Willow spp., were identified in the mid-19th century for the alleviation of pain, fever, and inflammatory responses. Study of this naturally occurring compound led to the discovery of aspirin, which was followed by the development of non-steroidal anti-inflammatory drugs (NSAIDs) due to the chemical advances in the 19th–20th centuries. Initially, the most of NSAIDs were organic acid, but the non-acidic compounds were also identified as NSAIDs. Although effective in the treatment of inflammatory diseases, NSAIDs have some undesirable and adverse effect, such as ulcers, kidney injury, and bleeding in the gastrointestinal tract. In the past two decades, anti-TNF biologics were developed. Drugs belong to this class include soluble TNF receptor 2 fusion protein and anti-TNF antibodies. The introduction of anti-TNF therapeutics has revolutionized the management of autoimmune diseases, such as RA, psoriatic arthritis (PsA), plaque psoriasis (PP), AS, CD and ulcerative colitis (UC). Nevertheless, up to 40% of patients have no response to anti-TNF treatment. Furthermore, this treatment is associated with some adverse effects such as increased risk of infection, and even triggered the de novo development of autoimmune diseases. Such harmful effect of anti-TNF treatment is likely caused by the global inhibition of TNF biological functions. Therefore, specific inhibition of TNF receptor (TNFR1 or TNFR2) may represent a safer and more effective treatment, as proposed by some recent studies. In this review article, the historical development of anti-inflammatory drugs after World War II as briefly described above will be reviewed and analyzed. The future trend in the development of novel TNF receptor-targeting therapeutics will be discussed in the context of latest progress in the research of TNF biology.Although initially described as an anti-tumor mediator, tumor necrosis factor-alpha (TNF) is generally considered as the master pro-inflammatory cytokine. It plays a crucial role in the pathogenesis of inflammatory diseases, such as rheumatoid arthritis (RA), inflammatory bowel disease, ankylosing spondylitis (AS), and psoriasis. Consequently, anti-TNF therapy has become mainstay treatment for autoimmune diseases. Historically, anti-inflammatory agents were developed before the identification of TNF. Salicylates, the active components of  Autoimmune inflammatory diseases affect approximately 7.6–9.4% of the world population, especially among the young and middle-aged women . FrequenNon-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease-modifying antirheumatic drugs (DMARDs) are traditionally used in the treatment of autoimmune inflammatory diseases. NSAIDs and glucocorticoids are effective in the alleviation of pain and inhibition of inflammation, while DMARDs have the capacity of reducing tissue and organ damage caused by inflammatory responses . More reWillow spp. responsible for the anti-inflammatory activity, which laid the foundation for the mass synthesis of acetylsalicylic acid in 1899 (Plant parts such as leaves of myrtle (Myrtus), bark of willow tree , bark of poplar (Populus), and meadow sweet (Spirea) were used in fork medicine for centuries in the treatment of fever, pain, and inflammatory responses . These m in 1899 . The pro in 1899 . After t in 1899 . Overall in 1899 .Salicylic acid was synthesized by the Gerland in 1853 for the first time, and acetylsalicylic acid was synthesized by Charles Gerhardt in 1853 . Until 1Non-steroidal anti-inflammatory drugs have the analgesic, antipyretic, and anti-inflammatory effect, frequently used for the treatment of conditions like arthritis and headaches . NSAIDs Based on the chemical structure, the traditional non-selective NSAIDs can be classified into different sub-types : (1) salThe anti-inflammatory effect of NSAIDs is mainly based on the inhibition of activity of cyclooxygenase (COX) enzymes . COX enzcis-stilbene with one of the pendant phenyl rings by different substitutes, termed as diarylheterocycles  were relatively more resistant to glucocorticoid-induced cell death  . AlthougAmong cDMARDs drug class, the methotrexate is still an ‘anchor drug’ for RA therapy, and the first choice for the most of DMARD-naïve patients . HoweverIn 1975, Lloyd and colleagues discovered an endotoxin-inducible serum factor named TNF had the capacity to cause necrosis of tumors . It is oFeldman’s group showed that a number of pro-inflammatory cytokines such as IL-1, TNF, IL-6, GM-CSF, and IFN-γ were expressed at a high levels in synovium samples from active RA patients . IntriguTNF is now generally considered as a master pro-inflammatory cytokine that plays a critical role in the pathogenesis of autoimmune inflammatory diseases . ConsequInfliximab is a recombinant chimeric antibody that is generated by mouse myeloma cells. The structure of infliximab contains a constant sequence of human IgG1 and variable regions of murine, which is specific for all forms of TNF in human and chimpanzees, and effectively blocks the binding of TNF to its soluble and membrane receptors . The maxEtanercept comprises soluble TNF receptor 2 and Fc portion of IgG1, which is a human recombinant protein . After sAdalimumab is a fully humanized IgG1 monoclonal antibody, which can specific block human TNF binding to its receptors . AdalimuAdalimumab is effective in alleviating the signs and symptoms of moderate to severe RA patients . AdalimuGolimumab is also a fully humanized IgG1 monoclonal antibody, which has a high affinity and specificity for human TNF . As showCertolizumab pegol (CDP870) is a humanized monoclonal antibody with the structure of polyethylene glycolated Fab fragment, which is a novel TNF inhibitor with a distinct mechanism of action compared with other TNF inhibitors . The uniCertolizumab pegol monotherapy could effectively control the symptoms of patients with RA . It is aTable 1) have been marketed  than those from patients responding to infliximab, and PBMCs isolated from patients who do not respond to infliximab produce higher levels of TNF and sTNFR2 than those from patients responding to infliximab . TherefoTNF plays a crucial role in host defense to invading pathogens . Anti-TNThere is also some evidence that anti-TNF treatment is associated with increased risk of malignancy . Howeverde novo induced by anti-TNF therapy. For example, psoriasis can be induced in IBD patients treated with anti-TNF biologics  and TNF-TNFR2 interaction preferentially activates and expands naturally occurring Tregs (+Foxp3- effector T cells (Teffs) can be induced and upregulated by TCR stimulation, and TNFR2 expression on Teff cells has a functional consequence (There is now compelling evidence that TNFR2 is constitutively expressed by immunosuppressive CD4ng Tregs . The immng Tregs . Selecting Tregs , and inhng Tregs . It was ng Tregs . These esequence . Thus, tTable 2, as a complementary and alternative biological treatment of anti-TNF therapy.In addition to anti-TNF agents, the biologics targeting other proinflammatory cytokines or immune competent molecules have also been extensively studied and actively developed. For example, abatacept, a fully humanized fusion protein of extracellular domain of CTLA-4 and Fc fraction of IgG1, has been approved for the RA patients with inadequate response to anti-TNF therapy . The majAlthough TNF plays a central role in acute and chronic inflammation, the anti-inflammatory agents were developed before the identification of TNF. After the World War II, agents from small molecules to anti-TNF antibodies have been developed, including NSAIDs, glucocorticoids, DMSADs, and anti-TNF biologics. The traditional non-selective NSAIDs are associated with severe gastrointestinal disorders, attributable to simultaneous inhibition of COX-1 and COX-2, which prompted the development of selective NSAIDs. Glucocorticoids have potent anti-inflammatory activity, accompanied by some severe adverse effects. Methotrexate remains the first-line drug, while the efficacy can be enhanced and adverse effects can be reduced by DMARDs combination therapy. Currently, five anti-TNF biologics have been approved for patients with autoimmune disorders. With great success in treating autoimmune diseases, anti-TNF biologics also represent the most profitable drug class in the history, exceeding $US 25 billion total sale in 2012 . Anti-TNAs compared to small molecule compounds, biologics have some clear advantages, such as higher safety profile or minimal toxicity, well-understood mechanisms, and more importantly, the target specificity . Due to PL drafted the work; YZ and XC revised the manuscript. PL, YZ, and XC contributed substantially to the conception or design of the work, approved the final version to be published, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Tursiops truncatus), are more limited. Herein, we describe the development of IFN-gamma (IFN-γ) and TNF-alpha (TNF-α) enzyme-linked immunosorbent assays (ELISAs) specific to bottlenose dolphins. Utilizing these assays, we monitored the immune status of bottlenose dolphins from a managed population over a period of eleven months. The ELISA assays developed for bottlenose dolphins were used to measure IFN-γ and TNF-α in serum or in culture supernatants from peripheral blood mononuclear cells (PBMCs) stimulated with varying concentrations of mitogens concanavalin A (ConA) or phytohemagglutinin (PHA). Induction of TNF-α in PBMC cultures was consistently highest with 1 μg/mL ConA, while 1 μg/mL PHA induced the highest secretion of IFN-γ. Serum levels of TNF-α and IFN-γ remained relatively constant for each animal over the time period examined. CBC and plasma chemistry variables measured concurrently in the bottlenose dolphins were then examined as independent predictors of cytokine levels. We found these clinical variables were more likely to predict linear changes in serum IFN-γ and TNF-α levels compared to concentrations of these cytokines in mitogen-stimulated PBMC culture supernatants. Cytokine assays developed will be of substantial benefit in monitoring bottlenose dolphin health as an adjunct to currently available diagnostic tests.Monitoring the immune status of cetaceans is important for a variety of health conditions. Assays to quantify cytokines, especially pro-inflammatory cytokines, could be employed, in addition to currently available diagnostic assays, to screen for alterations in the health status of an animal. Though a number of immunological assays are readily available for humans and mice, specific assays for many veterinary species, including cetaceans such as bottlenose dolphins ( Tursiops truncatus), a managed population that has been well studied. An increase in the number of geriatric dolphins in this managed population, as well as other managed populations, has led to increased incidence of diseases associated with aging including hyperinsulinemia, metabolic syndrome, and autoimmune disease [While much is known regarding the immune system of humans and mice, less is known about the immune system of many veterinary species. This is especially evident for marine mammals, such as cetaceans. The U.S. Navy Marine Mammal Program (MMP) is specifically interested in improved methods to monitor the health of their bottlenose dolphins  supernatant. We then determined whether CBC and plasma chemistry variables measured concurrently were predictive of cytokine levels in bottlenose dolphins.The MMP houses and cares for a population of dolphins in San Diego Bay, CA. The MMP is AAALAC-accredited and adheres to the national standards of the United States Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the Animal Welfare Act. The MMP’s animal care and use program is routinely reviewed by an institutional animal care and use committee and the Department of Defense Bureau of Medicine. The animal use and care protocol for MMP dolphins in support of this study was approved by the MMP’s Institutional Animal Care and Use Committee and the Navy’s Bureau of Medicine . Serum samples and PBMCs were collected between month 1 (June 2014) and month 11 (April 2015). For serum collection, whole blood was collected in serum separator tubes (SST). Tubes were spun at 3000 rpm for 10 min at room temperature. Serum was collected and placed at -80°C for future use.For PBMC isolation, blood samples were collected in BD Vacutainer CPT Mononuclear Cell Preparation Tube-Sodium Citrate . Tubes were spun at 1500 x g at room temperature for 30 min. Samples were then shipped at 4°C overnight. Upon arrival, the plasma layer was removed from the top of the gel plug and diluted with an equal volume of DPBS. The sample was spun at 1200 rpm for 10 min. The supernatant was removed and RBCs were lysed. After RBC lysis and a wash with PBS, the final cell pellet was resuspended in cDMEM  with 10% FBS, L-glutamine , Antibiotic-Antimycotic Solution , and MEM NEAA .6 cells/mL and 100 μL was added to a 96 well U-bottom plate. PBMCs were either stimulated with 1, 5, or 10 μg/mL of Phytohemagglutinin (PHA) Lectin from Phaseolus vulgaris (red kidney bean)  or 1 μg/mL of Concanavalin A from Canavalia ensiformis (Jack bean) (ConA)  or not stimulated (mock) in culture media for 48 h. ConA and PHA concentrations were based on previous studies examining mitogen stimulation of PBMCs from marine mammals [The PBMC cell count was adjusted to 2x10 mammals , 12. AftELISA assays were run using the bottlenose dolphin TNF-α (DIY0693P-003) or IFN-γ (DIY0690P-003) ELISA kits from Kingfisher Biotech, Inc. We optimized the protocol for each ELISA . Plates The following CBC and plasma chemistry variables (n = 48) were measured, on each bottlenose dolphin at each time point of the study, as part of routine care at the MMP using methods previously described : Total wSerum IFN-γ and TNF-α levels were used in the statistical models, as well as IFN-γ produced by stimulating PBMCs with 1 μg/ml PHA for 48h and TNF-α produced by stimulating PBMCs with 1 μg/ml Con A for 48h. All 48 CBC and plasma chemistry variables measured on each animal were included in a stepwise regression model to determine which were independent predictors (P ≤ 0.05) of each of the four cytokine measurements. Independent predictors were then assessed for linear relationships with cytokine measurements to identify CBC and plasma chemistry variables that were independent, linear predictors of IFN-γ and TNF-α levels in the study dolphins.As an adjunct to diagnostic assays to monitor bottlenose dolphin health, we sought to develop a set of data on pro-inflammatory cytokine levels through the establishment of specific cytokine/chemokine ELISAs. With this in mind, protein levels of TNF-α  and IFN-As can be seen in Figs Overall, 48 h PBMC stimulation with 1 μg/mL ConA induced the highest production of TNF-α, while 1 μg/mL PHA stimulated the uppermost levels of IFN-γ in all animals. We have limited data (not shown) where PBMCs were stimulated for 24 h. Based on the few samples collected, it appears that 24 h stimulation was not optimal to induce sufficient cytokine production for analysis. We therefore recommend a 48 h induction as shown in Figs Shown in CBC and plasma chemistry variables were more likely to predict linear changes in serum IFN-γ and TNF-α levels compared to PBMC-stimulated IFN-γ and TNF-α levels . Higher Cytokines play an important role in inflammatory processes, in the production of antibodies, and in cell mediated-immune responses. Thus, cytokine protein levels could be used in identifying modifications or alterations in the cetacean immune system as a result of environmental insults and serve as tools in characterizing immune responses to pathogens and/or vaccines. Here we developed and tested two bottlenose dolphin specific ELISA assays: TNF-α and IFN-γ. Each animal monitored herein exhibited a distinct cytokine pattern over the 11 months in both serum samples and in stimulated PBMC supernatants. To determine any deviation in health status for a specific animal, that individual’s baseline cytokine level under normal conditions would need to be determined. Overall, serum samples may be best to use for monitoring disease state over time. In addition to easier collection and storage, serum levels of TNF-α and IFN-γ seemed to remain steady over time. It would therefore be easier to observe a change in cytokine levels if an illness or other changes in health condition did occur.We and others have used cross-reactive antibodies specific for human, mouse, porcine, and bovine on cetacean samples , 13, 14.In collaboration with Washington State University Monoclonal Antibody Center, mAb production for specific cytokines has been initiated. These mAb’s will be utilized in sandwich ELISAs to further enhance the specificity and sensitivity of bottlenose dolphin-specific assays. We are continuing to develop dolphin-specific cytokine and chemokine ELISAs. Since the initiation of the present study, additional dolphin specific ELISA assays have become available: IL-1 receptor antagonist, IL-2, IL-4, IL-8, and IL-17A. The TNF-α and IFN-γ ELISA assays described herein and others that are available from Kingfisher Biotech, Inc. will be useful in moving forward with health monitoring and disease detection in captive, managed, and free-ranging bottlenose dolphins.In this study, to assess potential clinical relevance of IFN-γ and TNF-α levels in dolphins, elevated insulin was an independent predictor of elevated serum levels of cytokines IFN-γ and TNF-α in four dolphins monitored over 10 months. Elevated cholesterol also predicted higher serum TNF-α. There were no independent, linear associations identified between white blood cell counts and IFN-γ or TNF-α, suggesting that these cytokines were better indicators of subclinical, chronic changes than acute infections. Similar to humans, dolphins can develop insulin resistance with hyperinsulinemia and dyslipidemia with hypercholesterolemia as part of a cluster of conditions called metabolic syndrome . ChronicIn conclusion, we have developed ELISA assays specific for bottlenose dolphin cytokines. We have shown that TNF-α and IFN-γ protein can be detected in the developed ELISAs using either bottlenose dolphin sera or supernatants from mitogen-stimulated PBMCs. Longitudinal analyses indicated that each dolphin exhibited a distinct cytokine pattern over the 11 month period, with serum levels of TNF-α and IFN-γ remaining relatively level over time. Finally, we observed positive associations between increasing IFN-γ or TNF-α and insulin, as well as TNF-α with cholesterol, supporting similar roles of inflammation and elevated cytokines in bottlenose dolphins to that described for human patients. Cytokine ELISAs will be of substantial benefit as an adjunct to currently available diagnostic tests in monitoring bottlenose dolphin health.S1 Table(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


Polypyrrole (PPy)/cellulose (PPCL) composite papers were fabricated by vapor phase polymerization. Importantly, the vapor-phase deposition of PPy onto cellulose was assisted by employing different co-vapors namely methanol, ethanol, benzene, water, toluene and hexane, in addition to pyrrole. The resulting PPCL papers possessed high mechanical flexibility, large surface-to-volume ratio, and good redox properties. Their main properties were highly influenced by the nature of the co-vaporized solvent. The morphology and oxidation level of deposited PPy were tuned by employing co-vapors during the polymerization, which in turn led to change in the electrochemical properties of the PPCL papers. When methanol and ethanol were used as co-vapors, the conductivities of PPCL papers were found to have improved five times, which was likely due to the enhanced orientation of PPy chain by the polar co-vapors with high dipole moment. The specific capacitance of PPCL papers obtained using benzene, toluene, water and hexane co-vapors was higher than those of the others, which is attributed to the enlarged effective surface area of the electrode material. The results indicate that the judicious choice and combination of co-vapors in vapor-deposition polymerization (VDP) offers the possibility of tuning the morphological, electrical, and electrochemical properties of deposited conducting polymers. There is an increasing demand for high-performance electric supply devices with high energy and power density due to the increased social interest in environmentally friendly devices for next-generation energy systems. In representative terms, electrochemical capacitors have strong potential for excellent energy storage characteristics, such as high-efficiency, high-power, high-speed charging, and long life cycle, offering possibilities for creating advanced new techniques in the electronic, automotive, aerospace, and medical industries. Above all, advances in materials synthesis make it possible to devise new devices or to enhance the performance of existing devices.Conducting polymers have been extensively studied for a variety of electrochemical applications including capacitor electrodes due to their high conductivity, high energy density, and excellent power capability124567910The vapor-phase polymerization approach has been successfully used to deposit conducting polymers onto substrates, and the influence of vapor-phase over liquid-phase polymerization has been witnessed in the major characteristics of the deposited polymers1213141511Polypyrrole (PPy) is recognized as one of the most promising conducting polymers due to its high conductivity, good environmental stability, biocompatibility, and low cost. Cellulose, a natural polymer, is known to form microfibrils with inherent structural rigidity and high surface area17182021222324The weight variation of the deposited PPy was monitored during the VDP to examine how the co-vapor affects the polymerization rate, as plotted in −1, which reasonably fits into the conductivity range of only PPy28The conductivities of PPCL papers were measured by four probe method and the results are shown in +), respectively  analysis. The imine nitrogen atoms of PPy in the paper are protonated in whole or in part, to yield a range of oxidation states; and thus the oxidation level of the PPCL papers can be quantitatively differentiated, by scrutinizing the XPS spectra of nitrogen atoms around 400 eV. XPS N 1s spectra of PPCL papers prepared with different co-vapors were taken see  and eachectively 32. Posit−1. −1 are compared  vaporization of pyrrole, ii) orientation of forming PPy chains, iii) rate and efficiency of oxidative polymerization, iv) swelling of the substrate, and v) diffusion of pyrrole into the substrate during VDP, all of which lead to meaningful changes in the major characteristics of PPCL composite papers. Under our experimental conditions, unique surface porous structures were generated in the presence of benzene and toluene co-vapors, and relatively thin and uniform PPy coating on cellulose can be made with the aid of water and hexane. Using alcohol co-vapors leads to higher electrical conductivities in PPy. PPCL papers hold great potential for practical applications in various areas, such as sensors, catalysis, and energy333435363) (≥98%) was obtained from Merck. Alpha-cellulose paper (>98%) as a substrate was obtained from Whatman. Nickel plate was used as a working electrode, and carbon paste was employed as a binder. All solvents were used as received, and distilled water was used for all experiments.Pyrrole (98%) was purchased from Sigma-Aldrich, and ferric chloride (FeClPPCL composite papers with a diameter of 5 cm were obtained via VDP. Cellulose papers were impregnated with 2 M ferric chloride solution (0.4 mL) and then allowed to dry in vacuum oven at 25°C for 24 h. The cellulose paper was placed into a closed reaction chamber, and then an amount of pyrrole monomer was introduced into the chamber with co-vapors, in 1:1 molar ratio. The polymerization proceeded for 2 h at 70°C, at atmospheric pressure. Subsequently, the resulting PPCL papers were washed with excess distilled water to remove residual impurities, and then dried in vacuum oven for 12 h. Methanol, ethanol, water, benzene, hexane, and toluene were used as the co-vapor.2SO4 solution, with Pt counter electrode, and Ag/AgCl reference electrode. As a working electrode, PPCL papers of 0.5 cm × 1.5 cm dimensions were attached to a nickel plate. All electrochemical measurements were conducted using Metrohm Autolab B.V. PGSTAT101 potentiostat/galvanostat. The BET surface area and pore volume were calculated, using Fisons Instruments, Sorptomatic 1990.The morphological characteristics of PPCL papers were analyzed using JEOL JSM-7500F scanning electron microscopy. Specimens for SEM were coated with a thin layer of gold. The electrical conductivities were measured at room temperature by standard four-probe method. XPS was performed, by Thermo VG Scientific Multilab 2000 spectrometer with an Mg/Al twin-anode excitation source. Peak fitting of the collected spectra was conducted with VG Avantage software. CV and charge/discharge characteristics were examined, using a three-electrode cell containing 1 M HSupplementary Information


Aseptic loosening is a significant impediment to joint implant longevity. Prosthetic wear particles are postulated to play a central role in the onset and progression of periprosthetic osteolysis, leading to aseptic loosening of the prosthesis.We investigated the inhibitory effects of a lentivirus-mediated short hairpin RNA that targets the TNF-alpha gene on the particle-induced inflammatory and osteolytic changes via macrophages both in vitro and in vivo. An siRNA sequence targeting the mouse TNF-alpha gene from four candidates, transcribed in vitro, was screened and identified. A lentivirus vector expressing short hairpin RNA (shRNA) was then constructed in order to facilitate efficient expression of TNF-alpha-siRNA. Lentivirus-mediated shRNA was transduced into cells of the mouse macrophage line RAW 264.7. Ceramic and titanium particles were introduced 24 h after lentivirus transduction to stimulate cells. TNF-alpha expression, represented by both mRNA and protein levels, was quantified with real-time PCR and ELISA at all time intervals. Lentivirus-mediated shRNA suspension was locally administered into the murine calvarial model, followed by local injection of particles. A multi-slice spiral CT scan was used to evaluate the osteolysis of the calvaria by detecting the width of the cranial sutures.Macrophages developed pseudopods when co-cultured with particles. Lentivirus-mediated shRNA was shown to effectively inhibit the expression of TNF-alpha at both the mRNA and protein levels in RAW 264.7. The multi-slice spiral CT scan showed that the lentivirus-mediated shRNA significantly suppressed osteolysis of mouse calvaria.Our investigation highlighted the results that lentivirus-mediated shRNA targeting the TNF-alpha gene successfully inhibited particle-induced inflammatory and osteolytic changes both in vitro and in vivo. Therefore, lentivirus-mediated gene therapy may provide a novel therapeutic approach to aseptic joint loosening. Total joint replacement is a common procedure and has been proven to be highly successful for the treatment of advanced arthropathy. Internationally, nearly 1.5 million total arthroplasties are performed every year [Wearing a prosthesis is believed to play a leading role in the onset and progression of periprosthetic osteolysis. The particles resulting from internal wear induce osteolysis, as has been evidenced by in vitro studies, as well as in various animal models . TitaniuAseptic loosening is believed to be initiated by the interactions between periprosthetic cells and wear particles. It has also been highlighted that macrophages make up 60–80 % of the entire cellular population around loosening periprosthetic tissues . InflammAs a post-transcriptional gene silencing mechanism, RNA interference (RNAi) has demonstrated great promise for the study of human gene function, signal transduction research, and gene therapy . RecentlIn the present study, we aimed to find an efficient system by fabricating a recombined lentivirus vector encoding shRNA targeted against TNF-alpha and examining the TNF-alpha knockdown efficiency in mouse macrophages, as well as the in vivo effect on reducing the osteolysis and inflammation seen in mice with experimental particle-induced osteolysis.3 were purchased from Alfa Aesar , and alumina ceramic particles of 3.53 g/cm3 from Ceram Tec . All particles were disseminated in pure water and filtered through Millipore filter membranes  of different diameters (0.2 μm and 1.2 μm) to select particles with a target size between 0.2 μm and 1.2 μm. An image analysis system  was used to measure the exact size of the particles, and the results were 0.82 ± 0.12 μm and 0.84 ± 0.14 μm for the filtrated titanium and ceramic particles, respectively. All particles were rinsed with 70 % ethanol for 24 h at room temperature and then dried in an oven before being sterilized with ethylene oxide. Based on particle weight and density, particles incubated in phosphate buffered saline (PBS) were adjusted to 4 × 108 μm3/ml in concentration. The level of endotoxin in particle solutions was detected using the Limulus Amebocyte Lysate Assay , and the result was below the detection level of 0.01 EU/ml.Commercially pure titanium particles of 4.507 g/cm8 TU (titer unit)/ml.Four siRNA sequences against the mouse TNF-alpha gene (Gene Bank Accession No NM_004517.2) were designed using online siRNA software. Preliminary experiments indicated that the best performing TNF-alpha siRNA sequence with the highest inhibitory efficiency was 5’-GCAAACAGAGCATGGTCAA-3’. Meanwhile, a missense siRNA (MS siRNA): 5’-TAATCGTCGT AGACGGTTG-3’ was used as the control. Based on the siRNA sequences described above, an shRNA cassette was designed with 19 nucleotides (nt) of the target sequence, a loop sequence (TTCAAGAGA), the reverse complement of the 19-nt target sequence, and then a stop codon for the U6 promoter, as well as digestion sites for HpaI and XhoI. Following the design described above, the shRNA cassettes were synthesized along with their complementary strands. The shRNAs were inserted into the pFU-GW-iRNA lentivirus vector, which contained a CMV-driven EGFP reporter gene and a U6 promoter upstream from the restriction sites (HpaI and XhoI). All of the constructs were ascertained by sequence analysis. The vector was designed to co-express green fluorescent protein (GFP) cloned from a copepod. Bearing the shRNA sequences and pPACK Packaging Plasmid Mix (System Biosciences), the pFU-GW-iRNA lentivirus vector was co-transfected into 293TN cells with Lipofectamine 2000 (Invitrogen). After 48 h, viral supernatants were harvested and passed through 0.45 μm filters. The target lentivirus and the negative control lentivirus expressing TNF-alpha-siRNA and non-targeting sequence were denoted as TNF-alpha-RNAi-LV and NC-GFP-LV, respectively. Expression levels of GFP were studied by FACS analysis and PCR. In order to calculate the virus titers, the number of cells was multiplied by both the percentage of GFP-positive cells and the dilution factor, resulting in the formation of the titer units (TU)/ml. The concentration was 5 × 102 to improve the adherence of cells to the plates. Trypan blue dye exclusion was used for cell quantitation and the viability assays, with viability exceeding 98 % for all trials.RAW264.7, the murine macrophage cell line, was obtained from American Type Culture Collection (ATCC Number: TIB-71). The cells were seeded and cultured in 96-well tissue culture plates  in DMEM medium with 10 % fetal bovine serum (FBS) (Hyclone). Cells were incubated overnight in a humidified incubator at 37 °C with 5 % COFor lentivirus transduction, RAW 264.7 macrophages were infected with either TNF-alpha-RNAi-LV or NC-GFP-LV in serum-free growth medium with 5 mg/ml polybrene at multiplicities of infection (MOI) of 150, which was optimized from the toxicity curve before transduction. After 8 h’ of culturing, the medium was substituted by fresh DMEM complete medium, in order to remove debris and inactive lentiviruses before the cells were continuously incubated for another 72 h. Three days after transfection, the transfection efficiency was examined by fluorescent microscopy.3: cell number) and supplemented to cultures, whereupon the culture plates were gently agitated for 10 min. Experimental grouping was made up of seven groups, as follows:Control Group: free of any interference measure or particle stimulationTI Group: free of any interference measure, but stimulated with titanium particlesTI + TNF-LV Group: transfected with TNF-alpha-RNAi-LV and stimulated with titanium particlesTI + N.C-LV Group: transfected with NC-GFP-LV and stimulated with titanium particlesCE Group: free of any interference measure, but stimulated with ceramic particlesCE + TNF-LV Group: transfected with TNF-alpha-RNAi-LV and stimulated with ceramic particlesCE + N.C-LV Group: transfected with NC-GFP-LV and stimulated with ceramic particlesAfter lentivirus transduction, cells seeded in 6-well plates were digested. The particles were added to all of the experimental groups, except for the control group. They were then developed into suspensions at a final concentration of 100:1 , in order to determine interference efficiency by real-time PCR and ELISA.n fold difference relative to the negative control.An RNA-to-cDNA reverse transcription was performed with Primescript RT reagent . The mRNA levels were determined using real-time PCR on the Roche Light Cycler 480 System . With Primer 5.0, the following gene-specific primers were designed (5’ to 3’): GAPDH, TGTGTCCGTCGTGGATCTGA (forward) and TTGCTGTTGAAGTCGCAGGAG (reverse), as well as TNF-alpha, AGCCCCCAGTCTGTATCCTT (forward) and CTCCCTTTGCAGAACTCAGG (reverse). The product was detected with the SYBR green dye, SYBR Premix Ex Taq . The results of GAPDH amplification efficiency and TNF-alpha primers were 1.976 and 2.022, respectively. The comparative CT (threshold cycle) method with arithmetic formulas was used to determine the relative level of gene expression for both the target and house-keeping genes. Since the negative controls are referred to as the calibration factor, all of the other quantities are expressed as an The protein production of TNF-alpha in the supernatants was assayed with an immunoassay ELISA kit  as per the manufacturer’s instructions.Male C57BL/6 mice, aged 12 weeks, were purchased from the Center of Animal Experiments of Sun Yat-sen University . All animal handling and experiment procedures were performed in compliance with the guidelines of the National Institutes of Health for animal care, and all animal experiments were approved by The Memorial Hospital of Sun Yat-Sen University.Control: PBS was administered locally into the cranial sutures without lentivirus infection or particle stimulationTI: stimulated with titanium particles, free from interference measuresTI + TNF-LV: stimulated with titanium particles and transfected with TNF-alpha-RNAi-LV.TI + N.C-LV: stimulated with titanium particles and transfected with NC-GFP-LVCE: stimulated with ceramic particles, free from interference measuresCE + TNF-LV: stimulated with ceramic particles and transfected with TNF-alpha-RNAi-LVCE + N.C-LV: stimulated with ceramic particles and transfected with N.C control-lentivirus mediated shRNAIn this study, 49 mice were randomized into seven groups, with seven mice in each group:7 TU of TNF-alpha-RNAi-LV, NC-GFP-LV, or PBS alone was slowly injected into the intervals between the cranial sutures and the gelfoam, using a 1 ml injector. Since preliminary experiments have shown that particles injected locally on the surface of murine calvaria induced significant osteolysis by the 14th day, the mice in our study were sacrificed with carbon dioxide asphyxiation for CT scan analysis 14 days after transfection. All experiments involving the construction and in vivo evaluation of the lentiviral vectors were completed under bio-safety level II containment.Locally injecting wear particles in mice can establish a model of particle-induced osteolysis . A 1*1 cThe osteolysis of the calvaria was evaluated based on the width of the cranial sutures after 2 weeks using multi-slice spiral CT scans Fig. . The crap value of < 0.05. All statistical analyses were performed using SPSS .Results are shown as mean ± SD. ANOVA was performed to analyze differences between groups. Significance was set at a A scanning electron microscope  was used to observe the macrophages after particle stimulation, which showed that the macrophages in a stimulation-free environment basically maintained smooth and non-distorted contours Fig. . When coThe efficiency of lentivirus-mediated shRNA transfection into RAW264.7 macrophages was determined by GFP fluorescence. The numbers of cells and fluorescent spots in the same visual field were counted with light and fluorescence microscopes, respectively. The transfection efficiency of lentivirus-mediated shRNA via cationic liposomes was 85.2 ± 3.5 %  at any time point, and was insignificant between the Particle and Particle + N.C-LV groups (p > 0.05)  Fig. , b. The p < 0.05), and was insignificant between the Particle and Particle + N.C-LV groups (p > 0.05) .ELISA analysis of the cell supernatants demonstrated TNF-alpha gene silencing Fig. . As show05) Fig. . The inhp < 0.01) .The width of the cranial suture at the widest point was measured in order to assess the particle-induced osteolysis in the murine calvarial model. Significant narrowing of cranial suture width was revealed in the Particle + TNF-LV group compared with the Particle and Particle + N.C-LV groups (01) Fig. , suggestInflammation and the consequent osteolysis due to the presence of wear particles are both believed to be involved in the pathogenesis of aseptic loosening , which iMacrophages play a pivotal role in these processes . MacrophRNAi is a useful target for the analysis of gene function and a candidate for potential therapeutic strategies in various conditions . The delIn addition to the dose, size, and shape of the particle, its material property is considered to be a major parameter influencing the consequent immune response . With thUnder in vivo conditions, particles disperse randomly as they interact with the local tissue. The animal model used in our research was first described by Merkel, et al. . StudiesBy taking advantage of the strengths of murine calvaria, a lentiviral vector, and local administration, we explored the role of a novel gene therapy in inhibiting particle-induced inflammation and osteolysis. As shown by the experiments, a single dose of LV-mediated siRNA targeting TNF-alpha resulted in the hypoexpression of the corresponding mRNA by up to 80 %, and markedly compromised inflammatory responses in murine calvaria for at least 2 weeks. In addition, the fact that GFP fluorescence was found to be localized in the pouch area suggested that local administration was a safe and effective delivery route for the siRNA. Combined, these results lead to the conclusion that LV-mediated TNF-alpha shRNA remarkably inhibit particle-induced inflammation and osteolysis in the murine calvarial model. This finding serves as a building block for in vivo RNAi therapy in addressing aseptic loosening.In summary, our investigation shows that lentivirus-mediated shRNA targeting the TNF-alpha gene inhibits particle-induced inflammation and osteolysis both in vitro and in vivo. Conscious efforts have been made in this study to investigate an alternative therapeutic strategy for applying RNAi for the amelioration of this condition. Although the gap between this trial and clinical care for aseptic loosening has not yet been filled, this study has yielded insight into the potential therapeutic role of siRNA in this disease. In order to assess the osteolytic mediators in particle-induced aseptic loosening, the quantitative assessment of inflammatory cytokines on the murine calvarial surface is worthy of research.


Escherichia coli-GFP (E. coli-GFP). Monocyte phenotype, phagocytic activity, induction of apoptosis, and TNF-α/TNF-receptor (TNFR) -expression were analysed. In the course of infection TNF-α-secretion of CBMO was reduced to 40% as compared to PBMO (p<0.05). Neutralization of TNF-α by an αTNF-α antibody reduced apoptotic PICD in PBMO four-fold (p < 0.05 vs. infection with E. coli). PICD in CBMO was reduced 5-fold compared to PBMO and showed less responsiveness to αTNF-α antibody. CBMO expressed less pro-apoptotic TNFR1, which, after administration of TNF-α or infection with E. coli was internalized to a lesser extent. With similar phagocytic capacity, reduced TNFR1 internalization in CBMO was accompanied by lower activation of caspase-8 (p < 0.05 vs. PBMO). Stronger caspase-8 activation in PBMO caused more activation of effector caspase-3 and apoptosis . Our results demonstrate that TNFR1 internalization is critical in mediating PICD in monocytes after infection with E.coli and is reduced in CBMO.Phagocytosis-induced cell death (PICD) is diminished in cord blood monocytes (CBMO) as compared to cells from adults (PBMO) due to differences in the CD95-pathway. This may support a prolonged pro-inflammatory response with sequels of sustained inflammation as seen in neonatal sepsis. Here we hypothesized that TNF-α mediated induction of apoptosis is impaired in CBMO due to differences in the TNFR1-dependent internalization. Monocytes were infected with  Pre- and postnatal infections are triggers for a variety of diseases later in life, involving different organ systems. The common link between infection and its secondary sequels, organ damage, evidently is inflammation, and typical diseases of the preterm infant, such as bronchopulmonary dysplasia (BPD) , necrotiInflammatory reactions following infection are mediated by cytokines and effector cells. In addition to granulocytes, monocytes are part of the host’s rapid response component and generate a vigorous antibacterial reaction. The fast and effective elimination of bacteria is the basis for an effective antimicrobial defence and accompanied by cellular and humoral host cell signals. In sepsis of adults, a phenomenon called phagocytosis-induced cell death (PICD) plays a key role in the orchestration of an antibacterial host response, by provoking effector cell apoptosis and thus contributing to a controlled termination of inflammation .PICD is a prerequisite for both, neutralization of bacteria and termination of inflammation. An incomplete elimination of bacteria may result in additional damage to organs and tissues. Attenuation of PICD skews the situation to prolonged cytokine production which may lead to a permanent inflammation resulting in a systemic inflammation response syndrome (SIRS) .The latter clinical picture appears in postnatal sepsis of neonates, as well as in prenatal infection, and is termed fetal inflammatory response syndrome (FIRS). Therefore, effector cell apoptosis is tightly regulated and involves external and internal signalling pathways .in-vitro-system using the two most common agents of neonatal sepsis, group B streptococci (GBS) and Escherichia coli (E. coli), CBMO and PBMO showed identical phagocytic and intracellular degradation activities, however CBMO underwent PICD to a lesser extent than PBMO. Inhibition of phagocytosis by Cytochalasin D reduced the induction of PICD. Analyzing factors triggering apoptosis revealed a diminished secretion of CD95L, one of the death-ligands of the TNF-family, in CBMO .–22.E. coescribed . Therefond vs. 4th columns). These data, together with the observation that PICD of PBMO, but not CBMO could be greatly diminished by blocking TNF-α (st vs. 2nd column) and that TNF-α remained on the surface of CBMO (E.coli-induced PICD in cord blood (CBMO) remains to be elucidated; although the administration of anti-TNF-α antibody inhibited PICD of CBMO significantly , indicating that TNF-α initiates the downstream activation of effector caspase-3 (rd vs. 2nd columns) via caspase-8 (th columns), leading to apoptosis , and decreased the number of infected apoptotic PBMO (nd vs. 3rd columns). Adding TNF-α into this scenario (th columns) had no synergistic effect on the PICD of PBMO, but led to a significant increase of PICD of CBMO to the extent as comparable to infected PBMO.In this manuscript, we also addressed the question, whether shedding of TNF-α and its receptor TNFR1 interferes with pro-apoptotic signalling. Blockage with the metalloprotease-inhibitor GM6001  resultedin vivo.Besides TNF-α/TNFR1/TNFR2 and CD95L/CD95 other ligands/receptors may play a role in regulation of apoptosis in PBMO and CBMO. TRAIL receptors can be activated and also activate the extrinsic caspase signalling pathway. The TRAIL receptor expression was not found reduced in CBMO compared to maternal PBMO , but expS1 FigE.coli (MOI 25) and the PI  and the TNF-α secretion (B) measured. Induction of apoptosis in non-infected and infected whole blood of adults (white hatched bars) and neonatal (grey hatched bars) samples was also determined .Whole blood cells of adults (white bars) and neonates (grey bars) and MNC were infected with (TIF)Click here for additional data file.S2 FigPercentage of hypodiploid nuclei after addition of TNF-α was measured in monocytes exhibiting TNFR1 on the surface compare , n = 5.(TIF)Click here for additional data file.S3 FigE.coli infected PBMO with or without IETD treatment (compare lane 2 and 3). Note, that non-treated PBMO show different expression of cleaved RIP due to varied exposure time.Quantification of immunoblots comparing the expression of TRADD (A) and RIP (B) in PBMO and CBMO under indicated treatment . Representative immunoblots are shown below the charts. (C–D) One of three immunoblots comparing RIP (designated as RIP) cleavage in PBMO and CBMO under indicated treatment (C) and RIP cleavage in (TIF)Click here for additional data file.S1 TableTNFR2 expression on PBMO and CBMO was assessed by FACS staining .(XLSX)Click here for additional data file.S2 TablePBMO and CBMO received inhibiting TNFR2 antibody when indicated. Apoptosis was assessed by detection of hypodiplid nuclei and is given as percentage of apoptotic cells (n = 4).(XLSX)Click here for additional data file.


Tumor necrosis factor–α (TNF-α) blockers are a popular therapeutic choice in a number of inflammatory diseases. Thus far, five TNF- α blockers have been approved for clinical use . Despite being considered relatively safe, serious side effects associated with immune suppression have been reported, including central and peripheral nervous system (CNS) demyelinating disorders. It is still elusive whether these events are mere coincidence or a side effect of anti-TNF-α use. In this paper, we review the published case reports of CNS demyelination associated with anti-TNF-α therapy and present the follow-up of our 4 previously reported patients who developed neurologic symptoms suggestive of CNS demyelination after having received anti-TNF-α treatment. We also discuss the possible role of TNF-α blockers in demyelination. Tumor necrosis factor-α (TNF-α) blockers present a revolutionizing therapeutic choice for a number of inflammatory diseases such as Crohn’s disease (CD), ankylosing spondylitis (AS), and rheumatoid arthritis (RA). Several terms such as TNF-α blockers, anti-TNF-α agents, TNF-α antagonists, and TNF-α inhibitors are used interchangeably, all describing the same biological agents. Henceforth in our paper, we will use the term TNF-α blocker for clarity.These agents are more effective than traditional disease modifying drugs (DMDs), controlling disease activity and preventing underlying structural tissue damage. Although they are relatively safe, an increasing number of neurologic side effects have been reported in the literature, consisting of central and peripheral nervous system demyelinating events. These adverse events suggest a possible relationship between anti-TNF-α use and demyelination . HoweverIn this paper, we review the pathogenic and protective functions of TNF-α, the role of TNF-α blockers in CNS demyelination and the CNS demyelinating cases reported in the literature to be related to anti-TNF-α therapy. We also present the follow-up of our 4 previously reported patients  who deveTNF-α is a pleiotropic cytokine, with a wide range of functions: homeostatic, immune, and inflammatory. The beneficial homeostatic functions of TNF-α include defense against pathogens, development of lymphoid organ architecture, resolution of inflammation, tissue regeneration, immune regulation, and inhibition of tumor growth. The pathogenic functions of TNF-α comprise triggering of inflammation, stimulation of vascular endothelium, proliferation of immune cells, and tissue damage , 4. UndeTNF-α is produced initially as a transmembrane molecule (tmTNF). Subsequently, it is released from cells as a soluble cytokine (sTNF) via regulated cleavage of tmTNF by TNF-α converting enzyme (TACE). Both forms of TNF-α are biologically active and interact with two receptors (TNFR1 and TNFR2) with different affinity. sTNF has a higher affinity for TNFR1, contains a death domain, and mediates apoptosis and chronic inflammation . tmTNF hAt low levels in tissues, TNF-α exerts beneficial homeostatic functions, as it enhances host defense mechanisms against intracellular pathogens, particularly mycobacteria . At elevFive anti-TNF-α blockers are approved for clinical use: etanercept (circulating receptor fusion protein), infliximab, adalimumab, and golimumab , and certolizumab  . Both reCompared with the traditional DMDs, TNF-α blockers are more efficacious, with faster onset of action and more effective control of disease progression. These characteristics have made them an appealing option in refractory cases , 19.TNF-α blockers present a revolutionizing therapeutic choice for inflammatory diseases such as RA, AS, plaque psoriasis, psoriatic arthritis, juvenile polyarticular rheumatoid arthritis, and inflammatory bowel disease (CD and ulcerative colitis). TNF-α blockers are also used off-label for a number of other inflammatory conditions such as sarcoidosis, hidradenitis suppuritiva, Adamantiades-Behcet's disease, pyoderma gangrenosum, dermatomyositis, scleroderma, noninfectious uveitis, and others , 21. NovInitial studies in experimental autoimmune encephalomyelitis (EAE) animal models of multiple sclerosis (MS) showed beneficial effects of TNF-α blockers. Subsequently, given their anti-inflammatory effects, clinical trials of infliximab  and leneThese trials suggested that non-selective inhibition of TNF-α is harmful in MS and that TNF-α exerts both potent pro-inflammatory effects and essential protective functions in the CNS under pathological conditions ••.The efficacy of TNF-α blockers in inflammatory conditions has increased their use. Despite their relatively safe profile, well-known adverse events include injection site reactions, risk of infections , congestive heart failure, hemocytopenia, and T-cell lymphomas , 25. RepWith the widespread use of TNF-α blockers, a growing number of demyelinating events have been reported, including CNS demyelinating disorders , as well as peripheral nervous system disorders  , 30. It The pleiotropic functions of TNF-α often show contradictory effects, particularly in the CNS. TNF-α and its receptors can either promote neuroinflammation and secondary neuronal damage, or exert protective functions under pathological conditions. Furthermore, TNF-α exerts distinctive actions at different stages of autoimmune demyelination: sTNF, but not tmTNF, promotes inflammation and disease onset, whereas sTNF and/or tmTNF have protective functions in established disease by reducing the extent and severity of autoimmune inflammation. sTNF mediates the proinflammatory effects of TNF-α via TNFR1 signaling. TNFR1 plays a critical role for the onset of CNS autoimmune disease, through induction of a pro-inflammatory environment in the CNS, but subsequently suppresses local inflammation either indirectly, mediating neuroprotection, or directly, promoting repair processes ••. ElevaBesides being crucial in host defense and inflammation and accelerating acute demyelinating processes, TNF-α is also necessary for triggering remyelination , 39 and TNFRSF1A gene. This SNP in TNFR1 leads to expression of a soluble form of TNFR1 that inhibits TNF-α in humans and this may play a role in MS development in some individuals, possibly mimicking the effects of TNF-α blockers [Interestingly, MS susceptibility has been associated with a single nucleotide polymorphism (SNP) in a gene, encoding TNF receptor 1 (TNFR1), the blockers .TNF-α blockers cannot penetrate the intact BBB to suppress demyelination but they can enhance demyelination through increased ingress of peripheral autoreactive T-cells into the CNS (lack of entry theory). This theory provides a possible explanation for the failure of anti-TNF-α blockers in reducing demyelination and for their effect on aggravating MS , 47.TNF-α blockers may aggravate CNS demyelination by decreasing TNFR2 receptors, which are necessary for the proliferation of immature oligodendrocytes and myelin repair –54.TNF-α blockers could alter cytokine responses by downregulating interleukin-10 and upregulating interleukin-12 and interferon-γ, creating a profile similar to that of MS patients , 55•, 56TNF-α blockers may deactivate TNF-α systemically, but not within the CNS (due to BBB impermeability), leading to a high concentration of TNF-α in the CNS (“sponge effect”) , 47.There may be systematic dysregulation of TNF-α in patients with RRMS, as was shown in a recent study of Mausner-Fainberg et al ••, in whFinally, TNF-α blockers may unmask an underlying latent infection, which can lead to autoimmune demyelination , 58, 59.Since TNF-α is implicated in demyelinating processes, TNF-α blockers were considered as a potential therapeutic choice in MS. However, the negative outcomes of these agents in MS trials , 23 and We present the follow-up of our 4 previously reported patients, 2 with RA and 2 with AS who developed neurologic symptoms following anti-TNF-α exposure ••, 93.All cases are summarized in Table Our patients had been treated with TNF-α blockers previously or were taking them at the onset of symptoms; thus, a correlation between the treatment and demyelination was speculated. Anti-TNF-α treatment was discontinued as soon as demyelination was suspected and steroids were administered intravenously with consequent clinical improvement. During the 5-year follow-up, 2 patients remained stable without relapses or new MRI lesions, whereas the other 2, both male, had clinical relapses and radiological deterioration with new brain and spinal cord (SC) demyelinating lesions. One of them required pulsed steroid treatment, whereas the other had a minor relapse that subsided without therapy Fig. . It is rNone of our patients received specific MS treatment, although all of them were treated with immunesuppressants for their initial autoimmune disease (AS or PsA), after the presenting demyelinating event.TNF-α blockers are currently recommended for patients not responding to at least 2 different DMDs and seem to have an excellent and sustained therapeutic outcome: 80% of patients improve rapidly and 50% have complete remission , 95. PatAlthough CNS demyelination after treatment with TNF-α blockers is not necessarily associated with the duration of the therapy and drug discontinuation does not always lead to improvement , treatmeReported cases and pathogenic theories2.Miller criteriaThe large number of CNS and peripheral demyelinating disorders after TNF-α blocker administration published in the literature ••, 93 an3.Age of onsetSome of the published cases meet the Miller criteria , attribuThe peak age of MS onset is 20–40 years old, whereas the mean age of RA onset is 40–60 years old. In our review, the mean age of the patients who developed CNS demyelination was 45.47 years. This delayed onset of demyelination could suggest a possible association with anti-TNF-α use.IncidenceSeveral factors, on the other hand, challenge a causal relationship of anti-TNF-α treatment with the appearance of CNS demyelination2.However, it should be emphasized that demyelinating events could be silent and therefore under-reported, making it difficult to estimate the actual incidence of CNS demyelination after anti-TNF-α therapy . This isAlthough over 2 million patients with rheumatic and other chronic inflammatory diseases have been treated with anti-TNF-α therapies ••, the o3.Previous treatmentsPatients with autoimmune diseases could be genetically prone to develop another autoimmune disease , a fact 4.Rechallenge phenomenonTNF-α blockers are therapeutic options for patients who have already received treatment with a number of other immunosuppressive agents, most commonly methotrexate. Although methotrexate is an immunosuppressant extensively used in MS, it has been associated with demyelinating events at high doses and in combination with cranial radiotherapy .4.Rechal5.Time interval between therapy and symptom onsetThe rechallenge phenomenon could be simply explained by the relapsing-remitting nature of MS . On the 6.OutcomeThe interval between treatment administration and symptom initiation varies greatly, from 5 months up to 4 years . In the In most published cases, demyelination was either slowly progressing or stopped after anti-TNF-α discontinuation. This could suggest that anti-TNF-α therapy exerts a protective effect in patients already suffering from latent MS, or has a short lasting harmful impact.Anti-TNF-α blockers have reshaped the treatment of rheumatoid and autoimmune diseases, being the most effective drugs in our therapeutic armentarium. However, TNF-α functioning, especially in the CNS, is still incompletely understood, setting restrictions on their unlimited use. To date, the reported cases of demyelination address the possibility of a causal association. Our previous study raised the question whether the demyelinating events in patients who had previously received TNF-α blockers were the result of uncovering latent MS, onset of a novel demyelinating event (MS or MS-like syndrome), or were merely an accidental coexistence of the 2 disorders. Long-term follow-up of these patients is required to properly diagnose, determine the clinical course, and point out the potential differences from typical MS. Complete symptom remission after therapy withdrawal or a positive rechallenge phenomenon could prove causality, although the nature of MS, with relapses and remissions of symptoms, argues against such a relationship. The complexity of the biological functions of TNF-α further complicates the issue. Although several theoretical explanations of demyelination after TNF-α blockade have been proposed, the relation of these events with the use of TNF-α blockers remains to be elucidated.Nevertheless, according to the current guidelines, avoiding the use of anti-TNF-α therapy is recommended in patients with a history or familial occurrence of MS or other demyelinating diseases. Furthermore, in case of appearance of neurologic symptoms, the patient should undergo a thorough neurologic assessment (level of evidence III and a grade of recommendation B) . BesidesWe propose careful clinical evaluation of candidates for TNF-α blockers, including neurologic assessment before drug initiation and close neurologic monitoring, in order to diagnose a possible demyelinating condition in as timely a manner as possible, especially in high risk patients.Anti-TNF-α therapies undoubtedly are an excellent therapeutic choice in refractory inflammatory diseases. On the other hand, considering the possibility of demyelination, more specific TNF-α-targeting drugs might be safer. Anti-TNF-α therapies that either target TNFR1 while sparing TNFR2 signaling selectively, or inhibit sTNF (currently under investigation) could potentially minimize the adverse events in the CNS. Furthermore, based on the observation that TNFR2 antagonizes TNFR1 signaling, either TNFR2 agonists or increased expression of TNFR2 by gene therapy might be beneficial . The posWhether TNF-α blockers directly cause demyelination (either with a progressive or a monophasic course) or they trigger pre-existing demyelinating predisposition, still remains controversial. Unfortunately, only few patients with a satisfactory follow-up are reported in the literature. Systematic and long-term follow-up of affected patients might shed light to these still unanswered questions and help distinguish which patients will safely benefit from anti-TNF-α treatment for their inflammatory disease. Furthermore, elucidation of the exact mechanism of the apparently paradoxical response to TNF-α blockers in MS might improve our understanding of the pathophysiology of MS.


Several cellular functions such as apoptosis, cellular proliferation, inflammation, and immune regulation involve the tumor necrosis factor-α (TNF)/TNF receptor (TNFR) pathway. Human immunodeficiency virus 1 (HIV-1) interacts with the TNF/TNFR pathway. The activation of the TNF/TNFR pathway impacts HIV-1 replication, and the TNF/TNFR pathway is the target of HIV-1 proteins. A hallmark of HIV-1 infection is immune activation and inflammation with increased levels of TNF in the plasma and the tissues. Therefore, the control of the TNF/TNFR pathway by new therapeutic approaches could participate in the control of immune activation and impact both viral replication and viral persistence. In this review, we will describe the intricate interplay between HIV-1 proteins and TNF/TNFR signaling and how TNF/TNFR activation modulates HIV-1 replication and discuss new therapeutic approaches, especially anti-TNF therapy, that could control this pathway and ultimately favor the clearance of infected cells to cure HIV-infected patients. Tumor necrosis factor-α (TNF) was first described as a glycoprotein induced in response to endotoxin (the old name of which was cachectin) and has the capacity to kill tumor cells . Then, TMany cell types, especially immune cells, produce TNF in response to pathological conditions such as inflammation and infection, including: activated macrophages and T lymphocytes, natural killer (NK) cells, mast cells, and fibroblasts ,8. A memA 34 kDa adapter protein called TNFR-associated death domain (TRADD) interacts with the cytoplasmic death domain of TNFR1 through its own death domain in response to the binding of TNF to TNFR1  Figure . FurtherThe role of TNF in HIV-1 entry has been extensively described, especially in primary human macrophages  . In vitrSeveral intracellular signaling pathways, including NF-κB, JNK, and p38 MAP kinase pathways, are triggered by TNF  32]. Di. Di32]. Vpr encodes a 96-amino acid, 14 kDa protein, which is conserved among HIV-1, HIV-2, and simian immunodeficiency virus. Multiple cellular functions are targeted by Vpr, including the induction of cell cycle arrest in the G2 phase, transactivation of the LTR promoter, nuclear import of preintegration complexes, and modulation of apoptosis. Both pro- and anti-apoptotic roles of Vpr have been described. Vpr triggers apoptosis in infected T-cells through activation of caspases 3/7, 8, and 9 ,46 and bA close interplay between TNF and HIV-1 Nef is present throughout the progression of the disease . The mulThe HIV-1 Tat protein is critical for the elongation process during viral transcription, but also activates NF-κB, JNK, p38, and ERK1/2 pathways and thereby could mimic the effect of TNF on cellular gene expression ,69 Figu. Both MyThe production of TNF, but also of IL-6, IL-10, and interferon γ, has been reported after the interaction of gp120 with CD4 (Reviewed in ). The prThe interaction between macrophage-membrane bound TNF and TNFR2 present on CD8+ T- cells triggers CD8+ T-cell apoptosis . ChronicIn several chronic inflammatory diseases, such as rheumatoid arthritis, ankylosing spondylitis, juvenile arthritis, psoriatic arthritis, Crohn’s disease, and ulcerative colitis, the blockade of TNF signaling is commonly used. Thus, medications such as antibodies or receptors, which target the TNF/TNFR pathway for treating various inflammatory diseases, are now in preclinical or clinical phases of development ,83. In 1At all stages of HIV-1 infection, increased amounts of TNF can be detected either in the plasma or in the tissues ,86,87. EMoreover, the release of soluble TNF receptors (sTNFR1 and sTNFR2), following the membrane-bound TNFR shedding by proteolytic cleavage, can compete with the membrane-bound TNFRs for binding to TNF, thus inhibiting its activity. In addition, the cells could be transiently desensitized to TNF action, due to the decrease in the number of membrane receptor molecules as a result of receptor shedding ,96,97. NFor a complete viral eradication in HIV-1 infected patients, the persistence of HIV-1 reservoirs is a major challenge ,112,113.The ‘shock and kill’ paradigm has been challenged due to significant concerns about the feasibility and possible problems of this approach ,136. A nBoth immune activation and viral reservoirs are hallmarks of HIV-1 infection and involve the TNF/TNFR pathway. Thus, modulating TNF/TNFR pathway by new therapeutic approaches could limit immune activation and curtail the size of the HIV-1 reservoir in cART-treated patients with undetectable viremia, with the ultimate goal being to cure HIV-infected patients.


Microsoft Excel automatically converts certain gene symbols, database accessions, and other alphanumeric text into dates, scientific notation, and other numerical representations. These conversions lead to subsequent, irreversible, corruption of the imported text. A recent survey of popular genomic literature estimates that one-fifth of all papers with supplementary gene lists suffer from this issue.http://www.github.com/pstew/escape_excel), including a command line based Perl script, a Windows-only Excel Add-In, an OS X drag-and-drop application, a simple web-server, and as a Galaxy web environment interface. Test server implementations are accessible as a Galaxy interface (http://apostl.moffitt.org) and simple non-Galaxy web server (http://apostl.moffitt.org:8000/).Here, we present an open-source tool, Escape Excel, which prevents these erroneous conversions by generating an escaped text file that can be safely imported into Excel. Escape Excel is implemented in a variety of formats (Escape Excel detects and escapes a wide variety of problematic text strings so that they are not erroneously converted into other representations upon importation into Excel. Examples of problematic strings include date-like strings, time-like strings, leading zeroes in front of numbers, and long numeric and alphanumeric identifiers that should not be automatically converted into scientific notation. It is hoped that greater awareness of these potential data corruption issues, together with diligent escaping of text files prior to importation into Excel, will help to reduce the amount of Excel-corrupted data in scientific analyses and publications. Auto-conversion of gene symbols and database accessions by the spreadsheet software Excel  is a persistent issue in biomedical research. The first published account dates back to 2004, where gene symbols for the Septin family  and others were reported as automatically converted to date formats within the software  . Additioth column. Additionally, there is no option to select column data types when a text file is opened via "double clicking", "drag and drop", or "Open With" from the Windows file explorer or desktop. Thus, an automated method of escaping a text file prior to import in Excel, so as to protect vulnerable fields from unwanted auto-conversion, is necessary.In order to avoid unwanted auto-conversions, text files can be imported via the File->Open menu within the Excel program, which then guides the user through options that allow the user to decide which columns to treat as text and which to treat as other data types. However, this method is time consuming, becomes impractical for large numbers of columns, cannot handle columns of mixed data types, and does not allow for customization of any columns beyond the 256text to escape", is able to protect strings from auto-conversion on text file import.According to Microsoft, auto-conversion in Excel can be prevented by manually placing a single quotation (') in front of an entry as an escape character to force the cell to be interpreted as text . HoweverEscape Excel scans a tab-delimited text file for entries that need to be escaped. These entries are identified using regular expressions. All fields are first cleaned by removing leading/trailing spaces, enclosing quotes, leading single quotes, and UTF-8 byte order marks. These can interfere with correct parsing of the escaped output field by Excel, as well as interfere with regular expression pattern matching. Entries that are identified as likely to undergo unwanted auto-conversion by Excel are then encapsulated in a text string within an equation . Starting the entry with an equals sign tells Excel to treat the field as an equation, and the quotes tell Excel that the equation consists solely of a string. Once Escape Excel is run on a file, the resulting output can be opened in Excel without auto-conversion issues.Pseudocode for the algorithm is as follows: for each field within a line  # remove problematic characters  do   remove leading UTF-8 byte order mark   remove pre-existing Escape Excel escaping   remove enclosing double quotes   remove remaining leading double quotes   remove leading spaces   remove trailing spaces  while (any cleaning rule was applied)  # escape numbers  if field is a number   if escape leading zeroes flag setting    escape leading zeroes for integer numeric portion   end if   if escape scientific notation flag setting    escape scientific notation with ≥ 2 digits before decimal (i.e. 12E3)    escape numbers with > 11 digits before decimal   end if  # escape everything else, if paranoid option was set  else if paranoid flag setting   escape everything that is not a number  # escape only potentially problematic fields  else   escape leading single quote   escape leading equals sign   escape leading +/- if followed by non +/- characters   if escape dates flag setting    escape anything that looks like a date and/or time of day   end if  end if end forThe original implementation, from which all other implementations derive, is implemented as a Perl script. Thus, the Perl script requires a Perl interpreter to be installed, either on the server, or on the user's local machine, depending on the implementation. Usage information, along with non-default options for disabling certain classes of pattern matching rules, is displayed when the script is run on the command line with the—help option . If diffhttp://www.jrsoftware.org/isinfo.php). The Add-In does not require the installation of a Perl interpreter, as the included escape_excel.exe executable consists of a self-extracting archive that runs the script in memory from within an embedded Perl interpreter, packaged with the pp PAR Packer (http://search.cpan.org/dist/PAR-Packer/) using the Strawberry Perl interpreter (http://strawberryperl.com/). The included eol2eol.exe program, called from within the Add-In in order to support carriage return only end-of-line characters, output by default in OS X versions of Excel, was compiled with the i686-pc-mingw32-gcc compiler under Cygwin (https://www.cygwin.com/). The Excel Add-In will only run within Microsoft Windows versions of Microsoft Office, and requires a version of Office supporting the "Ribbon" menu interface (Office 2007 and newer). After installation, the Add-In can be run from the "Import with Escape Excel" icon in the newly-added right-most "Escape Excel" Ribbon tab, after which non-default options can be selected, followed by "Select File" to browse for the file to be imported . Once unzipped, there is no need to install the Escape Excel application, and it will run Escape Excel with default settings upon opening files with the application, or by drag and drop onto the application icon .An OS X only application (.app) was implemented by wrapping Escape Excel into an application bundle with an application binary to run the script using Platypus (ion icon . Non-defhttps://nodejs.org), using the Express web framework (https://expressjs.com/). The web server displays an HTML form that allows the user to enable the different command-line options via HTML checkboxes  are also impacted by scientific notation auto-conversions, such as 2610100E13, which largely stem from RIKEN identifiers.The ability of Escape Excel to prevent the auto-conversion of gene symbols is of especial interest to genomic and transcriptomic data, due to its high prevalence in the scientific literature , 2. UsinSome cleanup and alteration of the original input text is performed as part of the pre-processing step of the algorithm. Perl, as well as many other tools that import text files, are not aware of UTF-8 byte order marks, and thus treat them as literal characters. Some products, such as Microsoft SQL Server, export text files with UTF-8 byte order marks, which can lead to string corruption in software that is not aware of them. These superfluous byte order marks are thus removed to prevent potential problems in downstream applications, as well as regular expression matching. Pre-existing escaping from previous passages through Escape Excel, enclosing double quotes, leading double quotes, and leading/trailing spaces are also removed. This process avoids both problems with regular expression matching, as well as potential Excel parsing problems on either the escaped or unescaped output.The escaping pattern detection rules were all chosen due to Excel corruption issues that we have observed while analyzing various datasets during the course of our research. It is not uncommon, at least within databases at our institution, to use numerical identifiers which contain leading zeroes, zero-padded so as to reach a minimum field width (i.e. 001234). Loss of these leading zeroes during intermediate passages through Excel can result in many failed database queries, as the identifiers are no longer exact string matches to those within the database. Leading equals signs must be escaped to prevent Excel from attempting to evaluate the field as an equation. Excel will also attempt to treat fields such as "++ stain intensity" as an equation, rather than a text string representing the relative amount of staining on a tissue slide, so such fields must also be escaped. Excel will attempt to interpret anything that looks like a time of day  into an internal time representation. Escaping has thus been extended to cover combinations of dates and times that Excel is known to treat as dates, so as to preserve original date formats as-is, as well as prevent unwanted conversion of regular text into dates. Leading single quotes must also be escaped to prevent Excel from potentially removing them during text manipulation within Excel.Escaping of very large whole numbers, as well as less common forms of scientific notation, is required due to common sample/assay identifiers encountered when dealing with various biological data and assays. Escaping of scientific notation with ≥ 2 digits before the decimal point (i.e. 12E3) is necessary for protecting various assays involving plates with sample wells arranged in a grid, where the row identifiers are letters and the column identifiers are numbers. The reported sample identifier from such assays is commonly composed of plate # + row letter + column #. Plate 11, row A, column 9 would result in an identifier of "11A9". This becomes a problem with plates consisting of five or more rows. Plate 11, row E, column 9 would result in "11E9", which Excel would load as the number "1.1E10". Similar problems arise with chip / plate / assay barcodes consisting of long strings of digits, such as 52070900777217100409406332354780 . Excel automatically converts all whole numbers with ≥ 12 digits into scientific notation, resulting in irreversible identifier corruption unless reformatted to display non-scientific notation with zero decimal places prior to re-exporting to a text file .Most text-related functions in Excel will function properly on the escaped text equations, however, some features, such as Text-to-Columns, will require the escaped text to be copied and pasted as "Paste Values" to convert the equations back into regular text strings before they will work correctly. Although this additional step of converting the escaped text back into regular text is not frequently required, it would be desirable to escape the text in such a manner that the text-within-equations work-around is not required. One such possibility would be to output a Microsoft Office Excel XML file containing the escaped data with problematic fields specified as type "text". Predefinition of the type "text" for each problematic cell would prevent auto-formatting issues imposed by Excel. Although this could be used to import the problematic fields directly as text without going through an equation intermediate, there are several disadvantages to XML file output. The size of the file may increase significantly due to additional XML markup and formatting, which may be problematic for files of several hundred megabytes. The file would also no longer be in the form of a spreadsheet, which may reduce human readability. Although Escape Excel is envisioned to be run just prior to importing the file into Excel, thus obviating the need for compatibility outside of Excel, XML output would decrease compatibility with other software tools, including traditional UNIX row- and column- based command line utilities such as cut, paste, head, and tail, as well as other software that accepts tab-delimited input. We investigated implementation of an XML output version of the software, but encountered compatibility issues between different versions of Office, as well as introducing additional external XML writer library dependencies to the Perl and/or Python runtimes. Due to all of the aforementioned issues with XML output, we have decided to support only text-equation style field protection for now, although we may revisit XML support in future versions of the software.We have developed and implemented a method for preventing Excel from corrupting imported text files due to unwanted auto-conversions. After processing with the Escape Excel software, text files can be safely imported into Excel without auto-converting gene symbols into dates and other related auto-conversion silent data corruptions. This open source software is freely available, implemented in several different wrappers  for greater user accessibility. While this tool is unable to salvage existing data that has been corrupted by Excel auto-conversion, it is hoped that the availability of this tool will increase awareness of the problem and contribute to reducing its impact on published scientific data sets, analyses, and literature.S1 TableWhich fields should, or should not, be escaped are denoted by section header descriptions.(XLSX)Click here for additional data file.S2 TableSymbols were imported as forced-text, unescaped auto-converted fields, and escaped fields. Columns F and G indicate problematic symbols.(XLSX)Click here for additional data file.S1 File(PL)Click here for additional data file.


In spite of growing evidence of idiopathic normal-pressure hydrocephalus (NPH), a viewpoint about clinical care for idiopathic NPH is still controversial. A continuous divergence of viewpoints might be due to confusing classifications of idiopathic and adult-onset congenital NPH. To elucidate the classification of NPH, we propose that adult-onset congenital NPH should be explicitly distinguished from idiopathic and secondary NPH.On the basis of conventional CT scan or MRI, idiopathic NPH was defined as narrow sulci at the high convexity in concurrent with enlargement of the ventricles, basal cistern and Sylvian fissure, whereas adult-onset congenital NPH was defined as huge ventricles without high-convexity tightness. We compared clinical characteristics and cerebrospinal fluid distribution among 85 patients diagnosed with idiopathic NPH, 17 patients with secondary NPH, and 7 patients with adult-onset congenital NPH. All patients underwent 3-T MRI examinations and tap-tests. The volumes of ventricles and subarachnoid spaces were measured using a 3D workstation based on T2-weighted 3D sequences.The mean intracranial volume for the patients with adult-onset congenital NPH was almost 100 mL larger than the volumes for patients with idiopathic and secondary NPH. Compared with the patients with idiopathic or secondary NPH, patients with adult-onset congenital NPH exhibited larger ventricles but normal sized subarachnoid spaces. The mean volume ratio of the high-convexity subarachnoid space was significantly less in idiopathic NPH than in adult-onset congenital NPH, whereas the mean volume ratio of the basal cistern and Sylvian fissure in idiopathic NPH was >2 times larger than that in adult-onset congenital NPH. The symptoms of gait disturbance, cognitive impairment, and urinary incontinence in patients with adult-onset congenital NPH tended to progress more slowly compared to their progress in patients with idiopathic NPH.Cerebrospinal fluid distributions and disease progression were significantly different among the patients with adult-onset congenital NPH, idiopathic NPH and secondary NPH. This finding indicates that the pathogenesis of adult-onset congenital NPH may differ from those of idiopathic and secondary NPH. Therefore, adult-onset congenital NPH should be definitively distinguished from the categories of idiopathic and secondary NPH. Since Adams and Hakim et al. reported patients diagnosed with normal-pressure hydrocephalus (NPH) in 1965 , NPH hasRecent clinical studies in robust diagnostic criteria for idiopathic NPH have shown the beneficial effects of shunting procedures for a patient with idiopathic NPH –17. HoweThe study design and protocol were approved by the ethics committee for human research at our institute. Since we began to evaluate CSF distribution using T2-weighted 3D sampling perfection with the application of optimized contrast using the variable flip-angle evolution (SPACE) sequences in November 2013, and prospectively collected clinical data and conducted MRI examinations. Details of the clinical data collection, image acquisition, and segmentation and quantification of the ventricular and subarachnoid space are described in our previous publications . In brieAll MRI examinations were performed with a 64-channel 3-T MRI system . Volumetric data were delivered to a 3D workstation , and semi-automatically segmented into intracranial space, ventricles, and subarachnoid spaces. The subarachnoid spaces were segmented into the following three components: convexity subarachnoid space, Sylvian fissure and basal cistern and posterior fossa. Both, the mean volumes and volume ratios (in percentages), which were calculated as the measured volumes divided by the intracranial volume, were analyzed to eliminate the effect of head size.The Evans index was measured as the maximal width of the frontal horns of the lateral ventricles to the maximal width of the internal diameter of the cranium based on the X dimension, as shown in Figure P < 0.05. Missing data were treated as deficit data, and therefore did not affect other variables. Statistical analyses were performed using the R software .Mean values and SDs for age and other characteristics and indices were calculated in the three NPH groups. These parameters and CSF volumes in the patients with adult-onset congenital NPH were compared with those in the patients with idiopathic NPH or secondary NPH using the Mann–Whitney–Wilcoxon test, because the majority of them did not have normal distributions in the Shapiro–Wilk test. Statistical significance was indicated by Of the 130 patients who underwent CSF tap-tests and MRI examinations in our NPH center, 85 patients were diagnosed with idiopathic NPH, 17 with secondary NPH, and 7 with adult-onset congenital NPH. The other 21 patients who were diagnosed with Alzheimer disease or Parkinson disease and without any types of NPH were excluded from this study. Table The patients with idiopathic NPH showed lateral ventricles expanded to the vertex, enlarged Sylvian fissure and severely diminished convexity subarachnoid spaces Figure , whereasThe mean intracranial volume for the patients with adult-onset congenital NPH was 1,621 ± 128 mL, which was 100 mL larger than the volumes for patients with idiopathic NPH  and secondary NPH , as presented in Table The mean times on TUG and SWT before and after the CSF tap-test in the patients with idiopathic NPH were longer than those in the patients with adult-onset congenital NPH, although there was no statistical difference, because of large SDs Table . The meaThe morphological characteristics of intracranial CSF spaces in adult-onset congenital NPH are clearly distinguishable from those in idiopathic and secondary NPH. We summarize the clinical and radiological characteristics for the three NPH categories in Table The Japanese guideline for management of idiopathic NPH classifies congenital/developmental etiologies of NPH into secondary NPH subcategory in order to exclude it from the idiopathic NPH category . The intThe present study has several limitations. The classifications of idiopathic and adult-onset congenital NPH were only based on the brain imaging, because their symptoms resembled closely. Therefore, it was the logical result that CSF distribution pattern in adult-onset congenital NPH differed from that in idiopathic NPH. The diagnosis of adult-onset congenital NPH did not include confirmation of the congenital etiology, either by genetic analyses or by evidence of ventriculomegaly during childhood, although one patient with congenital NPH had a family history of NPH. Recent developments in genetic studies have facilitated detection of susceptibility genes for many late-onset genetic disorders. For example, panventriculomegaly with a wide foramen of Magendie and a large cisterna magna is associated with a DNAH14 mutation that contributes to motile cilia . AdditioFull name of the ethics committee is “Ethics committee of Rakuwakai Otowa Hospital.” The study design and protocol were approved by the ethics committee for human research at our institute. Authorization number is RAKUOTO-RIN-14-003. All participants in this study were provided written informed consent.Dr. Shigeki Yamada MD, PhD learned biostatistics at the Department of the Health and Environmental Sciences, Kyoto University School of Public Health from 2001 to 2004.SY had substantial contributions to the conception or design of the work, acquisition of data, statistical analysis, and interpretation of data. Statistical analysis conducted by SY. MI had substantial contributions to the study concept and design, critical revision of manuscript for intellectual content, and supervision. KY had substantial contributions to the acquisition of data and study supervision.SY received speaker’s honoraria from Johnson & Johnson, Nihon Medi-Physics, and Fujifilm Medical Systems. Masatsune Ishikawa received grant funding from Japan’s Ministry of Health and Welfare, as well as honoraria from Johnson and Johnson, Nihon-Medi-Physics and Medtronic Japan Co., Ltd. (Japan) for speaking at seminars. MI received grant funding from Japan’s Ministry of Health and Welfare, as well as honoraria from Johnson and Johnson, Nihon-Medi-Physics and Medtronic Japan Co., Ltd. (Japan) for speaking at seminars. KY declares no disclosures or conflicts of interest.