It is closely related to disease activity and anti-cyclic citrullinated peptide antibody levels. However, the main source of 14-3-3η and the mechanism of its release into the extracellular space remain unclear. Addressing these two points was the main goal of the current study.+ cells, and macrophages were selected as candidates among the various cell types in the synovial tissue. Phosphorylation of mixed-lineage kinase domain-like pseudokinase (MLKL) and cell death of macrophages were studied by phalloidin staining and electron microscopy after stimulation with an oxidative stress inducer (diamide) or tumour necrosis factor (TNF)-α. Extracellular 14-3-3η protein levels were examined by western blotting.The source of 14-3-3η was investigated by immunostaining RA synovial tissue. Fibroblast-like synoviocytes, CD4Macrophages from the synovial tissue from RA, but not osteoarthritis, showed dense and widespread cytoplasmic staining for the 14-3-3η protein, co-localized with peptidylarginine deiminase 4. Swelling and membrane rupture of macrophages were induced by treatment with TNF-α, but not interleukin (IL) 6/soluble IL-6 receptor (sIL-6R). Increased MLKL phosphorylation followed by necroptosis was also induced in TNF-α-stimulated macrophages. Necrostatin-1, a necroptosis inhibitor, antagonized MLKL phosphorylation. High levels of 14-3-3η were detected in the culture supernatants of macrophages stimulated with diamide and TNF-α, but not IL-6/sIL-6R.Macrophages that highly express 14-3-3η undergo TNF-α-induced necroptosis with damage to the cellular structure, resulting in the secretion of 14-3-3η into the extracellular space. The current study provides a novel mechanism for 14-3-3η level increase in the RA synovial fluid. Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovium and joint destruction –5. InitiChaperone 14-3-3η molecules, which are usually located intracellularly, are unexpectedly detected in the synovial fluid of RA patients and thus attracted scientific attention . 14-3-3ηThe importance of 14-3-3η in pathological processes relevant to RA was first proposed when 14-3-3η was detected in the serum of patients with early RA. Subsequently, the protein was found to be an early diagnostic marker of RA, showing complementarity with anti-citrullinated protein antibodies (ACPA) and rheumatoid factor , 18–20. Of note, 14-3-3η is detected in the central nervous system and synovial tissue , 23. How2 in RPMI-1640 medium . They were differentiated into macrophages by treating with 100 ng/ml macrophage colony-stimulating factor . Macrophages were cultured in RPMI-1640 medium containing 10% (v/v) foetal bovine serum  and antibiotics (1% (v/v) penicillin and streptomycin, Life Technologies, Carlsbad, CA), seeded at a density of 100,000 cells/cm2 in 6-well plates and incubated at 37 °C under 5% CO2 atmosphere, as described previously [Monocytes were purified from Ficoll-isolated peripheral blood mononuclear cells and cultured in 6-well plates at a density of 100,000 cells/cmeviously .The following antibodies were purchased and used for immunohistochemistry (IHC), immunofluorescence (IF), immunocytochemistry (ICC), and immunoblotting (IB) experiments: mouse monoclonal antibodies against 14-3-3η  and 14-3-3γ  for IHC, CD4  for IF, CD55  for IF, 14-3-3η  and phospho-Akt   for ICC, and β-actin  for IB; rabbit monoclonal antibodies against CD68 , PAD4 , and phospho-RIP3 (Ser227)  for IF and ICC, and MLKL  for IB; rabbit polyclonal antibodies against MLKL  for IB.Rhodamine-labelled anti-mouse IgG antibody , FITC anti-rabbit IgG antibody , 4′,6-diamidino-2-phenylindole , and rhodamine phalloidin  were used for IF. Anti-mouse IgG and anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (HRP) polymer for IB were obtained from GE Healthcare .TNF-α, IL-6, soluble IL-6 receptor (sIL-6R), and IL-21 were purchased from PeproTech, Miltenyi Biotech , R&D Systems , and PeproTech, respectively. Recombinant 14-3-3η, diamide, actinomycin D, and lipopolysaccharide (LPS) were from Abcam, Sigma-Aldrich, and Wako, respectively. Tofacitinib (TOF) and etanercept (ETN) were kindly provided by Pfizer ; ADA was provided by Abbvie . Necrostatin-1 (Nec-1) or zVAD-FMK was purchased from Abcam (ab141053) or Selleckchem (S7023) .The synovial tissue of patients  with RA and osteoarthritis (OA) was obtained during operation, fixed in 10% formaldehyde, and embedded in paraffin. Samples were subjected to histological examinations, including IHC and IF.IHC was performed as described previously, with slight modifications . BrieflyIF was performed as described previously . Biopsy ICC was performed as described previously, with slight modifications . Primary4, and dehydrated in a graded alcohol series. After freeze-drying in t-butyl-alcohol, the samples were examined under a transmission electron microscope (TEM) .The experiment was performed as described previously . Macrophg for 20 min at 4 °C. The supernatant obtained after the centrifugation was used as WCL. WCL proteins were separated by SDS-PAGE on 10% or 13% Tris-glycine gel and transferred onto nitrocellulose . The membranes were immunoblotted using mouse monoclonal antibodies specific against phosphorylated (p) MLKL or MLKL. Bound antibodies were visualized using anti-rabbit IgG secondary antibodies conjugated with HRP polymer  using chemiluminescence reagents .Whole-cell lysates (WCLs) prepared from primary macrophages were resolved by SDS-PAGE and then immunoblotting (IB), as described previously . BrieflyProteins in the gel were stained with Coomassie Brilliant Blue (CBB) R-350  according to the manufacturer’s instruction.WB protein bands were quantified by using ImageJ software as described previously .To examine 14-3-3η secretion by macrophages, macrophages were pre-treated for 30 min with or without nec-1, z-VAD-FMK (pan-caspase inhibitor), or TOF, and then stimulated with TNF-α, diamide, LPS, IL-6/sIL-6R, IL-1β, or IL-21 in 6-well plates for 24 h. Culture supernatants were concentrated using Microcon  according to the manufacturer’s instructions. 14-3-3η levels in culture supernatants were determined by WB, as described above.t test; P value < 0.05 was considered significant. Statistical analyses were performed using SPSS statistical software .All quantitative data are expressed as the mean ± standard deviation (SD). Differences between the two groups were tested for statistical significance by Student’s unpaired two-tailed The synovial tissue specimens from RA and OA individuals were analysed by IHC using an antibody against 14-3-3η  were next treated with an oxidative stress inducer diamide. The cytoskeletal protein phalloidin was then detected using a fluorescent-labelled antibody Fig. a. DiffusNext, the morphology of macrophages stimulated with various cytokines was observed by TEM Fig. a. UnstimNext, we examined whether the signalling pathway involved in necroptosis was related to one of the key necroptosis proteins, receptor-interacting serine/threonine-protein kinase (RIP) 3. We analysed the phosphorylation of RIP3 (p-RIP3) in macrophages stimulated with TNF-α, diamide, and LPS/zVAD-FMK by ICC. No p-Akt  was detected in macrophages treated with TNF-α  exocytosis and (2) spontaneous secretion of the 14-3-3η protein after cell death. First, macrophages derived from HD monocytes were cultured in vitro. No 14-3-3η was detected in the culture supernatants in the presence or absence of various cytokine stimuli, such as IL-1β, IL-6/sIL-6R, and IL-21, except for TNF-α. Therefore, we concluded that the mechanism of 14-3-3η secretion is independent of exocytosis, since exocytosis does not cause secretion of 14-3-3η into the extracellular space. Next, we focused on TNF-α, which is important for the RA pathogenesis and elicits different types of cell death  depending on the cellular circumstances. TEM analysis revealed that resting macrophages or macrophages stimulated with IL-6/sIL-6R or TNF-α were not apoptotic. Furthermore, p-RIP3 and p-MLKL, markers of necroptosis, were detected in macrophages stimulated with TNF-α, indicating the role of TNF-α in macrophage necroptosis. Collectively, macrophages stimulated with TNF-α may suffer from necroptosis.It has been reported that the level of glutathione is reduced in various cell types in patients with RA , 34. We In addition, in a previous clinical study, we showed that the TNF-α inhibitor ADA strongly reduces the 14-3-3η levels in the serum of RA patients even before the patients achieve remission . This suIt has been reported that a subset of genes, such as ones encoding TNF-α, IL-6, and MMP-3, is induced in the human monocytic cell line THP1 upon stimulation with recombinant 14-3-3η, resulting in inflammation . The extThe intracellular protein 14-3-3η is detected in the extracellular milieu in the synovial fluid of patients with RA. The goal of the current study was to identify the main source of 14-3-3η and the mechanism of its secretion into the extracellular space. Macrophages in the synovial tissue from RA, but not OA, exhibited widespread cytoplasmic staining for the 14-3-3η protein. Treatment with TNF-α, but not IL-6/sIL-6R, induced macrophage swelling and membrane rupture. This was followed by necroptosis. Further, high levels of 14-3-3η were detected in the culture supernatants of macrophages stimulated with TNF-α. These observations indicate that TNF-α promotes secretion of 14-3-3η by inducing necroptosis in macrophage, which constitutes a novel mechanism for 14-3-3η level increase in the RA synovial fluid.Additional file 1: Table S1. Baseline demographics and clinical characteristics of patients with RA and OA. ABT abatacept, ADA adalimumab, DAS28 Disease Activity Score based on 28-joints, DMARD isease modified anti-rheumatic-drugs, ESR erythrocyte sedimentation rate, ETN etanercept, GC Glucocorticosteroid, IFX infliximab, MTX methotrexate, PSL prednisolone, mPSL methylprednisolon, N.A. not applicable, OA osteoarthritis, RA rheumatoid arthritis, SDAI Simplified Disease Activity index, TCZ tocilizumab.Additional file 2: Figure S1. Endogenous 14-3-3η levels in macrophages are not affected upon treatment with IL-1β, IL-6/sIL-6R, and IL-21. Macrophages were cultured in the presence or absence of IL-1β , IL-6/sIL-6R , or IL-21  for 24 h. WCL prepared from macrophages were then analysed by IB using specific antibodies against 14-3-3η or β-actin. Figureure S2. Nec-1, but not TOF, blocks TNF-α–induced macrophage death. Macrophages were cultured with TNF-α  in the presence or absence of nec-1  or TOF  for 24 h and analysed by TEM. Representative images from three independent experiments are shown. Scale bar, 5 μm (upper panel), 2 μm (lower panel). Figure S3. TNF inhibitors do not block macrophage death caused by diamide or TNF-α. Macrophages were cultured with diamide  or TNF-α  in the presence or absence of ETN (100 μg/ml) or ADA (100 μg/ml) for 24 h and analysed by TEM. Representative images from three independent experiments are shown. Scale bar, 5 μm (upper panel), 2 μm (lower panel). Figure S4. TNF inhibitors do not block RA macrophage death caused by TNF-α. HD or RA macrophages were cultured in the presence or absence of TNF-α  with or without ETN (100 μg/ml) or ADA (100 μg/ml) for 24 h and analysed by TEM. Representative images from three independent experiments are shown. Scale bar, 5 μm (upper panel), 2 μm (lower panel). Figure S5. TNF-α induces phosphorylation of RIP3. Macrophages were cultured with or without TNF-α , diamide  (A), and LPS  in the presence or absence of zVAD-FMK . The cells were stained with specific antibodies against anti-RIP3 (phosphor S227) or phospho-Akt (Ser 473), or isotype control, and with DAPI. Representative images from three independent experiments are shown. Scale bar, 50 μm. Figure S6. IL-1β, IL-6/sIL-6R, and IL-21 fail to phosphorylate MLKL. Macrophages were cultured with or without IL-1β , IL-6/sIL-6R , or IL-21  for 24 h. WCL prepared from macrophages were analysed by IB using specific antibodies against the total or phosphorylated form of MLKL or β-actin. Quantification data for representative images from three independent experiments (n = 3) are shown. Scale bar, 5 μm (upper panel), 2 μm (lower panel). Figure S7. Nec-1 blocks phosphorylation of MLKL induced by TNF-α. Macrophages were cultured with or without TNF-α  in the presence or absence of nec-1 . WCL were then obtained, and total or phosphorylated form of MLKL, and β-actin were detected by WB. Representative images from three independent experiments are shown. Figure S8. 14-3-3η is detectable in culture supernatants of macrophages derived from HD and treated with TNF-α, diamide, or LPS. The culture supernatants of macrophages cultured in the presence or absence of diamide, TNF-α , or LPS , and with or without nec-1  or TOF  or zVAD-FMK , were analysed by WB. Recombinant 14-3-3η was used as a positive control. BSA was used as a loading control and was stained with CBB-R350. Representative images from three independent experiments are shown.


Background: Paclitaxel, a commonly used chemotherapeutic agent, is usually associated with peripheral neuropathy. Paclitaxel induced peripheral neuropathy (PIPN) can be dose limiting and may have detrimental influence on patients' quality of life. However, the mechanism of PIPN remains unclear. Medicinal herbs and their formulas might offer neuronal protection with their multitarget and integrated benefits in chemotherapy-induced peripheral neuropathy (CIPN). Siwei Jianbu decoction (J12) is a classic formula of traditional Chinese medicine which can promote blood circulation and treat diabetic nephropathy in clinical with the symptoms of weakness and pain. Methods: The effects of J12 were treated in C57BL/6 mice before injected with Paclitaxel.Behaviour studies: Measurement of mechanical hyperalgesia, thermal nociception and cold allodynia. On the last day at the end of week 6, DRGs were obtained from mice for western blot and immunohistochemical analysis containing NF-κB, p-ERK1/2 and p-SAPK/JNK protein expression. Quantitative real-time polymerase chain reaction: mRNA expression of NF-κB, IL-1β and TNF-α was analyzed. Additionally, the blood samples collected from the eye socket of the mouse were prepared to examine the levels of NF-κB, TNF-α, IL-6 and IL-1β using ELISA assay kits. Results: Hypersensitivity tests and pathology analysis have demonstrated that J12 could improve paclitaxel-induced peripheral pain. J12 acts by inhibiting the activation of  JNK,  ERK1/2 phosphorylation in (Mitogen-activated protein kinases) MAPK signaling pathway and the nuclear factor-κB (NF-κB) in C57BL/6 mice model, J12 also inhibits the production of inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6. Conclusion: The present study showed that J12 ameliorates paclitaxel-induced peripheral neuropathic pain. PaclitaNeuroinflammation is shown to be involved in several neuropathic pain models ,5. ChemoSiwei Jianbu decoction (J12) is a common clinical medicine which is proved to improve neurological function and symptoms of pain and edema of lower limbs . The forTherefore, we aimed to clarify the effects of J12 on paclitaxel-induced peripheral neuropathic pain and whether MAPK and NF-κB signal pathways induced by paclitaxel are associated with the preventive impact of J12 on PIPN.22.1Male C57BL/6 mice weighing 18–22 g (6–8 weeks) were used for these experiments . Free food and water were available. The animals were housed in a room with a normal 12 h light-dark cycle. All animals were habituated in the room for a week before experiments. All experiments were performed in accordance with protocols approved by the Animal Care and Use Committee at the Nanjing University of Chinese Medicine . All experiments were tested in a blinded manner.2.2Paclitaxel  was dissolved with Cremophor EL and ethanol in the radio of 1:1 at concentration of 6 mg/mL and then diluted to 2 mg/mL with 0.9% sterile saline. J12, containing four Chinese herbs, was purchased from Jiangsu Provincial Hospital  and deposited in Nanjing University of Chinese Medicine. The component herbs were decocted twice, each for 1 h. The decoction was filtered and the filtrates were combined and concentrated by rotary evaporation under reduced pressure to 120 mL which is equivalent to 1 g/mL of the original drug.2.3n = 10 per group). Mice were treated with paclitaxel intraperitoneally (i.p.) at a dosage of 20 mg/kg every other day after two weeks  to model paclitaxel-induced peripheral pain. Control mice were injected with vehicle only. There were two J12 treatment groups with different dosages of 5 g/kg (J12L) and 10 g/kg (J12H). J12 was injected i.g. daily. It was administered 1 hour before an injection of paclitaxel in the treated groups.Mice were randomly divided into four groups (2.4n = 10).The behavior test was measured after drug injection weekly and conducted at room temperature. The investigator was blinded to the treatment groups . Mice were placed in a plastic chamber to acclimate for half an hour prior to the test. Force was transferred to the hind paw at a frequency of 1 g/s. To minimize the damage, the cut-off force of 10 g was set. The nociceptive threshold was measured when paw withdrawal occurred. This was accessed on each hind paw three trials in total per mouse.2.4.2The thermal withdrawal thresholds were assessed on a plantar test . Mice were allowed to acclimate in the glass floor for 30 minutes before the test. An infrared source was located at the center of the hind paw of mice, which can transfer heat rapidly. The time of withdrawal was recorded when the source of heat was switched off. In order to avoid injury, a cut-off period of 20 s was maintained. The thermal withdrawal threshold was determined by the average of three treatments per mouse.2.4.3The mouse was placed in a fixing apparatus with the tail exposed outside only. The tail of the mouse was immersed in a water bath maintained at 4 °C until tail withdrawal. To avoid damage to the tail, the cut-off time of 20 s was set. The behavior test was repeated three times each mouse with a time interval of at least 15 minutes between two measurements.2.5On the last day at the end of week 6 DRGs were removed from mice for western blot analysis. DRGs were homogenized in RIPA buffer and PMSF (a protease inhibitor) with a ratio of 100:1. Then homogenates were centrifuged at 12,000 rpm for 15 min at 4 °C. Using a BCA protein assay kit, the supernates of protein were collected to be measured. Protein (20 µg) were separated by 10% SDS polyacrylamide gels and transferred onto PVDF membranes in transfer buffer for 60 min. The membranes were soaked in Tris-buffered saline and tween-20 (TBST) containing 5% defatted milk for 60 min at room temperature. They were incubated with rabbit polyclonal antibody against NF-κB , p-ERK1/2 , ERK1/2 , p-SAPK/JNK , SAPK/JNK  and mouse monoclonal antibody GAPDH  overnight at 4 °C. The membranes were then washed in TBST three times and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG  or horseradish peroxidase-conjugated goat anti-mouse IgG  for 60 min at room temperature. The membranes were washed in TBST three times again and the bands were detected by enhanced chemiluminescence. The gray values were quantified using image J software.2.6On the last day at the end of week 6, the L4 and L5 DRG were obtained from mice for immunohistochemical analysis. They were fixed in 4% paraformaldehyde overnight and then dehydrated with 30% sucrose solution. The DRGs were cut in 10 µm thickness and mounted on the glass slides. After treating with 0.5% TritonX-100 in PBS for 20 min and then washed with PBS three times quickly, the sections were blocked in 5% normal goat serum and 0.5% Tween-20 in PBS for 1 h at room temperature. The sections were washed with PBS three times again and incubated in primary antibodies containing NF-κB , p-ERK1/2  and p-SAPK/JNK  overnight at 4 °C. After washing with PBS three times, the sections were incubated in FITC-conjugated secondary antibodies (1:100) for 1 h in the dark at room temperature. Then the sections were washed three times and stained with DAPI . The immunostained DRGs were viewed under inverted fluorescence microscope.2.7−△△Ct relative quantification method was used to calculate the relative mRNA impression of the target genes.Total RNA was extracted from DRG tissues using TRIzol . RNA was used for the synthesis of cDNA with ReverTra Ace qPCR RT Master Mix with gDNA Remover . Real-time PCR was conducted with an Applied Biosystems 7500 RealTime PCR System  using TransStart Top Green qPCR SuperMix . GAPDH was used as internal reference and primers were synthesized by Shanghai Sangon Biotech. The primer sequences were as follows: GAPDH, (forward) 5'-GGT TGT CTC CTG CGA CTT CA-3', (reverse) 5'-TGG TCC AGG GTT TCT TAC TCC-3'; NF-κB, (forward) 5'-CTG GTG CAT TCT GAC CTT GC-3', (reverse) 5'-GGT CCA TCT CCT TGG TCT GC-3'; IL-1β, (forward) 5'-TTC AGG CAG GCA GTA TCA CTC ATT G-3', (reverse) 5'-ACA CCA GCA GGT TAT CAT CAT CAT CC-3'; TNF-α, (forward) 5'-GCG ACG TGG AAC TGG CAG AAG-3', (reverse) 5'-GAA TGA GAA GAG GCT GAG ACA TAG GC-3'. In all cases, the effectiveness of amplification was determined by the presence of a single peak in the melting temperature analysis and linear amplification throughout the PCR cycles. 22.8g for 15 min. The diluted samples and standard solutions were distributed in polystyrene 96-well plates. After incubation for 1h at 37 °C, plates were washed for 5 times. Then color development reagent was added to incubate for 15 min at 37 °C in dark. Stop solution was added to each well. After that, absorbance was recorded at 450 nm within 15 min.The blood samples collected from the eye socket of the mouse were prepared to examine the levels of NF-κB, TNF-α, IL-6 and IL-1β using ELISA assay kits  according to protocols. Briefly, the supernatants of blood samples were collected after centrifuged at 3000 × 2.9P < 0.05 was considered statistically significant.All of the experimental data were expressed as means ± S.E.M and analyzed with GraphPad Prism 5. The differences between groups were analyzed by two-way ANOVA followed by Bonferroni post-tests. 33.1p > 0.05). Body weights were measured weekly starting from the first injection of J12 through the end of the experiment.To examine whether J12 had effect on body weight in the paclitaxel-induced neuropathy model, body weight was evaluated in mice after paclitaxel treatment with or without J12. Paclitaxel-induced neuropathy model and drugs administration refer to Supplementary Figure S1. The assessment revealed that group of paclitaxel treatment showed no significant difference in body weight compared to vehicle control mice and administration of J12 did not induce any alteration or enhancement on the physiological body weight compared to model group  and persisted until week 6 in mechanical hyperalgesia test. Similarly, in model group, paw withdrawal latency time was significantly decreased from 9.67 ± 0.94 s to 8.90 ± 0.94 s at week 3  and to 7.44 ± 0.83 s at week 6 in thermal nociception tests. On the other hand, the paw withdrawal threshold was increased on the mice treated with J12 before paclitaxel compared with only paclitaxel-treated group for mechanical hypersensitivity (p > 0.05) which indicated that paclitaxel could not induce cold hypersensitivity in the mice.As shown in sitivity B and micsitivity D. On the3.3p < 0.01). Paclitaxel also significantly increased the protein levels of p-ERK1/2 (p < 0.05) and p-JNK in the DRG tissues compared with vehicle control group (p < 0.01). In addition, at a dose of 10 g/kg or 5 g/kg, J12 inhibited paclitaxel-induced NF-κB, p-ERK1/2 and p-JNK expressions. The injection of a low dose of J12 was more effective for the treatments. We next investigated NF-κB, p-ERK1/2 and p-JNK expressions in the DRG issues using immunostaining (To investigate the effects of NF-κB and MAPKs signal pathways in PIPN, we examined protein levels in the DRG tissues. Western blotting showed that the expression of NF-κB was increased after paclitaxel treatment (staining A–C. Withstaining D–F. The 3.4With Real-Time PCR assay, we examined the effect of paclitaxel on the levels of NF-κB and pro-inflammatory cytokines including IL-1β and TNF-α in the DRG issues. The data revealed that NF-κB, IL-1β and TNF-α mRNA expressions were increased in paclitaxel-treated mice compared with control group. In addition, J12 attenuated paclitaxel-induced increase in NF-κB, IL-1β and TNF-α mRNA expressions in the DRG issues A–C. Thes4Chemotherapy-induced peripheral neuropathic pain is a major dose-limiting toxicity caused by various types of chemotherapeutic agents. Paclitaxel is widely used as a chemotherapeutic drug for multiple solid tumor. However, paclitaxel-induced peripheral neuropathic pain causes loss of body function, significantly declines the quality of life and even leads to the cessation of treatment. Between 30% and 40% of cancer patients suffer from neuropathic pain ,18. SeveCurrent clinical experience showed that J12 mitigated the neuropathic pain induced by diabetic. Radix paeoniae rubra has an effect on activating blood flow and anticoagulation . Salvia To examine whether J12 is effective for attenuating paclitaxel-induced neuropathic pain including mechanical hyperalgesia and thermal nociception, mice were pretreated with J12 2 weeks that were injected with paclitaxel every other day at week 3. It was demonstrated in our study that J12 significantly increased paw pain thresholds of mice with a dose of 10 g/kg or 5 g/kg and the pain thresholds were similar to those at the baseline. Our results indicate that chronic administration of J12 is effective for ameliorating paclitaxel-induced neuropathic pain.It has been known that animals treated with paclitaxel caused mechanical hypersensitivity by activating phosphorylated ERK1/2 and JNK in the spinal cord . In addi−1 and 10 g kg−1 which both had the ability to inhibit increases.It has been reported in previous studies that Inflammatory and peripheral immune responses are involved in chemotherapy-induced peripheral neuropathic pain . SeveralThus, NF-κB and MAPK signaling pathways play a key role in paclitaxel-induced peripheral neuropathic pain, activated NF-κB contributes to stimulate the release of pro-inflammatory cytokines which generate behavioral hypersensitivity. J12 was demonstrated to have neuroprotective benefits that protected against paclitaxel-induced peripheral neuropathy pain in mice.5In summary, the current study demonstrated that injection of paclitaxel induced pain hypersensitivity, and J12 exerts anti-hyperalgesic effect on mechanical and heat hyperalgesia in mice by suppressing paclitaxel-induced activation of NF-κB and pro-inflammatory cytokines. Also, J12 alleviates neuropathic pain by blocking ERK and JNK pathways. Therefore, J12 has potential as new therapeutic agent targeting NF-κB and MAPK signaling pathway to ameliorate paclitaxel-induced peripheral neuropathic pain and decrease adverse reaction of anti-tumor drugs.The authors declare that they have no conflicts of interest.Figure S1. Paclitaxel-induced neuropathy model and drugs administration.https://doi.org/10.1051/rmr/200001.The Supplementary Material is available at


Currently, no specific treatment exists for heart failure with preserved ejection fraction (HFpEF). Left ventricular (LV) relaxation during diastole is a highly energy-demanding process, while energy homeostasis is known to be compromised in HFpEF. We hypothesise that trimetazidine – a fatty acid β‑oxidation inhibitor – improves LV diastolic function in HFpEF, by altering myocardial substrate use and improving the myocardial energy status.To assess whether trimetazidine improves LV diastolic function by improving myocardial energy metabolism in HFpEF.N-terminal pro-brain natriuretic peptide levels, and quality of life.The DoPING-HFpEF trial is a randomised, double-blind, placebo-controlled cross-over intervention trial comparing the efficacy of trimetazidine and placebo in 25 patients with stable HFpEF. The main inclusion criteria are: New York Heart Association functional class II to IV, LV ejection fraction ≥50%, and evidence of LV diastolic dysfunction. Patients are treated with one 20-mg trimetazidine tablet or placebo thrice daily  for two periods of 3 months separated by a 2-week washout period. The primary endpoint is the change in pulmonary capillary wedge pressure during different intensities of exercise measured by right heart catheterisation. Our key secondary endpoint is the myocardial phosphocreatine (PCr)/ATP ratio measured by phosphorus-31 magnetic resonance spectroscopy and its relation to the primary endpoint. Exploratory endpoints are 6‑min walk distance, The DoPING-HFpEF is a phase-II trial that evaluates the effect of trimetazidine, a metabolic modulator, on diastolic function and myocardial energy status in HFpEF.  Heart failure with preserved ejection fraction (HFpEF) is a growing healthcare burden and its prevalence is increasing: it currently accounts for approximately half of all new heart failure cases . Similar2+-ATPase (SERCA) pumps, and adenosine triphosphate (ATP) is also required for cross-bridge detachment [31P‑MRS) [LV relaxation is a highly energy-demanding process. During diastole, calcium ions are actively transported back into the sarcoplasmic reticulum of the cardiomyocyte by sarcoplasmic/endoplasmic reticulum Catachment . In the tachment . In HFpEtachment . This istachment , 11. Inttachment . The myo31P‑MRS) .Myocardial energy deficiency in HFpEF can be explained by mitochondrial dysfunction. Mitochondrial dysfunction in HFpEF is considered mainly to be a consequence of chronically increased oxidative stress, overexpression of proinflammatory cytokines, and other factors common in HFpEF known to be associated with mitochondrial abnormalities such as aging, renal dysfunction, and insulin resistance Fig. , arrow 1Another important contributing factor in HFpEF is the severely attenuated peak myocardial oxygen delivery during exercise, possibly related to endothelial dysfunction and impaired vasodilator capacity of the microcirculation . When thTo test the ‘energy depletion’ hypothesis , we propN-terminal pro-brain natriuretic peptide (NT-proBNP) as well as an improvement in symptoms, quality of life, and exercise tolerance after 3–6 months of treatment [Multiple small randomised trials with trimetazidine in ischaemic and non-ischaemic cardiomyopathy have been performed in HFrEF patients: meta-analysis of these HFrEF trials demonstrates improvement in systolic and diastolic function resulting in a reduction in reatment . Studiesreatment . Until nThis study will be conducted in accordance with the guidelines for Good Clinical Practice and with the Declaration of Helsinki. The study has been approved by the Medical Ethical Committee of VU University Medical Center, Amsterdam, The Netherlands (NTR: NL7830). Recruitment began in May 2019, and the study is expected to end in December 2020.Our primary endpoint is to assess the effect of a 3-month trimetazidine treatment in patients with HFpEF on the change in exercise pulmonary capillary wedge pressure (PCWP) at specified exercise workloads.31P‑MRS, and its relation to the primary endpoint. Additionally, we assess safety (frequency of adverse events) and explore NT-proBNP levels, C‑reactive protein level, 6‑min walk distance, and quality of life .Our key secondary endpoint is to evaluate the change in the myocardial PCr/ATP ratio measured by DoPING-HFpEF is a phase II (proof-of-principle) single-centre, double-blind, placebo-controlled, cross-over intervention study Fig. . The stuThe DoPING-HFpEF study will enroll patients with the diagnosis and symptoms of HFpEF, without contra-indications for the study drug or procedures (see Tab. 2). It has a safe pharmacological profile: most important but rare side-effects are parkinsonism and restless legs syndrome. No important interactions have been reported and no safety concern has been observed in the elderly population.Trimetazidine is administered orally by (encapsulated) tablets. Trimetazidine is contra-indicated in severe renal impairment (estimated glomerular filtration rate <30 ml/min/1.73 m31P‑MRS and positron-emission tomography for this dosage in HFrEF patients [The dosage of trimetazidine 20 mg three times daily (TID)  was chosen for our current study for the following reasons: (1) clinically relevant metabolic effects on the heart have been demonstrated by patients , 20; (2)patients , allowinVia a jugular venous approach, right heart catheterisation (RHC) is performed with a fluid-filled, balloon-tipped, flow-directed 7F Swan-Ganz catheter , 22. Zer1H) signal reception, and include the following acquisitions: standard cine series of LV long-axis and short-axis views covering the whole heart; myocardial native T1 and T2 mapping, and localised 1H‑MRS of the septum. Next, localised 31P‑MRS of the left ventricle is performed using a 14-cm-diameter 31P surface coil positioned over the heart with the subject in a supine position, as described previously [1 and T2 are quantified using commercially available software packages. Stroke volume and LV ejection fraction will be calculated. Myocardial triglyceride and total creatine levels are estimated relative to the total water signal by fitting the 1H‑MR spectra in jMRUI [31P‑MR spectra, corrected for partial saturation [Magnetic resonance (MR) examinations are performed with subjects positioned supine in a 3 T MR system  equipped with vendor-supplied 16-element anterior and 12-element posterior coil arrays for proton  of ∆ = 3.2 mm Hg with a standard deviation σ = 5.0 mm Hg [n = 18 . To compensate for potential drop-out, we increased the total number of patients for this study to n = 25.The primary endpoint will be tested by a repeated measures ANOVA. However, a power calculation based on this statistical test would require imputation of multiple unknown variables, making it less reliable. Therefore, we simplified the power calculation and derived it from a paired .0 mm Hg . Based o.0 mm Hg , we expet-test. Fragasso et al. reported a change in the myocardial PCr/ATP ratio after trimetazidine treatment of (mean ± σ) 1.35 ± 0.33 to 1.80 ± 0.50 [n = 13, which is less than the total number of inclusions planned for this study.Our key secondary endpoint, change in myocardial PCr/ATP, will be tested by a paired 0 ± 0.50 . Based oThe DoPING-HFpEF trial is a mechanistic study that aims to evaluate the relevance of mitochondrial dysfunction in HFpEF. The proposed intervention builds on the paradigm that has been proposed by Paulus et al., which assigns systemic inflammation and endothelial dysfunction a central role in the disease process of HFpEF, with oxidative stress and mitochondrial dysfunction as consequences . RecentlAlmost all HFpEF-trials, including TOPCAT and PARAMOUNT, used high natriuretic peptide levels and echocardiographic evidence of LV dysfunction to define the study population . We, and31P‑MRS in our study protocol. Localised 31P‑MRS allows for a non-invasive assessment of the myocardial energy status [Because we primarily aim to improve LV diastolic function, we choose ‘change in exercise PCWP’ as our primary endpoint, which has been shown to correspond to conventional heart failure outcome measures –26. Morey status , 20, whi31P‑MRS) will provide mechanistic insights. Ultimately, if the results of this trial are encouraging, data of this study will be used to design a large multi-centre, randomised clinical trial.In conclusion, this study will test a novel approach in the treatment of HFpEF patients with trimetazidine, a metabolic drug with a favourable pharmacological profile that is readily available. Sophisticated measurements (exercise RHC and


Quantifying soccer players’ performance using different types of technologies helps coaches in making tactical decisions and maintaining players’ health. Little is known about the relation between the performance measuring technologies and the metrics they measure. The aim of this study is to identify and group the different types of technologies that are used to track the health-related performance metrics of soccer players.We conducted a systematic search for articles using IEEE Xplore, PubMed, ACM DL, and papers from the Sports Medicine Journal. The papers were screened and extracted by two reviewers. The included papers had to fall under several criteria, including being about soccer, measuring health-related performance, and using technology to measure players’ performance. A total of 1,113 papers were reviewed and 1,069 papers were excluded through the selection process.We reviewed 44 papers and grouped them based on the technology used and health-related metrics tracked. In terms of technology, we categorized the used technologies into wearable technologies (N=27/44) and in-field technologies (N=14/44). We categorized the tracked health-related metrics into physiological metrics (N=16/44) and physical metrics (N=44/44). We found out that wearable technologies are mainly used to track physical metrics (N=27/27) and are also used to track physiological metrics (N=14/27). In-field technologies are only used to track physical metrics (N=24/24).Understanding how technology is related to players’ performance and how it is used leads to an improvement in the monitoring process and performance outcomes of the players. As soccer is gaining popularity around the world, soccer organizations and researchers are attempting to improve the overall performance of the players, maintain their health, and win more matches . The perSoccer coaches are interested in technical and health-related performance metrics  as both To monitor the players’ performance, soccer organizations and researchers are using different types of tracking technologies that are capable of tracking different metrics of the players. These technologies replace the manual process where specialized observers review taped videos of the matches and code players’ activity patterns . These tThe aim of this study is to identify the different technologies used to track performance metrics of soccer players in recent literature. The study also identifies and categorizes the different health-related performance of soccer players. Identifying and linking the various technologies to health-related metrics help coaches and health practitioners in the sports field to focus on many aspects of the player to maintain players’ health conditions, which leads to enhance the overall performance of the players.We conducted a systematic search and included studies up until the 31st of March, 2019. We identified the articles from IEEE Xplore, PubMed, and ACM DL. In order to have more specialized journals in the sports field, we searched within SJR’s  list of highly ranked journals under the “Sports Science” category and chose the Sports Medicine Journal, which is one of the most highly ranked international journals. Then, we identified the papers using the “Springer Link” database search engine.We searched IEEE Xplore, ACM DL, and Springer Link databases using the combination of the following keywords: (1) “soccer”, “football”; (2) “technology”, “system”; (3) “athlete”, “player”; (4) “performance”. We connected the terms with the “OR” term within each of the four combination groups. We combined the four combination groups using the “AND” term. The exact phrasing of the search keywords is:In the Pubmed database, we used the “MeSH”  approach, which is the national library of medicine’s controlled vocabulary thesaurus that is used for indexing and organizing articles. Each article is associated with MeSH terms that describe the content of the citation. Our MeSH search term included “Soccer” and “Athletic Performance” as two mesh phrases joined by the “AND” keyword. Then that, we separated the keywords “technology” and “system” by the “OR” keyword. The exact phrasing of the search keywords is:The selection criteria was based on four main requirements. First, the study had to be about soccer only. We used the keyword “football” since it is used to refer to soccer in some countries, and excluded any study that discussed American football, rugby, or futsal (indoor soccer). Second, the study had to focus on players’ performance. As such, we excluded studies that assessed the performance of a particular technology. Third, the study had to be about monitoring players’ performance through technology. Thus, we excluded studies that monitored players’ performance through manual monitoring techniques. Finally, the paper had to be published within the last ten years (i.e. 2009-2019). To visualize our search process, we used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), as illustrated in Fig. After completing the basic search, we had a total of (N=1113) papers. Then, we started excluding papers in three phases. In the first phase, we excluded papers (N=380) due to the following reasons: non-English papers (N=8), papers published before 2009 (N=366), and duplicate papers (N=6).In the second phase, we, the two assessors (JM an AT), independently assessed a total of (N=733) papers using the title and abstract of the article. We excluded a number of papers (N=644) based on the following exclusion criteria. We excluded: (1) response or review papers (N=131); (2) papers related to sports other than soccer, such as American football, rugby, and futsal (N=210); (3) papers that used manual performance tracking approaches or papers that did not specify the used performance tracking method (N=107); (4) papers that were not related to player’s performance, such as player’s injury, strategy, or recovery (N=210); and (5) papers that were not related professional players, such as referees and home training (N=6).In the last phase, we included a total of 69 papers (N=69) for full-text review. After reviewing the full text, we excluded some papers (N=25) based on the following criteria. We excluded: (1) studies that analyzed a whole team, but not individual players within the team (N=1); (2) papers related to players’ technical performance, such as the number of passes (N=8); (3) studies that used manual performance analysis or used existing datasets without involving technology in the analysis (N=2); (4) technical papers that did not discuss players’ performance metrics (N=11); (5) studies that did not focus on soccer only but rather discussed other sports (N=1); and (6) papers that the assessors could not access (N=2).Figure Table The age of the players is addressed in the papers using 2 representations; standard age range in years and the soccer notation, such as senior (players in the first league) and U23 (under 23 years old). The age of the players in the studies ranges from 9 to 30 years old. We categorized the ages of the participating players into three main groups: (1) Senior: players above 23 years old; (2) Youth: players between 18 and 23 years old; and (3) Young: players below 18 years old. More than 54% of the papers (N=24/44) focused on senior players, around 23% of the papers studied young players (N=10/44), and around 20% of the papers studied youth players (N=9/44). One paper did not specify the age of the studied players (N=1/44).The duration of the studies ranged from 1 day to 7 soccer seasons. Out of the 44 papers, 34 papers specified the duration of the conducted studies (N=34/44), whereas 10 papers did not specify (N=10/44). We grouped the duration of the studies into three groups: (1) one-day-study: studies conducted in one day (N=2/34); (2) weeks: studies conducted over weeks (N=5/34), which ranged from 3 to 39 weeks; and (3) seasons: studies that lasted for one or more soccer seasons (N=24/34). In the seasons group, 66% of the papers were conducted during 1 season (N=16/24), and the rest of the papers lasted between 2 and 7 seasons (N=8/24).We divided the studies into two groups according to the match type used for data collection: studies that were conducted on players during official matches and studies that were conducted on players during unofficial matches. Approximately 91% (N=40/44) of the papers specified the match type. Out of the 40 studies, 40% (N=16/40) of the papers conducted their experiments with players during official matches, whereas 60% (N=24/40) experimented with players during unofficial sessions. The rest of the studies (N=4/44) did not specify the match type of the conducted experiments.We extracted different types of technologies used to track the players during matches. Global Positioning System (GPS) is used to measure the position of the players for each second in time , 9. Locawearable technologies include the devices that are attached to the players to monitor their performance. Approximately 68% of the papers (N=30/40) used wearable technologies to track the performance of the players. Approximately 90% of those studies (N=27/30) used GPS technology. The other wearable technologies are GS, DC, MEMS, LPMS, HRM, ACC, and sensors, which complement the GPS to get more specific and accurate data about the players. Second, in-field technologies include the devices that are installed in the soccer arena to monitor the players. Approximately 39% of the studies (N=24/44) used in-field technology to track the performance of the players. The in-field technology refers to the MCS. Only 9% of the studies (N=4/44) used both wearable and in-field technologies to track the performance of the players.As shown in Table Different health-related metrics were extracted from the papers. The Heart Rate (HR) of the players is usually measured in predefined time intervals . The MetWe categorized the performance metrics into two main categories, as shown in Table Table A few papers N=4/44) used both wearable and in-field technologies to track the performance of the players. In two studies , 33, the/44 used Our paper summarized the findings in the literature to identify and group the different health-related performance metrics tracked for soccer players along with the technologies used to track these metrics in official and unofficial matches. All of the papers we reviewed conducted experiments with soccer players using different technologies to assess different performance aspects without relating the use of technology to the performance aspects. Our contribution in this paper filled this gap by linking each technology used with the performance metrics it measures. We also identified the type of match that the technology is used in. To the best of our knowledge, no similar work has been done to fill this gap.Based on this review, there were only three papers that studied the performance of soccer players in official matches using wearable technologies. These studies were conducted in 2016 and 2017. In 2015, the International Football Association Board (IFAB) approved the use of wearable technologies in official matches . Before In-field technologies are only used to track the physical performance of the players as MCS can automatically track the CD, SP, and AD of the players. However, studying the physiological aspects of the players has to be done through direct interaction with the player’s body, which cannot be achieved using the in-field multi-cameras system. The wearable technologies are used to track all different types of health-related performance metrics required by the researchers. There are a few papers that used both types of technologies in their studies. However, their main purpose was to compare the results of both technologies.The physical metrics were tracked by all the papers in our study. We discovered that there are three main physical metrics that were tracked in the papers: (1) distance covered, (2) speed, and (3) acceleration and deceleration. Tracking the physical performance is crucial since it is directly related to the performance of the team during a match, which also affects the technical performance of the players and eventually winning a game. Both in-field and wearable technologies were used to track the physical performance of the players. Mainly, wearable technologies were used to track the players’ physical performance during unofficial matches, whereas in-field technologies were used to track the physical performance during official matches.The physiological performances of the players were tracked using wearable devices only in the studied papers. We identified three main physiological metrics: (1) HR, (2) MP, and (3) MSF. All the included studies that analyzed physiological performance were on unofficial matches. We believe that more work has to be done regarding the physiological performance during official matches since players tend to have higher intensity activities compared to unofficial matches. Studying the physiological performance during official matches will allow coaches and researchers to understand and improve players’ performance during official matches accordingly.Most of the studied papers focused on the physical performance of the players in the first place, which is due to the ease of measuring the physical performance using the different types of technologies. Also, monitoring the physical performance of the players does not require special skills to analyze and understand the readings by coaches, players, and technical staff. However, monitoring the physiological performance requires more specialized practitioners and needs specific skills to interpret the results.This scoping review examined papers written in English; other languages were not included, which might have excluded some studies conducted in other parts of the world like South America. A limitation might be the gap between the time the research was done and the time the work was submitted, which will exclude published papers during that period. We conducted a systematic search in the academic databases to include all published papers in this area, but we might have missed some. Although we tried to discuss all the findings in the literature, it is impossible to detail all the findings found in the papers.There is a need to compare the performance of the in-field technologies with the wearable technologies to track the physical performance of the players. Also, future research should focus more on the physiological aspects of the players as it helps in understanding the players’ health status. More studies are required on female players due to the lack of studies conducted on them compared to male players. It is also important to link the monitored metrics to the health of the players.Technology has automated the process of measuring soccer players’ performance. Two types of technologies are used to monitor health-related performances. Players’ physiological performances are determined primarily using wearable technologies. The physical performance of the player is measured using both wearable and in-field technologies. Understanding the relationship between technology and performance as well as how and where it is being used helps in improving the monitoring process, which leads to improving the overall performance of the players.


Wearable sensors are becoming increasingly popular for complementing classical clinical assessments of gait deficits. The aim of this review is to examine the existing knowledge by systematically reviewing a large number of papers focusing on the use of wearable inertial sensors for the assessment of gait during the 6-minute walk test (6MWT), a widely recognized, simple, non-invasive, low-cost and reproducible exercise test. After a systematic search on PubMed and Scopus databases, two raters evaluated the quality of 28 full-text articles. Then, the available knowledge was summarized regarding study design, subjects enrolled , sensor characteristics  and body placement, 6MWT protocol and extracted parameters. Results were critically discussed to suggest future directions for the use of inertial sensor devices in the clinics. The 6-minute walk test (6MWT) is a simple, non-invasive, low-cost and reproducible exercise test used to evaluate endurance during self-paced, submaximal walk by measuring the distance walked within 6 minutes (6MWD) along a flat, straight course with a hard surface . The 6MWAccelerometers, gyroscopes and magnetometers are the most common wearable sensors used in human motion analysis and physical activity monitoring ,13. AcceSeveral MIMU-based methods have been proposed in the literature for the detection of gait events  and the Despite the increasing body of evidence for the use of wearable sensing during gait, the use of MIMUs during standard clinical tests such as the 6MWT is not widespread. The well-defined setting of the 6MWT and its long duration compared to other walking tests represent favorable conditions to extract valuable information from both turn and straight walking, potentially increasing test efficiency. This study aims at reorganizing the existing knowledge by systematically reviewing a large number of papers focusing on the use of wearable inertial sensors for the assessment of advanced gait features during the 6MWT. It was not the interest of the authors to review papers focusing on energy expenditure, physical activity estimation or basic gait metrics extraction . The objectives of this work are: (1) to select and perform a quality check on papers that assess gait during the 6MWT using wearable inertial sensors; (2) to identify the characteristics of the populations included in the studies; (3) to describe the features of the sensors used in terms of technical characteristics, number and location on the human body; (4) to indicate the main parameters extracted; (5) to suggest future directions for the use of inertial sensor devices in the clinics, especially fostering their testing and validation in the pediatric population.For this review the authors followed the guidelines of the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement , and thePubmed and Scopus search engines were accessed in November 2019 to identify articles measuring advanced quantitative parameters associated to gait with MIMU wearable sensors. The queries used for the database search within the title and/or abstract were the following:  AND . In addition, cross-referencing was applied to all included papers, in order to identify additional relevant studies. The literature search was conducted by F.S.After completion of the preliminary electronic database search, one rater (F.S.) screened titles and abstracts and evaluated the suitability of each paper for inclusion in the present review. Papers were excluded if they: (i) Did not use a wearable inertial sensor during the 6MWT; (ii) were an abstract and/or were conference proceedings; (iii) were a review article or a case study; iv) were a study protocol; (v) were not written in English; (vi) were not ranked on Thomson Reuters; or (vii) were published before 2010. In addition, papers were excluded from further quality assessment if they were out of topic with respect to the aims of the present review i.e., the study of advanced gait parameters using wearable inertial sensors during the 6MWT. For example, studies were excluded if they focused on energy expenditure and physical activity intensity or used wearable sensors only to extract basic information such as the number of steps walked during the 6MWT.Full-text publications that met the inclusion criteria were downloaded into Mendeley Desktop 1.19.4. Then, a quality assessment was performed for each article. The quality assessment checklist was based on similar published checklists used for systematical and/or meta-analysis reviews ,30,31 anThe following information was collected from each paper: (i) aim, (ii) study design, (iii) subjects enrolled , (iv) sensor characteristics , (v) sensor body placement, (vi) length of the 6MWT walkway, (vii) calibration and filtering of raw data, (viii) parameters extracted, and (ix) synthetic conclusions.A flowchart of the systematic review process is reported in Internal, external and statistical validity of the 28 included papers were evaluated by two authors (F.S and E.B.). The summary of the quality assessment process is shown in The aims of the studies reflected the predominant exploratory and preliminary nature of most articles. Based on the information reported in the reviewed articles, most of the papers were pilot (n = 12) or validation studies (n = 12). In addition, there were 2 feasibility studies and 2 cohort studies.Populations with a variety of disease characteristics were investigated in the reviewed papers, the following being the most represented patient populations: people with multiple sclerosis , chronic obstructive pulmonary disease , symptomatic lumbar spinal stenosis  and cancer (2 papers). Healthy elderly and young healthy individuals were reported as studied populations in 2 papers each. Of the 28 papers, 12 (43%) included a healthy control group. Further details are reported in The authors of the papers included in the review used a variety of sensor types and supports to collect data, including research-grade MIMUs, triaxial accelerometers or gyroscopes, consumer-based sensors and smartphones with embedded sensors. All of the 28 included papers collected signals from at least one triaxial accelerometer. In general, 14 papers (50%) processed data collected through triaxial accelerometers only, 8 papers (28.6%) also collected signals from triaxial rate gyroscopes, and 6 papers (21.4%) used full MIMU data. Mostly, sensors were embedded in commercial research-grade sensors . Some authors used sensors embedded in commercial smartphones or similar devices  or used in-house manufactured devices . The sampling frequencies (SF) at which the sensors collected data varied widely across papers. The most recurring SF was 60 Hz , followed by 128 Hz and 100 Hz . SFs of 400 Hz and 50 Hz were reported by two papers each (7.1%). SFs of 1000 Hz, 200 Hz, 32 Hz, 30 Hz and 10 Hz were reported by one paper each (3.6%). Finally, 3 papers (10.7%) reported more than one SF, and 4 did not report any (14.3%). The majority of the papers did not report any sensor range . The accelerometer sensor range was reported in 9/28 papers, the rate gyroscope range was reported in 5/14 papers, while the magnetometer range was reported in 1/6 studies.The relative majority of the papers  reported the use of a single sensor. Of the remaining 15 papers, 2 papers (7.1%) reported the use of 2 sensors, 4 papers (14.3%) reported 3 sensors, 2 papers (7.1%) reported 4 sensors, 1 paper (3.6%) reported 5 sensors, 2 papers (7.1%) reported 6 sensors, 3 papers reported 7 sensors (10.7%) and 1 paper (3.6%) reported the use of 17 sensors. Overall, the majority of the studies  reported placing at least one wearable sensor on the lower back, with the preferred locations being at the level of the lower back . Shanks and ankles were also frequent locations chosen by researchers, with 8 (28.6%) and 7 (25.0%) papers, respectively. Further details on the placement of the wearable sensors used in the included articles are summarized in All 6MWT were performed indoors along a flat straight corridor with 180° turns, apart for one study that used a rectangular set of corridors . Most stThe assessed papers reported a wide variety of outcome parameters obtained from MIMU data. Pre-processing of the signals involved a number of different techniques. Calibration procedures were reported by only 6 papers ,56,58,59Spatio-temporal parameters were reported by the majority of the papers , acceleration descriptive statistics were reported by 10 papers (35.7%), 5 papers reported frequency and kinematics measures (17.9%), 3 papers reported stability indexes or other type of measures (10.7%), gait events were reported by only one study. A summary of the outcome parameters with a brief description are shown in This work demonstrated that a significant number of papers  have been published assessing gait using wearable inertial sensors during the 6MWT. We obtained substantial inter-rater agreement for the assessment of the quality of the papers analyzed (Cohen’s kappa = 0.69) . When peTechnical advances in MIMU research are allowing this technology to become a valid alternative to classic laboratory-based and clinical assessments. While many methods and metrics still need to be validated, in the near future it will be possible to extend quantitative measures outside the classic clinical environment, even including free-living data to inform diagnosis and treatment of diseases ,66.In the authors’ opinion, the benefits of using MIMUs and wearable sensors in general during the 6MWT are evident: a thorough and comprehensive assessment of gait can be performed without adding further burden to the patient. They provide an alternative to traditional gait analysis and postural control assessments which require expensive equipment, are time consuming and can provide detailed information only for a very limited number of consecutive gait cycles . ConsideOf the reviewed articles, the majority of the pilot studies aimed at comparing gait features between a clinical population and a healthy control group. The preliminary outcomes gathered from these studies will serve to inform larger scale studies. Validation studies aimed generally at addressing accuracy, reliability and robustness of outcome measures against a clinical scale or a validated gold standard. The most represented clinical population were patients with MS, where wearable inertial sensors were used to inform on additional aspects of gait, especially walking-related fatigue. The reviewed papers showed that gait fatigability measured using both traditional and novel outcome parameters was significantly correlated with physical fatigue, gait disability, and fall history ,56,62. INot surprisingly, accelerometers were the most common source of raw data, due to their low power consumption and widespread use in comparison to rate gyroscopes and magnetometers . ResearcOverall, the reviewed articles followed the technical guidelines for the 6MWT . RegardiThe most frequently used outcome measures were those in the spatio-temporal domain, especially cadence. This is not surprising since a large amount of literature concerning the development of algorithms for step detection and step counting has been published in the last decades ,81,82,83In the reviewed articles, the first quartile of Fourier transform and the ratio of even/odd harmonics were the most frequent outcome measures in the frequency domain. Both metrics have been linked to instability and higher fall risk ,85. The Among acceleration descriptive statistics, root mean square (RMS) was one of the most reported outcome measures. RMS is a statistical measure of the magnitude of acceleration, it is simple to compute and of clear clinical meaning; however, its high correlation with walking speed needs to be taken into account when experiments are performed at self-selected pace. To overcome this limit, Sekine and colleagues recently proposed the RMS ratio, which represents the ratio between RMS in each direction and the RMS vector magnitude .Non-linear analysis of inertial sensor data has also been proposed in recent years. Local dynamic stability of the gait pattern measured by means of accelerometers was assessed by some of the reviewed articles using short-term Lyapunov exponents ,63, whicThe most common kinematic indexes reported in the reviewed articles were angles computed at the lower limb joints. These metrics were generally compared against a gold standard, such as optical motion capture system ,45. MeasOther measures have been proposed that do not fit into the previous categories, for example Dandu and colleagues propose dynamic time warping (DTW) and warp scores, outcome measures specifically designed and fitted to the 6MWT to quantify progressive changes to walking patterns . Byrnes Gait has been acknowledged as a biomarker in many pathological conditions and the variety of gait parameters provided by inertial sensors can help clinicians to enhance assessments of interventions, disease progression and rehabilitation programs.This review showed that wearable sensors are a technical solution that can be adopted in clinical settings to complement traditional assessments of gait deficits. The 6MWT is a well-suited test for this purpose because of its relatively long duration. Reporting additional insights contributes to inform clinical decisions and benefits the health care system by increasing test efficiency.For example, parameters obtained using the methods reviewed in the present study were sensitive to disability in post-stroke patients , were suNone of the reviewed papers included a pediatric population. This may be surprising because the 6MWT is a common functional outcome measure for chronic pediatric conditions . HoweverAfter a systematic literature search and a quality assessment, we reviewed the current knowledge on the assessment of gait during the 6MWT using wearable MIMUs to extract advanced gait parameters, highlighting the main objectives of the studies, describing the preferred sensors and body location, and the outcome measures used in the studies. The results suggest that MIMUs could be successfully used to obtain additional information of clinical significance and increase the meaningfulness of the 6MWT evaluation. Future works should focus on extending the clinical populations studied, with particular attention to the pediatric population, on the validation of additional outcome measures and on extending the use of machine learning approaches.


Heart failure with preserved ejection fraction (HFpEF) is a relatively new disease entity used in medical terminology; however, both the number of patients and its clinical significance are growing. HFpEF used to be seen as a mild condition; however, the symptoms and quality of life of the patients are comparable to those with reduced ejection fraction. The disease is much more complex than previously thought. In this article, information surrounding the etiology, diagnosis, prognosis, and possible therapeutic options of HFpEF are reviewed and summarized.2FPEF score and the HFpEF nomogram are recently validated highly sensitive tools employed for risk assessment of subclinical heart failure.It has recently been proposed that heart failure (HF) is rather a heterogeneous syndrome with a spectrum of overlapping and distinct characteristics. HFpEF itself can be distilled into different phenotypes based on the underlying biology. The etiological factors of HFpEF are unclear; however, systemic low-grade inflammation and microvascular damage as a consequence of comorbidities associated with endothelial dysfunction, oxidative stress, myocardial remodeling, and fibrosis are considered to play a crucial role in the pathogenesis of a disease. The HDespite numerous studies, there is still no evidence-based pharmacotherapy for HFpEF and the mortality and morbidity associated with HFpEF remain high. A better understanding of the etiological factors, the impact of comorbidities, the phenotypes of the disease, and implementation of machine learning algorithms may play a key role in the development of future therapeutic strategies. The spectrum of disorders involving myocardial dysfunction with typical signs and symptoms has since been referred to as heart failure (HF) , 2. EchoHeart failure has been increasingly recognized as an epidemic and various possible etiologies have now been identified. These include coronary artery disease, valvular heart disease, hypertension, cardiomyopathies, and adverse effects of drugs and toxins . In deveThe complex interplay between various factors involved in the etiopathogenesis and potentiation of heart failure has sparked a new drive for heart failure classification based on various  parameters and biomarker profiles , 7•. IndThe prevalence of HF is estimated to be 1.1–5.5% in the general population . It is ap = 0.05, p < 0.001, and p < 0.001, respectively). On the contrary, patients with HFpEF are less likely to have a myocardial infarction or left bundle branch block (LBBB) . Compared to patients with HFrEF, patients with HFpEF have significantly higher blood pressure (p = 0.04), lower resting heart rate, and lower levels of potassium in the plasma [The etiology and pathophysiology of HFpEF are still being uncovered. Firstly, the etiological factors affecting HFpEF and HFrEF seem to be different . The Frae plasma . Many ste plasma , 18, 19.e plasma .Furthermore, comorbidities seem to play a pivotal role in the pathophysiology of HFpEF. The most common are obesity, diabetes, atrial fibrillation, metabolic syndrome, chronic obstructive pulmonary disease, sleep-disordered breathing, renal dysfunction, and anemia •, 21–27.2+) operating level and increased L-type calcium channel current, which contrasts with the suppressed Ca2+ cycling state typical for HFrEF [Although the pathophysiology of HFpEF is yet to be understood, systemic low-grade inflammation, mediated through tumor necrosis factor (TNF) alpha and transforming growth factor (TGF) beta 1, was proposed as a cause of disease , 30. Howor HFrEF .The initial presenting symptoms of HFpEF may be included in the broad category of heart failure. Dyspnea is the most common manifesting symptom among them. Shortness of breath can manifest in various ways, whether it be upon exertion or at rest as in paroxysmal nocturnal dyspnea or orthopnea. Other non-specific symptoms such as fatigue are present. The typical heart failure symptoms such as ankle edema and jugular venous distention are often not present. Other possible presentations include decreased exercise tolerance, chest pain, or discomfort.2FPEF score and the HFpEF nomogram are recently validated highly sensitive tools employed for risk assessment of subclinical heart failure. These tools are based on clinical and echocardiographic parameters, including body mass index (BMI) > 30 kg/m2 (H); use of 2 or more antihypertensive medications (H); the presence of atrial fibrillation (F); pulmonary hypertension (pulmonary artery systolic pressure > 35 mmHg) (P); elderly with an age > 60 years (E); and elevated filling pressures (E/e′ > 9) (F). The H2FPEF score determines the probability of HFpEF by assigning a number for each item  guidelines for the diagnosis of HFpEF include left ventricular EF (LVEF) ≥ 50%, evidence of either diastolic dysfunction or structural heart disease, signs and/or symptoms of heart failure, and elevated natriuretic peptides . Given tSince HFpEF may also share similar clinical characteristics with valvular heart disease, pericardial disease, and high-output HF , diagnosAging phenotypeObesity phenotypePulmonary hypertension (PH) phenotypeCoronary artery disease (CAD) phenotypePhenotypic presentations of HFpEF may vary widely across patients and determine the choice of diagnostic tests and targeted management plan ••, 43–46Although this classification acknowledges the heterogeneity and need for individualized approach, the biological phenotypes seem to better describe the underlying pathomechanisms of HFpEF. Shah et al. proposed a classification created by the use of machine learning , 48:Natriuretic peptide deficiency syndrome—younger subjects with moderate diastolic dysfunction and relatively normal BNPExtreme cardiometabolic syndrome—obese, diabetic subjects with a high prevalence of obstructive sleep apneaRight ventricle-cardio-abdomino-renal syndrome—older subjects with significant chronic kidney disease and cardiopulmonary comorbiditiesThe three identified biological phenogroups are as follows:Worse outcomes are observed in phenogroups 2 and 3.It is generally thought that the heterogeneity in the clinical presentation of HFpEF may be explained by underlying comorbidities in individual subjects. Thus, phenogrouping enables risk stratification and the institution of better-targeted therapies as opposed to BNP-based stratification alone .Since symptoms of HFpEF are non-specific, diagnosis might be elusive. The majority of patients complain of exertional dyspnea, which is a common cause of hospital admission. As such, there are multiple differential diagnoses to consider, including pulmonary and cardiovascular causes, or vocal cord conditions . DiffereIn recent years, a new paradigm of HFpEF has been suggested, implying that it is a very heterogeneous disease. It can be caused by comorbidities through systemic endothelial inflammation leading to structural and functional remodeling of the heart .The most significant comorbidities are obesity, diabetes, metabolic syndrome, chronic obstructive pulmonary disease, sleep-disordered breathing, renal dysfunction, and anemia •, 21–27.Chronic maladaptive neurohumoral activation leading to sustained systemic arterial hypertension has been implicated in the course of HFpEF . Studies99mTc) labeled diphosphonopropanodicarboxylic acid (99mTc-DPD), pyrophosphate (99mTc-PYP), or methylenediphosphonic acid (99mTc-MDP), once used as a bone scintigraphy, provide a novel, non-invasive diagnostic approach with relatively high sensitivity (> 90%) and specificity (86%) [99mTc-DPD in the myocardium with lower or absent uptake in the bones suggests ATTR amyloidosis. Positive bone scintigraphy in patients without monoclonal gammopathy characterizes 100% specificity [Two types of amyloid commonly infiltrate the myocardium—immunoglobulin light chain (AL or primary systemic) amyloid and transthyretin (TTR) amyloid. Transthyretin-related amyloidoses (ATTR) may be either hereditary  or acquired (due to misaggregation of wild-type transthyretin). ATTR amyloidosis is an increasingly common cause of HFpEF and must be excluded in patients suspected of HF , 64. Thety 86%) , 85. Int% , 85. Icificity .There is no evidence that medications, which are known to be effective at alleviating symptom burden and reducing mortality in patients with HFrEF, are equally effective for patients with HFpEF. It may be due to the disparateness of the disease as well as multifactorial pathophysiology of the disease . The numInterestingly, therapy with chlorthalidone has been found to prevent the occurrence of new-onset HFpEF in hypertensive patients . FurtherThe treatment of TTR amyloidosis is based on tafamidis, a drug that has been approved for use in patients with TTR polyneuropathy. In this condition, it has a significant impact on reducing symptoms and stabilizing TTR tetramers, and has been well-tolerated . Finding2 in HFpEF after 12 weeks of exercise therapy. In contrast, no improvement was observed in the group with HFrEF [Emphasis is now being placed on the benefit of exercise therapy for patients with heart failure. This is in direct response to exercise intolerance being the primary symptom of patients with chronic HF and a major factor decreasing quality of life (QOL) in these patients . Studiesth HFrEF . The Intth HFrEF , 105.p = 0.0029), while the incidence of HFpEF is on the rise (p < 0.001). These trends were noticed from 1990 to 2009 [Due to the complex pathophysiology of HFpEF, multiple treatment strategies are still needed and will be required to target specific mechanisms of disease. As described in the Framingham Heart and the Cardiovascular Health Studies, the incidence of HFrEF has been declining B-type natriuretic peptideNT-proBNPDiabetesGrowth factor 15 (GDF-15) –123The identified prognostic factors in patients with HFpEF are as follows:Compared to patients with HFrEF, patients with HFpEF show lower levels of both B-type natriuretic peptide and NT-proBNP. However, in both cases, they are an important prognostic factor , 124. FaHeart failure with preserved ejection fraction (HFpEF) is defined by a left ventricular ejection fraction ≥ 50% in the presence of clinical signs and/or symptoms of heart failure, diastolic dysfunction, or structural abnormality of the left ventricle (LV). However, the system of classifying HF according to LVEF has been recently challenged. Symptoms classically associated with HF include dyspnea, paroxysmal nocturnal dyspnea, orthopnea, and fatigue. Natriuretic peptides are elevated.The most common underlying causes of the disease are coronary artery disease, valvular heart disease, and hypertension, while the most common comorbidities in this population include obesity, diabetes, atrial fibrillation, metabolic syndrome, chronic obstructive pulmonary disease, sleep-disordered breathing, renal dysfunction, and anemia. Amyloidosis, specifically ATTR amyloidosis, is also an increasingly common cause of HFpEF and must be excluded in patients suspected of HF. While the pathophysiology of HFpEF is still being uncovered, the role of systemic low-grade inflammation and microvascular damage related to endothelial dysfunction, oxidative stress, and myocardial remodeling and fibrosis seem to be important components. As the percentage of HFpEF grows, relative to all cases of HF, it is a diagnosis, which clinicians need to be cognizant of.2FPEF score and the HFpEF nomogram may be utilized. As there may be other diseases that mimic or share clinical characteristics, diagnostic algorithms are useful in making the diagnosis of HFpEF. The existence of different phenotypes of HFpEF becomes important when deciding which diagnostic strategies to employ.Due to the fact that several pathophysiological processes may lead to dyspnea, the differential diagnosis is necessary to exclude the non-cardiac etiologies. Not all cases of HFpEF will present acutely. To screen for subclinical heart failure risk, the HCurrently there is no proven pharmacotherapy specifically for HFpEF. Current pharmacotherapy includes angiotensin-converting enzyme inhibitors/aldosterone receptor blockers (ACE-inhibitors/ARBs), calcium channel blockers (CCBs), and beta-blockers. These medications are being used among HFpEF patients because of the high cardiovascular risk and concomitant diseases seen in this population. Treatment with spironolactone, however, seems to be promising. Finally, exercise therapy is being studied for its possible role in the treatment of these patients.Due to a lack of evidence-based treatment strategies for HFpEF, the mortality and morbidity associated with the disease have remained high. The 5-year survival rate among patients with HFpEF is 35–40% after hospitalization. Further studies, especially with the use of machine learning, are warranted to investigate other underlying processes that lead to HFpEF as well as targeted pharmacotherapy for patients with HFpEF.


Crohn’s disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) characterized by intestinal inflammation. Increased intestinal levels of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) are associated with disease activity and severity. Anti-TNF-α therapy is administered systemically and efficacious in the treatment of IBD. However, systemic exposure is associated with adverse events that may impede therapeutic treatment. Clinical studies show that the efficacy correlates with immunological effects localized in the gastrointestinal tract (GIT) as opposed to systemic effects. These data suggest that site-specific TNF-α inhibition in IBD may be efficacious with fewer expected side effects related to systemic exposure. We therefore reviewed the available literature that investigated the efficacy or feasibility of local TNF-α inhibition in IBD. A literature search was performed on PubMed with given search terms and strategy. Of 8739 hits, 48 citations were included in this review. These studies ranged from animal studies to randomized placebo-controlled clinical trials. In these studies, local anti-TNF-α therapy was achieved with antibodies, antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA) and genetically modified organisms. This narrative review summarizes and discusses these approaches in view of the clinical relevance of local TNF-α inhibition in IBD. Ulcerative colitis (UC) and Crohn’s disease (CD) are immune-mediated types of inflammatory bowel diseases (IBD) affecting the gastrointestinal tract (GIT). IBD is a chronic disease with a course characterized by remission and relapse. Disease symptoms include chronic diarrhea, abdominal pain, weight loss and bloody stools. The severity combined with the chronic nature of the disease results in a decreased health-related quality of life, disability and frequent hospitalizations. Whereas the continuous and diffuse inflammation of the mucosa in UC is typically limited to the rectum and may extent proximally, the granulomatous transmural inflammation in CD affect most commonly the ileo-colonic region ,2,3.Although the exact pathogenesis of IBD is unclear, research shows that a combination of genetics, environmental factors and the microbiome play a prominent role in the onset of gut epithelial dysfunction. Consequently, increased exposure of the gut wall to luminal antigens trigger an aberrant acute inflammatory response driven by the innate immune system. Secretion of proinflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) results not only in tissue damage, but the activation of the adaptive immune system as well. Tissue damage in turn may result in an increased exposure of the gut wall to luminal antigens inducing a stronger activation of both the innate and adaptive immune system, which perpetuates the inflammatory state resulting in chronic inflammation  2,3,4,5,54,5,6,7TNF-α is a pleiotropic proinflammatory cytokine implicated in a wide range of cellular processes including cell proliferation, survival and death. In addition, TNF-α signaling is associated with the regulation of several inflammatory pathways including the cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) pathways ,9,10,11.Anti-TNF-α therapy aims to antagonize the effects of TNF-α. Examples of anti-TNF-α therapies which are or have been used in the clinical setting of IBD are infliximab (IFX), adalimumab, golimumab, certolizumab, etanercept, onercept and CDP571 . These bResearch shows that the local immunological environment in the GIT correlates with IBD disease activity ,43,44,45The objective of this narrative review was to evaluate the available literature on PubMed with regards to local TNF-α inhibition in IBD. First, animal studies investigating the efficacy or feasibility of local TNF-α inhibition in IBD are discussed. These studies investigated formulations containing antibodies, antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA) and genetically modified organisms. Subsequently, clinical studies ranging from case reports to randomized placebo-controlled clinical trials that investigated the efficacy or feasibility of local TNF-α inhibition in IBD are discussed. This review aims to summarize the available literature on local TNF-α inhibition with macromolecules intended for the treatment of IBD.This is a narrative review. However, in view of finding relevant citations in the medical literature, the following search strategy was used on PubMed. The search term for citations with regards to local biologic therapy was ” AND  AND (Crohn’s disease OR inflammatory bowel disease OR ulcerative colitis OR proctitis OR pancolitis OR colitis OR IBD)”. This yielded 8339 hits, published from June 1985–May 2020. The citations published up to 1 May 2020 were included in this review.The search term for citations with regards to gene-silencing therapy was “ AND  AND (Crohn’s disease OR inflammatory bowel disease OR ulcerative colitis OR proctitis OR pancolitis OR colitis OR IBD)”. This yielded 400 hits, published between July 1993–May 2020. The citations published up to 1 May 2020 were included in this review.All the titles and abstracts of the citations were read. The reference sections of the included citations were read for additional relevant citations that could be included in this review. Citations that investigated local TNF-α inhibition in human or animal studies were included. Studies that investigated systemically administered anti-TNF-α therapy with experiments aimed to elucidate the local effects in gastrointestinal (GI) regions were included as well. Citation that conducted in vitro studies without any in vivo investigations were excluded.Out of a total of 8739 citations, 31 animal studies are summarized in Table 1 and discussed in Anti-TNF-α formulations have been investigated in animal models . These aSeveral antibodies were investigated in IBD animal models in the context of local TNF-α inhibition. These antibodies were or were not produced by a host carrier. For instance, prokaryotic or eukaryotic carriers of a vector that produce anti-TNF-α antibodies may secrete the antibody in the GIT of the host in view of local TNF-α inhibition. Alternatively, the carrier may be used to deliver a vector to gut epithelial cells that express the protein after genetic transformation. These complex processes impose great challenges in order to achieve reproducible and therapeutic local TNF-α inhibition since drug levels are dependent on many factors that are variable such as the host microbiome, carrier growth rate, transformation efficiency, drug expression rate by the carrier or transformed host cells and drug stability in the GIT. These factors may be subjected to inter- and intraindividual fluctuations as a result of the dynamic GI environment and in turn correlate with fluctuations in efficacy.Nucleotide formulations have been investigated as well. The investigated formulations were ASO, siRNA, miRNA or chemical modifications thereof to increase the stability and/or efficacy. ASO are single-stranded nucleotides that are typically 10–50 nucleotides long whereas siRNA are typically 15–25 nucleotides long. Both can modulate gene expression by a variety of mechanisms which are out of the scope of this review. Simplified and generally speaking, ASO can bind to complementary pre-mRNA or mRNA and alter splicing or induce degradation by endogenous RNase H, respectively, whereas siRNA binds to endogenous RNA-induced silencing complex and thereby induces mRNA degradation. Both approaches aim to silence target genes  compared to IV administration was investigated in a mouse model of acute DSS colitis . As expeV565 is a 115 amino acid 12.6 kDa single domain antibody . In vitrThe same research group  investigAVX-470 is an orally administered antibody against TNF-α derived from the colostrum of cows that have been immunized with TNF-α . The in Avian-anti-TNF-α is an oral formulation containing polyclonal anti-TNF-α antibodies derived from the yolks of immunized hens . An in vdb/db mice, known for the expression of TNF-α in their adipose tissue [−/− mice investigating prophylactic as well as therapeutic treatment regimens with SC doses ranging from 0.01–10 mg/kg, reductions in histopathology scores and TNF-α as well as interferon-gamma (IFN-γ) levels measured in colonic organ cultures were seen. Since ISIS 25302 was administered systemically in these studies, off-target, systemic anti-inflammatory effects contributing to the favorable response cannot be ruled out [ISIS 25302 is an ASO targeting murine TNF-α. In the first animal study investigating the efficacy of SC doses ranging from 0.25–12.5 mg/kg, a dose-dependent decrease in disease severity and colonic TNF-α mRNA expression was reported . Anothere tissue , showed e tissue . Furtheruled out ,112.In a follow up study, ISIS 25302 was associated with galactosylated low molecular weight chitosan  to form a nano-complex . The galAnother formulation using ISIS 25302 is GGG-ASO. However, GGG-ASO is a microspheric oral formulation (~650 µm) in which ISIS 25302 is complexed in a glucomannan–gellan gum mixture. Due to this polysaccharide mixture, the formulation has a time-dependent release mechanism targeting the colon. Furthermore, the mannose entities of glucomannan aid in macrophagic phagocytosis of the formulation, which express mannose receptors . In vitrCAL-ASO is also a formulation using ISIS 25302, which is complexed with lentinan and encapsulated in a chitosan–alginate hydrogel . The comSPG-ASO is an enema containing ASO against TNF-α complexed with the polysaccharide schizophyllan, which is a β-(1–3) glucan . The comASO-miR-301a is an enema containing an ASO against miRNA 301a (miR-301a) , which iGal-LMWC-pre-miR-16 is a formulation containing miR-16 precursor complexed with galactosylated low molecular weight chitosan in view of macrophagic targeting by the MGL . StudiesOf note, the observed anti-inflammatory effects of the investigated miRNA’s may have been partly the result of the miRNA’s interfering with other targets than TNF-α and IL-12p40 expression since both are expressed and regulated in a wide variety of cells ,127,129.PACC-siRNA-TACE is an IV formulation consisting of siRNA against TACE complexed in disulfide-linked poly arginine–cysteine complex (PACC) . This biGTC-siRNA is an intracolonic administered formulation consisting of nanoparticles that contain siRNA against TNF-α complexed with galactosylated trimethyl chitosan–cysteine . In macrLipoplex-siRNA-2 is an enema containing liposomal, chemically modified siRNA against TNF-α . ChemicaCycD-siRNA is an enema containing nanoparticles (~240 nm) consisting of siRNA against TNF-α complexed with amphiphilic cyclodextrin . This coco-glycolic acid) (PLGA) coated with polyethyleneimine (CaP-siRNA is an enema containing nanoparticles (~150 nm) of siRNA against TNF-α loaded on calcium phosphate, which is then encapsulated in poly containing siRNA against TNF-α encapsulated in a reactive oxygen species (ROS)-sensitive material . The ROSGalC-siRNA is an oral formulation consisting of nanoparticles (~250 nm) containing siRNA against TNF-α loaded on PLGA after which a galactosylated chitosan layer is added for macrophagic targeting by MGL . In vitrNanoparticle-in-microsphere oral delivery system (NiMOS) is also an oral formulation containing nanoparticles (~210 nm) entrapped in microspheres (~3 μm). This system can be used to encapsulate siRNA against TNF-α (NiMOS-siRNA) . After oCA-siRNA is an oral colon-targeted formulation containing nanocomplexes of siRNA against TNF-α encapsulated in chitosan–alginate . This foTo increase the targeting performance, the same research group synthesized CA-Fab’-siRNA . This foGal-siRNA-IL-22 is an oral formulation as well and consist of nanoparticles containing the cytokine IL-22 and siRNA against TNF-α in galactosylated PLGA, which is further encapsulated in a chitosan–alginate hydrogel . A combiLactococcus lactis, subspecies cremoris MG1363, carrying an eukaryotic expression plasmid coding for a single-chain fragment variable (scFv) antibody against TNF-α in view of transforming the epithelial cells of the host [9 colony-forming units (CFU) giving for four days resulted in significant effects on DAI, loss of body weight, colon length, histological scores and CRP. Furthermore, significant downregulation of colonic mRNA expression of TNF-α (~50%) and proinflammatory cytokines was observed. No adverse effects were reported. Taken together, these observations show that Lacto-scFv was able to deliver the eukaryotic plasmid for expression in the host’s cells of the GIT, and thereby, ameliorated colitis in vivo.Lacto-scFv is the prokaryote the host . This prthe host . In an aL. lactis, the exact same strain was modified to secrete bivalent single domain antibody fragments (nanobody) against TNF-α (Lacto-Nanobody) [9 CFU resulted in an average of 4 × 108 CFU in the entire colon of colitic mice producing approximately 10 ng nanobody per entire colon. These nanobodies were detected in the mucosa and lamina propria while no nanobodies could be detected in the systemic circulation. Positive effects on histopathologic scores and MPO activity was seen in two chronic colitis models. However, TNF-α, cytokines, inflammatory markers or clinical scores were not analyzed and therefore the effects of this formulation on chronic inflammatory processes is unclear.In another study investigating the anti-inflammatory effects of engineered anobody) . This naPRX-106 is an oral formulation containing plant–cell-expressed (BY-2 cell line) recombinant fusion protein of sTNFR2 fused to the Fc part of human immunoglobulin (Ig)G1. The DNA sequence of this protein expressed by the plant cell line is the same as for the commercially available etanercept . In vitrThe studies that investigated local TNF-α inhibition in humans were mostly limited and small pilot studies . FurtherIn the first study investigating local anti-TNF-α administration in perianal fistulizing CD, a dose of 20 mg IFX divided by several SC injections was administered at the site of inflammation in 9 patients. This study included patients that did not have a sustained response to IV IFX or other systemic drugs. A clinical response was seen in 7 patients of which 4 showed complete healing of the fistulas in 4 weeks. During the 6-month follow-up period no development of ADA was observed . In anotn = 33) after a median number of 9 injections and median follow-up of 11 months. These patients responded without the need for other medical therapy. However, 24% failed to respond to therapy and one adverse event (complication not stated) was reported [n = 9) of which resolution of the fistula was observed in 78% of patients after an average therapy period of 23 week [Local adalimumab injections combined with surgical therapy has also been investigated in perianal fistulizing CD. One study investigating 40-mg injections every 15 days reported complete closure of fistulas in 40% of patients  [Endoscopy-guided local IFX injections in CD has been investigated as well. A study with 8 patients investigated the feasibility and effectiveness of IFX injected locally during colonoscopy in localized inflamed regions (<5 cm) . The dosn = 4)  investign = 4) ,178.n = 3) investigated locally injected IFX (90–120 mg) in colonic strictures [t = 0, t = 2 and t = 4 weeks showed a response of 100% in 6 patients. No adverse events or relapses were reported during a follow-up period of 6 months [n = 2) [The effectiveness and feasibility of locally injected IFX in structuring CD has also been reported. One case series (rictures . One pat6 months . In addi [n = 2) . HoweverIn our search we found that topical anti-TNF-α therapy has only been investigated in one case report regarding a UC patient . The patPRX-106 is an oral formulation containing plant–cell-expressed (BY-2 cell line) recombinant fusion protein of soluble TNF receptor 2 (sTNFR2) fused to the fragment crystallizable region (Fc) part of human IgG1. This formulation is also discussed in n = 37) with an established diagnosis of UC with colonic involvement were enrolled of which approximately 50% had a history of pancolitis. Treatment consisted of 0.1 g, 0.78 g or 1.17 g of oral AVX-470 twice daily for 28 days. Therapy was well tolerated in all three treatment arms with no serious drug-related incidents. Pharmacokinetic analysis showed no substantial systemic AVX-470 absorption or the development of antibodies against bovine Ig, which was a surrogate marker for the immunogenicity of AVX-470. However, stool bovine Ig increased with increasing dose suggesting that AVX-470 transited through the entire GIT, which is consistent with the observed GI stability of orally administered Ig [AVX-470 is an orally administered antibody against TNF-α and is also discussed in tered Ig ,78. Phartered Ig  and fountered Ig ,105,109,V565 is a 115 amino acid 12.6 kDa single domain antibody and is also discussed in Local TNF-α inhibition in IBD is feasible. Animal studies showed that topical antibody or nucleotide therapy could penetrate into the inflamed sites of the GIT and exert a pharmacological effect. This observation was consistent for antibodies that were investigated in clinical studies.Experimental animal studies showed the feasibility and efficacy of local TNF-α inhibition in IBD models. Nonetheless, several challenges remain for these experimental therapies due to the relatively unconventional mechanism of action. For instance, appropriate dose regimens, dose escalation algorithms, and mechanisms to terminate oligonucleotide as well as prokaryotic therapy should be available before these therapies reach the clinical settings. Furthermore, methods to evaluate the site-specific targeting performance of oligonucleotide formulation are of great value since mucosal concentrations may not always correlate with the intracellular drug concentrations of the targeted cells.The clinical studies showed the effectiveness of local TNF-α inhibition realized by local injections, topical treatment or oral therapy. For some patients not responding to conventional therapy, which included systemic anti-TNF-α therapy, local TNF-α inhibition was effective. However, most clinical studies were inherently limited, biased, and used a patient unfriendly route of administration. A prerequisite for adequate therapy and patient compliance is a patient friendly formulation with the objective to inhibit TNF-α locally. An effective oral formulation targeted to the site of inflammation meets these requirements. Several formulations discussed in this review aim to achieve this treatment approach.The different approaches discussed in this review aimed to target the anti-inflammatory therapy to the site of inflammation in view of maximizing the local efficacy while minimizing the systemic effects. Appropriately designed clinical trials are necessary to investigate the efficacy of this approach in IBD. Additionally, it would be interesting to compare local TNF-α inhibition to gold standard anti-TNF-α therapies in these trials. These data would give insight into the efficacy and feasibility of local TNF-α inhibition as well as the mechanism of action of these compounds and TNF-α in IBD.