejection fraction with decreased compliance as well as impaired relaxation, most often due to hypertrophy . This tyRheumatoid arthritis treatment and cardiovascular riskFirst-line standard treatment for patients with RA involves the use of disease-modifying anti-rheumatic drugs (DMARDs), such as methotrexate, a dihydrofolate reductase inhibitor, to decrease inflammatory response and slow disease progression . HoweverWhile TNF alpha inhibitors result in inflammatory protection in most organs by decreasing levels of chronic inflammation and inhibiting the apoptotic pathway, studies have found that TNF alpha inhibitors may worsen HF in patients with systolic or diastolic dysfunction, especially at higher doses . In partTNF alpha receptors and rheumatoid arthritis treatmentAs previously noted, RA is managed using anti-inflammatory agents such as nonsteroidal anti-inflammatory drugs (NSAIDs), DMARDs, glucocorticoids, and TNF alpha inhibitors . DMARDs In the clinical trial ATTACH, patients given the higher dose of 10 mg/kg of infliximab experienced a greater rate of cardiac-related hospitalization . This maTNF alpha inhibitors have been effective in controlling and managing patients with RA, but the risk of elevated cardiovascular risk continues to be debated. We have found that while inhibition of TNFR1, an apoptotic receptor, is cardioprotective, the greater inhibition of TNFR2, which is a cardioprotective receptor, may be the cause of the cardiovascular risks associated with TNF alpha inhibitors. Based on our findings and the role TNFR1 and TNFR2 receptors play, we recommend further study of the biochemical pathways of TNF alpha, which will further allow for the synthesis of selective TNFR1 inhibitors or TNFR2 agonists as treatment modalities for patients with RA and concomitant cardiovascular disease. In the meantime, proper patient risk factor analysis, specific testing protocols, and greater communication between cardiologists and rheumatologists should improve the management of patients with RA and at cardiovascular risk. Treatment decisions should be based on this risk and managed closely to prevent serious medication-induced cardiovascular complications.