Syzygium aromaticum essential oil. It is known to have anti-inflammatory and chemoprotective properties as well as a potent anti-oxidant activity due the presence of its phenolic group. In this study, we examined the effects of eugenol on neutrophil superoxide production, a key process involved in innate immunity and inflammation. Superoxide anion generationin human neutrophils was measured by cytochrome c reduction assay. Western blotting was used to analyze the phosphorylation of, p47phox, MAPKinases (p38 and ERK1/2), MEK1/2 and Raf, key proteins involved in the activation of NADPH oxidase. Pretreatment of neutrophils by increasing concentrations (2.5 µg/mL–20 µg/mL) of eugenol for 30 min, inhibited significantly (p < 0.001) superoxide anion generation induced by the chemotactic peptide formyl-Met-Leu-Phe (fMLF) with an IC50 of 5 µg/mL. Phorbolmyristate acetate (PMA)-stimulated O2− production was affected only at the highest eugenol concentration (20 µg/mL). Results showed that eugenol decreased the phosphorylation of p47phox onSer-345 and Ser-328, the translocation of p47phox to the membranesand the phosphorylation of Raf, MEK1/2 and ERK1/2 proteins. Taken together, our results suggest that eugenol inhibits the generation of superoxide anion by neutrophils via the inhibition of Raf/MEK/ERK1/2/p47phox-phosphorylation pathway.Eugenol is a polyphenol extracted from  These molecules are mainly derived from food and medicinal plants. A wide range of non-enzymatic antioxidants can be produced by plants and they have the ability to attenuate ROS-induced oxidative damage2. These natural molecules are especially polyphenols and carotenoids, exhibit various biological activities3. Eugenol (C10H12O2), a phenolic compound, is the major constituent of Sysygium aromaticum (L.) Merr. & L.M. Perry, Myrtaceae), it represents between 45 and 90% of the essential oil4. Eugenol is a colorless or a light yellowish fluid, slightly soluble in water and soluble in organic solvents5. As a natural product, eugenol has gained a great deal of attention in topical applications. Many reports indicate that it is endowed with many pharmacological activities including anti-bacterial6, anti-fungal7, local-anesthesic8, anti-tumoral9 and anti-inflammatory effect10. However, the effect of eugenol on inflammatory immune cells is less documented.Natural exogenous molecules have been used as alternative therapeutic approaches for treating oxidative stress-related health problems, such as atherosclerosis, diabetes, cancer and inflammatory diseases11. Their essential activity is the phagocytosis and subsequent killing of microorganisms12. Stimulated neutrophils producehigh amounts of reactive oxygen species (ROS) through the activation of NADPH oxidase complex13. This activation occurs through a complex series of protein interactionsleading to the assembly of membrane proteins  and the cytosolic components . Thus, catalyzing a rapid electron transfer from the NADPH via the complex to oxygen generating superoxide anion (O2−)14. Superoxide acts as the precursor of other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) generated by heme enzyme myeloperoxidase15. At physiological concentrations, ROS are involved in the host defense response and acts as signaling molecules that regulate cell growth, adhesion toward other cells, differentiation, senescence, and apoptosis16. Further, enhanced ROS generation is considered central to the progression of inflammatory diseases17. The mechanisms implicated in the activation of NADPH oxidase are complex and diverse. However, two major steps are well established to be required for NADPH oxidase activation, the phosphorylation of p47phox on several serines and the translocation of the cytosolic subunits to the membrane18. In this context, our work aimed to study the effect of eugenol on neutrophil superoxide production and the signaling pathways implicated in this process.Polymorphonuclear neutrophils (PMN) are the first cells recruited to sites of infection or inflammation attracted by chemoattractants such as the complement fraction C5a, Interleukin-8, platelet activating factor and the bacterial peptide N-formyl-methionyl-leucylphenyl-alanine (fMLF)50 value of 5 µg/mL. However, the inhibition was less important in neutrophils stimulated with Phorbolmyristate acetate (PMA), an activator of neutrophils functions via direct action on PKC  inhibited superoxide anion production by neutrophils stimulated by fMLF  which are involved in microbial killing. ROS generation requires the activation of NADPH oxidase via phosphorylation of p47phox on various residues. Phosphorylation of serines Ser-328 and Ser-345 of p47phox subunits is critical for the activation of neutrophils NADPH oxidase and its ability to mount a potent oxidative burst in response to activating signal. For this, we examined the effect of eugenol on this process. Neutrophils were incubated with increasing concentrations of eugenol (2.5 µg/mL–20 µg/mL) and then stimulated with fMLF or PMA. The phosphorylation of p47phox residues  were detected by western blotting using specific antibodies. Results showed that eugenol decreased the phosphorylation of Ser-328 and Ser-345 in neutrophils stimulated by fMLF Fig.  and at lThe phosphorylation of p47phox on several serines induces its translocation from the cytosolic compartment to the membranes a keyprocess for NADPH oxidase activation. We thus examined the effect of eugenol on this process. Neutrophils were incubated with eugenol at concentration of 20 µg/mLand then stimulated with fMLF or PMA, neutrophils were lysed and cytosol and membranes were separated by ultracentrifugation. The translocation of p47phox was detected by western blotting using specific antibodies. Results showed that eugenol decreased significantly the translocation of p47phox subunit in neutrophils stimulated by fMLF Fig.  without To investigate whether the inhibition of superoxide anion generation by eugenol is mediated through modulation of MAPKs pathway, we further examined its effects on phosphorylation of ERK1/2 and p38MAPK. We found that fMLF-Fig.  and PMA-As eugenol inhibited ERK1/2 phosphorylation, we investigated the effect on the phosphorylation of the upstream kinases MEK1/2 and Raf in human neutrophils using SDS-PAGE and Western Blot. Results show that compared with the control, the phosphorylation levels of MEK1/2 and Raf were dramatically decreased in Eugenol-treated neutrophils. The inhibition was significantly dose dependent from a concentration of 2.5 µg/mL in fMLF- Fig.  and PMA-19. Excessive ROS production and accumulation seem to play a pivotal role in tissue injury and development of inflammatory diseases20. Thus, the inhibition of ROS over production and neutrophils hyperactivation represent a major research interest. Phytochemicals such as flavonoids, present in several plants, have been reported to have antioxidant and immunomodulatory propreties21. Clove (Syzygiumaromaticum) a well known spice, is characterized by several biological activities especially antioxidant and anti-inflammatory effects. Its biological activities have been attributed to eugenol (EUG), the most abundant constituent of essential oil (45–90%) of Syzygium aromaticum22. Several studies have recently demonstratedthe importance of eugenol as abioactive molecule with various pharmacological properties. In this work, we showed that eugenol inhibits significantly and dose dependently the strong fMLF-induced superoxide anion production by human neutrophils with IC50 of 5 µg/mL. The inhibitory effect of eugenol on superoxide anion production by PMA-stimulated neutrophils, was observed with 20 µg/mL (p < 0.05), the highest concentration used in this study. The difference in the inhibitory effect of eugenol on fMLF- and PMA-stimulated superoxide anion production could be explained by the different mechanisms of neutrophil activation. Thus, we can suggest that low concentrations of eugenol had no effect on PKC activity. Our findings are in agreement withKim et al., whostudied the antioxidant activity of eugenolusing ABTS and DPPH free radicals. At concentration of 20 µg/mL, eugenol performed the elimination of 76.9% and 90.8% of ABTS and DPPH free radicals, respectively23. Superoxide anion, the precursor of other reactive oxygen species, is frequently produced byactivated neutrophils through the NADPH oxidase complex24. The activation of this enzyme requires the phosphorylation of its cytosolic component p47phox on serines located between Ser-303 and Ser-37925. It is known that stimulation of neutrophils by high concentrations of the chemotactic peptide fMLF or by the PKC agonist PMA induces complete phosphorylation of p47phox14. In the current study, we haveshown that eugenol, at concentration of 5; 10 and 20 µg/mL, reduceddose dependentlythe phosphorylation of both Ser-328 and Ser-345 residues in neutrophils stimulated by fMLF and PMA. A pronounced inhibition was observed withSer-345 for both fMLF and PMA studies. Since, the same amount of p47phox was loaded into each well, these results suggest that the inhibition of superoxide anion production by stimulated neutrophils is due in part to reduced phosphorylation of p47phox. Moreover, it is known that the NADPH oxidase is tightly regulated by the translocation of p47phox from the cytosol to the membranes, we demonstrated that eugenol at 20 µg/mL reduces the translocation of p47phox to neutrophils membranes and prevented the assembly of a functional NADPH oxidase enzyme. To the best of our knowledge this is the first report describing the effect of eugenol on p47phox translocationThese results all together suggest that eugenol reduce the phosphorylation of p47phox on specific serines, and consequently reduce its translocation to the membrane and the assembly of active NADPH oxidaseNeutrophils are the most abundant white cells in humans. They are the first line of host defense in organisms. During infection, they play a key role in destroying infectious agent by oxidative and non oxidative mechanisms26. MAPKinaseis a family of Mitogen-activated protein kinases implicated in many biological processes. Extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinases p38 are the most studied families of MAPKs27. ERK1/2 and p38 MAPK are activated in neutrophils in response to a wide variety of pro-inflammatory stimuli including growth factors, phorbol esters, fMLF and cytokines. They are implicated in the respiratory burst, adherence, exocytosis and priming28. Ourresult showed that eugenol inhibits phosphorylation of ERK1/2 dose dependently as low as 2.5 µg/mL. However, the phosphorylation of p38 MAPK was decreased significantly at the two highest tested concentrations, 10 µg/mL and 20 µg/mL. p38 MAPK and ERK1/2 are required for the phosphorylation of p47phox on Ser-345. This phosphorylation is critical for NADPH oxidase hyperactivation and priming29. On the basis of our observations, we can suggest that the inhibition of phosphorylation of Ser-345 and Ser-328 is due to the decrease of MAPKs phosphorylation and activities. Similar data were reported by Deepak et al., in a model of osteoclastogenesis where by a pretreatment of RAW264.7 macrophages with 200 µM of eugenol, for 4 hours, inhibited the phosphorylation of ERK, JNK and p3830. Another in vitro study showed that two synthetic derivates of eugenol, glyceryl-isoeugenol and eugenolol, attenuated LPS-induced phosphorylation of ERK, p38 MAPK and JNK in macrophages31. Phosphorylation and activation of ERKs is accomplished by MEK1 and MEK2, two relevant specific MAPKs, which are, to date, the only validated physiologically substrates of Raf serine/threonine kinases32. Several studies reported the implication of Raf and MEK in many diseases essentially in cancer development33. For this, Target-based therapies are considered as advanced approaches of cancer treatment and there isstrong interest in developing pharmacological inhibitors of Raf and MEK as a means to block ERK signaling34. Our results show clearly that eugenol inhibits the phosphorylation of Raf and MEK in fMLF and PMA stimulated neutrophils. These results suggest that eugenol could be a promising inhibitor of Raf/MEK/ERK cascade.The activation of NADPH oxidase via the phosphorylation of p47phox subunits is performed by kinases such as protein kinases C (PKC) andMitogen-activated protein kinases (MAPK)Taken together, this study demonstrates that eugenol may suppress fMLF-mediated production of superoxide anion by down regulating the activity of NADPH oxidase via the inhibition of Raf/MEK/ERK cascade and the phosphorylation of p47phox on activators residues (Ser-345 and Ser-328). Our findings demonstrate the molecular basis for the antioxidant proprieties of eugenol and show its potential therapeutic role in the treatment of oxidative stress-related diseases.35. HRP-conjugated goat anti-rabbit, HRP-conjugated goat anti-mouse, AP-conjugated goat anti-rabbit antibodies and ECL (enhanced chemiluminescence) reagent were from Santa Cruz Biotechnology Inc .Eugenol, Hank’s Balanced Salt Solution (HBSS), Phosphate Buffered Saline (PBS), Phorbolmyrisate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLF), mouse monoclonal anti-β-actin antibody, phosphatase and proteases inhibitors were from Sigma Aldrich . Dextran T500 and Ficoll were purchased from GE healthcare . SDS-PAGE and western blot reagents were from Bio-Rad Laboratories . Anti-phospho-Raf, Anti-phospho-MEK1/2, Anti-phospho-p38 and Anti-phospho-ERK 1/2 antibodies were from cell signaling Technology . The anti-phospho-p47phox antibodies (Ser-328 and Ser-345) were generated by our lab as previously described36. Red blood cells were removed by hypotonic lysis. To prevent activation, isolated neutrophils were resuspended in HBSS without Ca2+/Mg2+. A cell count was performed and cell viability was determined using the trypan blue exclusion method.The clinical Research Committee at the Xavier Bichat hospital  approved all methods, performed in accordance with the internationals guidelines and regulations. Venous blood was collected from healthy volunteersafter obtaining written informed consent. The density gradient separation method was used to isolate human neutrophils from whole blood using 2% Dextran (T500) and ficoll centrifugation6 cells) were incubated in absence or presence of increasing concentrations of eugenol (0–100 µg/mL) for 30 min at 37 °C. The trypan blue dye test is used to determine the number of viable cells. Results are presented as the percentage of viable cells compared to untreated cells.Isolated neutrophils (1 × 106 cells) were suspended in 1 mL of HBSS containing 1 mg/mL cytochrome c in the presence or absence of increasing concentration of eugenol (2.5 µg/mL–20 µg/mL). After incubation in the thermostated chamber of a spectrophotometer  for 30 min at 37 °C, cells were stimulated with fMLF (10−6 M) or PMA (100 ng/mL). Changes in absorbance were measured at 550 nm for 10 min.Neutrophils (1 × 1037. Isolated neutrophils (50 × 106 per mL) were incubated for 30 min at 37 °C in the absence or presence ofeugenol at 20 µg/mL. Cells were then stimulated by fMLF (10−6 M) or PMA (100 ng/mL). The stimulation was stopped by addition of ice cold PBS, followed by centrifugation at 400 × g for 10 min. The cell pellet was resuspended and sonicated on icein the described relaxation buffer37, then centrifuged at 400 × g for 10 min. The lysates were then ultracentrifuged at 150 000 × g for 45 min at 4° on a sucrose gradient. The cytosolic and membrane fractions were collected and washed in the same lysis buffer, denaturated in Laemmli sample buffer and storedat −80 °C until use.The experiment was performed as previously described−6 M for 15 seconds) or PMA (100 ng/mL for 8 min). The reaction was stopped by adding 5× concentrating Laemmli sample buffer supplemented by 12.5 mM Na3VO4, 25 mMNaF, 6.25 mM p-NPP, 12.5 mM EDTA, 12.5 mM EGTA, 50 µg/mL pepstatin and 50 µg/mL aprotinin. Samples were vortexed immediately and denaturated for 3 min at 100 °C. Following sonication, the lysates were subjected to sodium dodecyl-sulfate polyacrylamide (SDS-PAGE) (10%) to separate the proteins. The proteins separated through electrophoresis were transferred onto nitrocellulose membranes followed by blocking the membranes with 5% non fat dry milk in a mixture of tris-bufferd saline and tween-20. The membranes were incubated overnight at 4 °C in solution containing specific relevant primary antibodies; anti-phospho-S328-p47phox (1:2500), anti-phospho-S345-p47phox (1:10000), anti-phospho-Raf (1:1000), anti-phospho-MEK1/2 (1:1000), anti-phospho-ERK1/2 (1/2000), anti-phospho-p38 (1:2000) and p47phox (1:5000), following by incubation in horseradish peroxidase conjugated secondary antibodies . An enhanced chemilumiscence solution ECL  was added to the membranes and proteins were detected and quantified using the biospectrum imaging system (Amersham Imager 600).Human neutrophils were treated or not with increasing concentrations of eugenol (2.5 µg/mL–20 µg/mL) for 30 min at 37 °C and subsequently stimulated with fMLF (10All experiments were performed in at least triplicates per group and data are reported as mean ± SEM. One way analysis of variance followed by Newman-Keuls multiple comparisons test was used for statistical analysis. p values less than 0.05 were considered as statistically significant.Related Manuscript File


A receptor which is different from the GABA binding site, thus increasing the chloride current. Benzodiazepines like diazepam bind to GABAA receptors containing the α subunits α1, α2, α3, or α5, most likely in abgabg combinations.Anxiety disorders affect some 19 million people in the U.S. alone, costing $46.6 billion, or one third of the nation’s total mental health bill in 1990. Benzodiazepine tranquilizers like the prototypic diazepam are among the most widely used anti-anxiety agents. In addition to their anxiolytic action, they also induce sedation and may impair motor coordination, both of which are undesired side effects when they are used as anxiolytics. Not surprisingly, road traffic accidents may be increased for patients on classical benzodiazepines. In addition, these drugs carry the risk of dependence liability. Benzodiazepines augment the action of the inhibitory neurotransmitter g-aminobutyric acid (GABA) at contact points between two nerve cells called synapses, points at which information is transmitted from one nerve cell to the next. Synaptically released GABA binds to postsynaptic GABAA receptors, thus causing an influx of negatively charged chloride ions into the postsynaptic neuron. This leads to a hyperpolarization and thus functional inhibition of the postsynaptic cell. Benzodiazepines bind to a site on the GABA


The accuracy of robot-assisted pedicle screw placement is unstable and remains controversial. The purpose of this study was to determine the risk factors for unsatisfactory Renaissance robot-assisted pedicle screw placement. This was a retrospective study of prospective data. From January 2017 to March 2018, 136 robot-assisted pedicle screw placements were performed in our department for spinal diseases, and a total of 874 screws were evaluated. All screws were assessed by the Gertzbein and Robbins classification. A and B were defined as satisfactory. C, D, and E were defined as unsatisfactory. Intraoperative registration failures due to nontechnical reasons or intraoperative adjustment were also defined as unsatisfactory. According to the evaluated results, the screws were divided into the satisfactory group (Group A) and the unsatisfactory group (Group B). The satisfactory rate was defined as satisfactory screws (the screws in Group A)/total screws, and the accurate rate was defined as accuracy screws (the screws in Group A)/the screws implanted by the robot . The age, sex, BMI, and BMD as well as the type of disease, the degree of vertebral rotation and the type of screw placement (percutaneous implantation or open implantation) were compared between the two groups, with the assessment of potential risk factors for unsatisfactory robot-assisted screw placement using logistic regression. A total of 874 screws were evaluated; there were 759 screws in Group A and 115 screws in Group B. The satisfactory rate was 86.8% (759/874), and the accuracy rate of the robot-placed screws was 94.4% (759/804). After logistic regression analysis, the independent risk factors were identified as obesity , osteoporosis, vertebral rotation and the presence of congenital scoliosis , particularly for severe osteoporosis (T < −3.5) and severe vertebral rotation (III-IV). According to the results of this study, obesity, osteoporosis and congenital scoliosis are risk factors for unsatisfactory robot-assisted screw placement. Furthermore, for surgeons in the initial stage of using a robot, we suggest avoiding cases in which a single risk factor or multiple risk factors exist to ensure the safety of the operation and to help augment the confidence of the surgeons. Therefore, the key to the success of the pedicle screw fixation technique is to improve the accuracy of pedicle screw fixation. With the development of medical imaging technology and computer technology, image navigation and robot technology have been gradually applied to pedicle screw placement. It has been reported that robot-assisted pedicle screw placement has higher accuracy, lower radiation exposure and fewer complications in some studies5. However, the accuracy of robot-assisted pedicle screw placement is unstable and remains controversial. A prospective randomized study showed that robot-assisted pedicle screw placement did not have better accuracy of screw placement6. A meta-analysis by Marcus et al. also showed that robot-assisted placement did not show higher accuracy than free hand placement4. These results may be due to differences in the diseases. The Renaissance robot system is the first technique approved by the FDA for robot-assisted spinal surgery7. Global spine surgeons were widely concerned about the accuracy and safety of this system. However, while the focus of the previous studies was to evaluate the accuracy of the screws, few studies have focused on the successful performance of robot-assisted screw placement. The spinal surgical robot is a semi-autonomous robot, and most steps of the procedure require manual operation by the surgeon. This procedure may affect the accuracy of the screw because of differing conditions. With the increase in use of the application, many scholars have noticed that several factors may affect screw placement. However, as far as we know, there has been no research focused on the risk factors that affect the failure or inaccuracy of robot-assisted placement. This study was a retrospective analysis of the risk factors for failure of Renaissance  robot-assisted pedicle screw placement.Since it was invented, the technique of pedicle screw fixation has been widely used in spinal fractures, spinal degenerative diseases, etc.; it is a milestone in the development of spinal surgery. However, there are some risks and complications with pedicle screw fixation due to the adjacent nerve roots, spinal cord and blood vessels around the pedicleThis was a retrospective study of prospectively collected data. From January 2017 to March 2018, 136 robot-assisted pedicle screw placements  were performed in our department for spinal diseases, and a total of 874 screws were evaluated. All cases were selected from 3 surgical teams trained by simulated robot-assisted screw placement, and the first 10 cases were excluded for each team. X-ray, CT  and MRI examinations were performed before the operation. The preoperative examination showed no obvious contraindications as well as definite operative evidence. The age, sex, BMI , and BMD  as well as the type of disease , whether or not there was revision surgery, the degree of vertebral rotation  and the type of screw placement (percutaneous implantation or open implantation) were compared between groups to assess potential risk factors.The preoperative plan was based on the optimal location and length of the screws in the preoperative CT scan plan. The preoperative plan was performed by the surgeon and the assistant surgeons to ensure that the design was reasonable.After the patient was anaesthetized, the robot workstation was connected to the data line of the C-arm X-ray machine , and the CT data from the patient were imported. The clip or the fixed needle was used to install the metal tag attachment to the correct position. Some cases needed to be adjusted and positioned several times.The intraoperative fluoroscopy image (AP and oblique images) data from the C-arm were registered and matched with the preoperative CT data. On the robot computer, the screw placement points and the needle angles were adjusted appropriately.When the appropriate “bridge” was selected and installed to ensure stability, the robot was installed on the back of the patient’s back support, and the proper installation of the specified manipulator was selected according to the preoperative planning and the robotic manipulator; then, the channel on the robot was selected according to the robot computer. The robot adjusted the position automatically according to the screw which needed to be implanted; then, the surgeon bored a hole along the channel and placed the needle.The AP and lateral images from the C-arm were performed, and the surgeon determined whether the position of the needle should be adjusted or not according to experience. For the open operation, a manual adjustment of the screw canal was performed. The pedicle screw was placed after the safety of the screw canal was confirmed. For the percutaneous cases, the screw, which needed to be adjusted, was implanted under fluoroscopy guidance Fig. .Figure 1Surgical procedures:8. A and B were defined as satisfactory. C, D, and E were defined as unsatisfactory. Intraoperative registration failure due to non-technical reasons or due to intraoperative adjustments were also defined as unsatisfactory. According to the evaluated results, the screws were divided into the satisfactory group (Group A) and the unsatisfactory group (Group B). The satisfactory rate was defined as satisfactory screws (the screws in Group A)/total screws, and the accurate rate was defined as accuracy screws (the screws in Group A)/the screws that were implanted by the robot . A surgeon on our research team collected radiological data; however, radiological data were evaluated by two other surgeons, and if the results of the two evaluators were inconsistent, the final decision was made according to the opinion of the third surgeon.After the removal of the drainage tube, a CT scan was performed on the fixed segments to assess the accuracy of each screw . All the screws were assessed by the Gertzbein and Robbins classification with the following grades: A, the cortical layer of the pedicle is complete; B, C and D, penetrates the cortical layer of the pedicle but is less than 2 mm, 4 mm and 6 mm, respectively; E, breaches the cortical layer of the pedicle in any direction by more than 6 mm or is outside of the pedicleThis work was supported by the National Key Research and Development Plan of China (Precision treatment solution of high-risk complex orthopaedic diseases Grant No. 2016YFC0105804). We confirmed that all methods were carried out in accordance with relevant guidelines and regulations of our licensing committee. We confirmed that all experimental protocols were approved by the licensing committee of Xi’an HongHui Hospital and that informed consent was obtained from all patients. Informed consent for publication of identifying images has been approved by each relevant patientStatistical analyses were performed with SPSS 22.0 software . The chi-squared test was used for analysis of intergroup differences, and the Student’s t-test for independent samples was used to compare the two sets of data that were normally distributed. P < 0.05 was considered statistically significant. The significance of each of the potential risk factors was assessed using the stepwise logistic regression test. P < 0.05 was considered statistically significant.A total of 874 screws were evaluated, and 39 screws were adjusted during the operation. There were 31 failed registration screws in total, and 19 screws could not be registered in 2 patients with congenital scoliosis. There were 45 C, D and E screws evaluated after the operation. There were 759 screws in Group A and 115 screws in Group B. The satisfactory rate was 86.8% (759/874), and the accuracy rate of the robot-placed screws was 94.4% (759/804).The chi-squared test showed no significant difference for sex (p = 0.07); however, other variables had significant differences Table . The varIn the stepwise logistic regression analysis, the independent risk factors identified by stepwise logistic regression were obesity , osteoporosis, vertebral rotation and the presence of congenital scoliosis , especially for severe osteoporosis (T < −3.5) and vertebral rotation (III-IV) . We analysed sample sizes for every factor, and found that except for severe rotation (III-IV) with 25 screws, the sample size of every other factor was more than 80 screws. At the same time, we reviewed previous studies of robot-assisted spinal surgery and found that our study had larger sample sizes of patients and screws. We determined that the present sample size has enough power to support the conclusions of this study.2 OR 5.357 [95% CI 2.897–9.906]), and vertebral rotation (I-II OR 6.724 [95% CI 2.514~17.985]); III-IV 14.004 [95% CI 4.881~40.182]) as well as the presence of congenital scoliosis (OR 9.835 [95% CI 4.279–22.604]). We found that inaccurate placement was more common in obese patients, especially for lower lumbar surgery. Potential reasons for this outcome may be that the soft tissue was too thick and that the tissue tension was high. In patients with soft congenital scoliosis, the robot occasionally failed at the step of registration. The reasons for this may be related to muscle relaxation after anaesthesia, which may have caused a difference between the intraoperative position and the preoperative CT scan. We found that in the condition of severe rotation of the vertebral body, with robot assistance, some screws were difficult to place because of the limited range of the robot and the shielding of surrounding tissues. At the same time, poor bone quality (lower BMD) may lead to the failure of matching.This study included all the screws that were adjusted intraoperatively or that experienced registration failure due to non-technical reasons. The results showed that the satisfactory rate of pedicle screw placement was 86.8% (759/874) and that the accuracy rate of the robot-placed screw was 94.4% (759/804); the robot still showed excellent results. The independent risk factors identified were osteoporosis , obesity  may lead to the failure of matching. Tsai et al. also noted that the robot has a high requirement for body position; compared with the preoperative CT scan, the intraoperative body position cannot be changed too much because otherwise it is difficult to match17. Previous studies have found that there was a problem of “casing slip” on the surface of a slope, which led to unsatisfactory screw placement18. One research study also found that cases of abnormally steep slope could give rise to a lateral skidding of the cannula at the screw entry point due to the un-consistently reliable anchorage of the cannula6. Moreover, such patients are older, which often increases the risk of inaccurate placement when combined with osteoporosis and obesity. Hu et al.19 reviewed 102 cases with robot-assisted screw placement. They found that 89.5% of the patients had spinal deformity or needed revision surgery, and the results showed that the real success rate of the screw placement was 87.5% (949/1085), with as many as 110 screws replaced manually in the robot-assisted cases and 15 screws abandoned. In our initial cases, we found that in patients who underwent open surgery, especially those with thick soft tissue of the back, it was sometimes difficult to keep the channel led by the robot in the wound, and the surgeon often had to prolong the incision for percutaneous screw placement. At the same time, for cases undergoing lower lumbar revision surgery combined with obesity, the above situation would also occur; because of the outer screw entry point and larger angle of the screw, we had to elongate the incision or place a percutaneous screw from a new incision. However, both approaches added unnecessary injury, especially where accuracy and minimal invasiveness are required (Fig. et al., it was found that for the accuracy of screw placement, the robot did not show an advantage in placing lower lumbar and sacral screws6. Upon review of the literature, we found that most of the articles did not seriously discuss this problem, and these failed cases were also excluded from the screw evaluation19. We believed this exclusion was not conducive to the correct application of the robot and that it greatly reduced the guiding meaning for peers. The robot-assisted technique has brought some changes to spine surgery; however, this technique still has some limitations. We need to choose appropriate cases to utilize the advantages of robots. The purpose of this study was to determine the related risk factors for unsatisfactory robot-assisted screw placement. Furthermore, for surgeons in the initial stage of using a robot, we suggest avoiding cases in which a single risk factor or multiple risk factors exist to ensure the safety of the operation and to help augment the confidence of the surgeons.Some previous studies have reported that factors such as BMD and BMI may be causes for the inaccuracy of robot-assisted placement and the failure of registrationIn our experience, for cases with severe vertebral rotation, the robot-assisted placement application should be avoided and computer-navigation assisted placement is a better choice. For patients with higher BMIs and thick soft tissue of the back, the incision should be lengthened appropriately and the assistant should pay additional attention to protecting the operation of screw implantation. At the same time, for complex cases, multiple technologies can be used simultaneously. Exploiting the advantages and avoiding the weaknesses is the key to efficient utilization of any technology.This study still has some limitations. First, this was a single-centre, retrospective study; so there is a need for a multicentre prospective study with a larger sample size to verify our findings. Second, our study included three different surgeon groups. However, all the surgeons were trained before performing operations, and the study excluded the first 10 cases in each group. Finally, due to limited sample size, several factors, such as malformed vertebrae, that may contribute to unsatisfactory pedicle screw position have not been assessed in the study.According to the results of this study, robot-guided pedicle screw placement is a well-established technique, and we found that obesity, osteoporosis and congenital scoliosis are risk factors for unsatisfactory robot-assisted screw placement. We suggest that it is better to avoid cases containing the above risk factors in the early stages of robot applications.


Gadus morhua and Scomber japonicus models, determined their β-PV isoform composition and analyzed the interaction of the IgE from fish-allergic patient sera with these different conformations. We found that each fish species contains a major and a minor isoform, with the total PV content four times higher in Gadus morhua than in Scomber japonicus. The isoforms showing the best IgE recognition displayed protease-sensitive globular folds, and if forming amyloids, they were not immunoreactive. Of the isoforms displaying stable globular folds, one was not recognized by IgE under any of the conditions, and the other formed highly immunoreactive amyloids. The results showed that Gadus morhua muscles are equipped with an isoform combination and content that ensures the IgE recognition of all PV folds, whereas the allergenic load of Scomber japonicus is under the control of proteolysis. We conclude that the consideration of isoform properties and content may improve the explanation of fish species allergenicity differences.Most fish-allergic patients have anti-β-parvalbumin (β-PV) immunoglobulin E (IgE), which cross-reacts among fish species with variable clinical effects. Although the β-PV load is considered a determinant for allergenicity, fish species express distinct β-PV isoforms with unknown pathogenic contributions. To identify the role various parameters play in allergenicity, we have taken  Changes in fishery policies, aquaculture, processing and eating habits have modified the landscape of fish consumption, increasing the worldwide fish allergy prevalence4. The major allergen of fish has been identified as β-parvalbumin (β-PV), a Ca2+-binding acidic protein that is abundant in muscle7. Most allergic patients react to more than one fish species due to the cross-reactivity of their β-PV chains, but despite the presence of anti-β-PV, IgE often tolerates a variety of species12. The tolerance of cartilaginous fish results from the absence of cross-reactivity of the basic α-lineage of the expressed PV12. In contrast, differences in the allergenic potency of bony fish species have been related to both the content and the pattern of the expressed β-PV isoforms15. However, the molecular signatures and the relation between these features are still unknown.Fish is one of the eight major foods causing type I food allergy, an immunoglobulin E (IgE)-mediated hypersensitivity disease resulting from the breakdown of immune tolerance17. These chains display a complex pattern of IgE linear epitopes that involve intermotif spacers , acid loops  and a salmonid-specific region (residues 1–18)25. Of these, the segment containing residues 33–44 has been described as highly immunoreactive in β-PV from several fish species25. Ca2+ binding stabilizes the proteins in a helical globular fold, as shown by the 3D structures of cod (Gad m 1) and mackerel (Sco j 1) major β-PVs, among others 27. Impairment of this fold in the carp β-PV Cyp c 1 by mutation of both acid loops reduces the IgE binding by 95%, supporting the existence of conformational epitopes28. However, cation removal by either gastric pH or chelates at neutral pH triggers Gad m 1 aggregation into amyloid assemblies29. Gad m 1 amyloids are formed by the polymerization of the intermotif linker regions and cause the amplification of IgE binding30. This sequence and concentration-dependent conformational transitions are not unique to this β-PV, having been described for a large number of proteins, some of which, such as β-lactoglobulin (Bos d 5), ovalbumin  and κ-casein (Bos d 8), are food allergens32.Fish β-PVs are made up of a single polypeptide chain of approximately 109 amino acids that contains three EF-hand motifs , of which only two (the CD and EF) have functional cation-binding acid loopsClupeiformes , Cypriniformes (carp), Gadiformes , Perciformes , Pleuronectiformes (sole and whiff), and Salmoniformes 34. Fish species from these orders differ in the total content of β-PV, the pattern of the expressed isoform and the tolerance in allergic patients39. Of them, cod (Gadus morhua), mackerel (Scomber japonicus) and tuna  are examples of fish species with high (>2.5 mg/g muscle), medium (0.3–0.7 mg/g muscle) and low (<0.5 mg/g muscle) β-PV content, respectively41. In addition, for a given species, the β-PV concentration varies with fish part, and it is higher in dorsal regions than in ventral regions and in the rostral than in the caudal regions40. Examination of the UniProtKB sequence base shows that the Gadus morhua β-PV family is composed of gmPV1 , gmPV2  and single residue variants of each of the chains (Q90YK9 and Q90YL0). Of these isoforms, gmPV1 appears to govern the IgE-binding properties of the population isolated from cod muscle37. For Scomber japonicus displaying heat-sensitive allergenicity, two chains have been described so far: conventional sjPV1  and sjPV2 , which have been detected at the transcriptional level42. For Thunnus albacares, UniProtKB uniquely reveals the C6GKU3 sequence (Thu a 1). Of these three species, mackerel and tuna are more highly tolerated than cod12.Commonly consumed fish are restricted to a few orders, including Gadus morhua and Scomber japonicus models given their difference in allergen load and the availability of two protein sequences and analyzed the interaction of the IgE of fish-allergic patient sera with the denatured, globular and fibrillary folded states of the β-PVs. The results obtained provide novel variables that can be included in predictions of clinically relevant cross-reactivity from diagnostic tests.To simultaneously evaluate the role that content and β-PV isoforms play in the PV allergenic potency of fish species, we chose Gadus morhua  and Scomber japonicus (sjPV1 and sjPV2) are shown in Fig. Gadus morhua to 81.6% in Scomber japonicus. Sequence differences were mainly found at the N-terminal half of the chains, overlapping with the predicted PARV1936 epitope, the most common IgE immunoreactive region (IgE-I)43, the N-terminal EF-hand (AB region) and the amyloid forming segments of gmPV130. Despite the sequence differences, the AmyloPred2 algorithm revealed several regions with aggregation potential in all chains , which shares 99% sequence homology with sjPV1, was taken as a model of mackerel (data not shown). Analysis of muscle extracts prepared in TBS by SDS-PAGE using gmPV1 as a standard for quantitation of the monomer band showed that the PV content (mg PV/g tissue) amounts to 3.1 ± 0.4 and 0.6 ± 0.2 in Gadus morhua and Scomber scombrus, respectively  and overestimated the amounts for Scomber japonicus (increased affinity of sjPV1).To gain insight into the sequence factors involved in the interaction with IgE, the β-PV chains were denatured under reducing conditions and analyzed by immunoblot Fig. . To alloity Fig. . When thity Fig. . Screeniity Fig. . It must43. Therefore, residues 37–38 seem to play a key role in dictating the affinity (SG>AA) and allowance  of the IgE interaction. Notwithstanding, unknown contributions from the unfolded states of the proteins, such as local refolding and aggregation upon SDS removal, cannot be ruled out.Binding of IgE was then assayed using sera from patients with confirmed fish allergy Fig. . As show2+-bound globular forms. Figure 2+-bound proteins cooperatively folded with predominant α-helical secondary structures and hydrodynamic radii close to the RHT of spherical monomers (1.6 ± 0.1 nm). These observations agree with the available 3D structures of gmPV1 and sjPV143. However, despite these similarities, Ca2+-bound sjPV1 and gmPV2 unfolded at a significantly lower temperature than the other chains  under neutral  and acidic  aggregation conditions, and the changes in the amyloid-probe fluorescence were monitored , whereas those formed by sjPV2 contained solely β-oligomer epitopes (A11 specificity)  Fig. . These dTogether, these data showed that isoforms differ in the stability and protease sensitivity of the globular fold , in the capacity to form amyloids (sjPV1 is amyloid prone), in the concentration requirements for assembly (gmPV2 << gmPV1 and sjPV2) and in the conformation (OC vs A11 reactivity) and protease sensitivity of the aggregated state. Additionally, the coaggregation of isoforms from the same fish species is discarded as a possibility.). This assay avoids the interference of events such as precipitation, the dissociation of oligomers, pH-induced conformational changes and differential adherence during the ELISA coating step and allows the direct quantitation of protein loads by the Ponceau red staining of duplicates. As observed with the denatured states, serum IgE interacted the most with the Ca2+-bound globular monomers of gmPV2 and sjPV1. Preheating of Ca2+-bound globular monomers caused a signal reduction but preserved the trend of interaction of the unheated monomers. Removal of the bound Ca2+ from the monomers by the addition of EDTA in dilute solutions, both in the absence and presence of heat treatment, reproduced the previous recognition pattern, indicating that the allergenicity of the globular fold is likely governed by independent sequential Ca2+-binding epitopes. Furthermore, the absence of a significant effect of the preheating treatment on sjPV1 immunoreactivity and the lack of IgE recognition of sjPV2 suggested that the reported heat sensitivity of mackerel allergenicity might be linked to the lack of protease resistance of the major isoform42. On the other hand, the analysis of the IgE recognition of gmPV1, gmPV2 and sjPV2 amyloids showed immunoreactivity only in gmPV1 aggregates  showed a 1.3-fold higher β-PV allergenicity for Gadus morhua than for Scomber japonicus/scombrus. Furthermore, assuming that all the insoluble β-PV shown in Fig. Gadus morhua was 17-fold higher than that of Scomber japonicus/scombrus.To gain insights into the relative β-PV allergenic potency of fish muscle extracts, IgE binding was simulated as a linear combination of content and serum average immunoreactivity of the component isoforms for the different structural states Fig. . Under r48, to experimentally test the simulation results, the immunoreactivity of the extracts and their soluble and insoluble fractions was analyzed by dot blot. Figure Gadus morhua as having the best immunoreactivity, largely due to the anti-amyloid fibrils OC antibody-reactive insoluble fraction.Although fish muscle extracts can contain allergens other than β-PVa priori since 70–95% of its cases are elicited by the fish muscle protein β-PV49. Despite this apparent simplicity, β-PV is not a single entity but rather a complex collection of isoforms in each fish species tailored to accomplish muscle relaxation rate modulation during swimming50. In addition to the physiological implications, the sequence differences among β-PV isoforms define their capacity to act in sensitized individuals as IgE ligands and consequently to trigger IgE-mediated pathogenic events4. For the muscles of fish species such as Gadus morhua and Scomber japonicus, which were taken as models of high and intermediate β-PV content, sequence banks provide two major sets of isoforms with pairwise sequence identities that are above 70%. Here, we have identified and quantified the isoforms in fish muscles, addressed how the diversity impacts the recognition by serum IgE from fish allergic patients using the three major conformations of β-PVs and reconstructed the allergenic potency combining the content and the properties.Fish allergy constitutes a good model to study food allergens Gadus morhua will contain approximately 263.5 and 46.5 mg of gmPV1 and gmPV2, respectively, whereas a Scomber filet of similar weight will contain approximately 51 and 9 mg of sjPV1 and sjPV2, respectively. If all isoforms would interact similarly with serum IgE, then the Gadus morhua having higher allergenicity than Scomber japonicus could have been solely explained in terms of allergen dose, as previously proposed51. However, sequence and concentration differences impact folding, stability and interaction properties. Of the four β-PV isoforms studied, only gmPV2 and sjPV1, with a similar abundance, displayed optimal linear epitopes, resulting in the best IgE recognition of both the unfolded and globular states. However, their high allergenicity was counteracted by the low thermal stability and protease sensitivity of their globular folds and the lack of amyloid formation or the generation of non-immunoreactive amyloids. Then, their contribution to IgE binding depends largely on the intrinsic proteolytic extent of extracts. On the other hand, gmPV1 and sjPV2 both formed temperature-stable and protease-resistant globular folds and exhibited amyloid assembly capacity. Of these chains, sjPV2 was not recognized by serum IgE in any of the structural states considered, and gmPV1, displaying a weak interaction when denatured and globularly folded, exhibited the same level of IgE recognition as gmPV2 and sjPV1 upon amyloid assembly, which is ensured by its increased abundance. Indeed, anti-amyloid fibril OC-reactive species were detected in the insoluble fractions of Gadus morhua extracts. In sum, Gadus morhua muscles are equipped with an isoform combination and content that ensures IgE recognition of all PV folds. In contrast, the Scomber japonicus allergenic load is under proteolytic control. It must be noted that immunological benefits, if any, arising from the hypoallergenic sjPV2 will be reduced based on the protease sensitivity of its aggregates.Quantitative analysis showed notable differences in the relative abundance of these isoforms in muscle extracts, such as those used in allergy diagnosis tests. For 100 g muscle samples, 11. These findings were interpreted in terms of cod being the primary sensitizer due to the dietary traditions of the group under study, and contributions from unknown differences in β-PV from cod and mackerel were also considered. Here, we have demonstrated using sera from pediatric patients sharing hake and megrim as offending fish species that these differences exist and that the obtained IgE reactivity pattern supports a lower probability of tolerance for Gadus morhua than for Scomber japonicus in fish-sensitized patients. Notwithstanding, it must be noted that increasing the number of fish species and the use of a patient cohort with a varying geographical distribution is needed to strengthen the overall conclusion.Previous tolerance studies using open food challenges have reported lower eliciting doses and higher symptom severity for cod than for mackerel51. Additionally, the protease sensitivity and aggregation capacity of the distinct isoforms must be taken into account as a source of IgE reactivity variability. In this sense, a recent analysis has shown a large variation in the β-PV content in the commercial fish extracts used for skin prick testing, suggesting the possible interference of uncontrolled intrinsic proteolysis 48. In other grounds, the clinical determination of fish-specific IgE levels is performed using a variety of extracts lacking accurate definitions of β-PV isoforms, content and properties. Our results suggest that expanding the extract conditions to include all possible isoform conformations and their properties may provide a better understanding of the clinically relevant IgE reactivity.Contribution of both content and isoform composition to the relevance of β-PV cross-reactivity underlines the importance of the definition of common methodologies and actions. A reevaluation of the β-PV isoform composition and content of fish species using actual proteomic tools may be helpful for resolving this issue. The development of novel monoclonal antibodies universally recognizing β-PV will prevent the requirement of correction factors for the use of PARV19 in β-PV quantificationsAll experimental protocols and methods were performed in accordance with the Hospital Universitario La Paz and the Consejo Superior de Investigaciones Científicas guidelines. Approval from the Ethics Committee of Hospital Universitario La Paz (PI1950) and the written informed consents of the patient parents were obtained.TM , and positive skin prick tests to distinct fish extracts: cod and tuna ; swordfish and salmon ; and hake (1.25 mg protein/mL) and megrim   following the Ethics Committee of the Hospital Universitario La Paz guidelines. Serum samples from 7 fish-allergic patients  with specific IgE antibodies to cod and tuna PV and volume availability (at least 4 ml) were selected. All patients had a history of symptoms suggestive of immediate hypersensitivity elicited by eating fish, specific IgE antibodies to fish as determined by CAP-System FEIAGadus morhua and Scomber scombrus were purchased from local markets and genetically identified using a fish ID Kit . For total PV quantifications, 0.5 g of muscle was homogenized in 1 mL of 25 mM Tris-HCl pH 7.4, 137 mM NaCl, 2.7 mM KCl (TBS) using an OMNI TH International homogenizer (2 cycles of 10 strokes in ice), followed by centrifugation at 4,000 x g for 5 min at 4 °C. Supernatants were collected, and their protein content was determined by the Bradford assay (see below). The soluble and insoluble fractions of extracts were obtained by 1 h ultracentrifugation at 100,000 x g at 4 °C using an Optima TmMax ultracentrifuge . Samples containing 10 μg of total protein were analyzed by SDS-PAGE and Coomassie Blue staining (see below). The amount of PV was determined from the band of the 11–12 kDa monomer using gmPV1 as a standard for quantification. Samples were analyzed using two distinct extracts in duplicate. For isoform analysis, the sarcoplasmic protein extraction was carried out by homogenizing 1 g of white muscle in 1 mL of MilliQ water for 30 s on ice in a sonicator device  as described39. β-PV extracts were acidified with 0.1% formic acid in 50% acetonitrile and analyzed using an LTQ FT Ultra mass spectrometer . For mass spectrometry, 50 µL of sample was analyzed by direct infusion at a flow rate of 1 µL/min. Ions were generated by positive ion mode using 4.5 kV for spray voltage. Proteins were analyzed with full-scan MS in the positive mode from 400 to 1,200 m/z applying 100,000 full widths at half mass of mass resolution. Data were collected for 5 min. The obtained signals were deconvoluted within XCalibur software  using the Xtract feature in the Qualbrowser program. All samples were analyzed in triplicate.Fish belonging to Gadus morhua (Atlantic cod) and Sco j 1  from Scomber japonicus (chub mackerel) were obtained from Genscript (USA) in pET15b constructs. All proteins were produced in Escherichia coli BLD21 (DE3), isolated and purified as described previously45. N-terminal His-tag removal was performed using a Thrombin CleanCleaveTM kit as recommended by the manufacturer . Before their use, protein solutions were extensively dialyzed against 5 mM HEPES pH 7.5 containing 0.1 mM CaCl2, concentrated using 10 kDa-pore size Amicon Ultra-15 filters and centrifuged at 16,000 x g for 20 min at 4 °C to remove any possible aggregate.Synthetic genes corresponding to the sequences of Gad m 1.0202  and Gad m 1.0102  from 2) were diluted with distinct buffers to 14–150 µM protein and supplemented with 10 μM ThT. The buffers for the reactions were 25 mM Tris-HCl, pH 7.5, 0.1 M NaCl containing 4 mM either EDTA or CaCl2 (control for no aggregation) and 50 mM Gly, pH 1.6. Assays were initiated by placing the sealed 96-well plate at 37 °C in a POLARstar  microplate reader. The ThT fluorescence was measured through the bottom of the plate every 30 min with a 450 nm excitation filter and a 480 nm emission filter. All measurements were performed in duplicate, and the experiments were repeated twice with different protein batches. When required, aggregates were harvested from the reaction mixtures by a 100,000 x g centrifugation for 1 h using an Optima TmMax ultracentrifuge . The resulting pellet and supernatant fractions were used for protein concentration determination. Before use, pellet suspensions were sonicated in a bath sonicator for 5 min.The kinetics of amyloid formation was followed using a 96-well format Thioflavin T (ThT) binding assay. Cleared protein stocks  measurements were performed with a DynaPro spectroscatter  using 100 µM protein solutions as described previously29.Circular dichroism (CD) measurements were performed with a Jasco J-820 spectropolarimeter  as described previously2 were either left at 25 °C or heated at 95 °C for 15 min and then cooled to 25 °C for 10 min. Heated and unheated globular monomer samples were then diluted in either 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl or 0.1 M Gly, pH 1.6. Similarly, aggregated protein samples at 4 mg/ml in 5 mM HEPES, pH 7.5, were diluted in either 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl or 0.1 M Gly, pH 1.6. Solutions at pH 7.5 were incubated for 30 min in the absence and presence of proteinase K  at 37 °C, and the reactions were stopped with phenylmethylsulfonyl fluoride . Solutions at pH 1.6 were incubated for 30 min in the absence and presence of pepsin  at 37 °C, and the reactions were stopped with 0.2 M Tris-HCl, pH 8.5. Reaction products were analyzed by SDS-PAGE.Solutions of globular monomers at 4 mg/ml in 5 mM HEPES, pH 7.5, and 0.1 mM CaClProtein solutions were diluted in Laemmli sample buffer (2x) containing 2-mercaptoethanol, heated for 10 min at 100 °C and loaded on a 15% tris–glycine gel . Precision Plus protein standards  were used as standards for the molecular weights. Electrophoresis was carried out using a Mini-PROTEAN®  system at 100 V. For SDS-PAGE, proteins were visualized by Coomassie Brilliant Blue R-250  staining. For Western blotting, proteins were electrotransferred to PVDF membranes at 150 V for 1 h using prechilled 25 mM Tris, 192 mM glycine, pH 8.3, and 10% methanol transfer buffer in a Mini Trans-Blot® Cell. Membranes were immersed in methanol, allowed to air dry and stored at −20 °C until use. For development, membranes were first blocked for 1 h in TBST-BSA . Immunodetection was performed by a 2 h incubation with PARV19  and sera from patients allergic to fish (1:10 dilution) prepared in TBST-BSA. After 3 x 10-minute washes, membranes were incubated for 1 h with horseradish peroxidase-labeled goat anti-mouse IgG  or mouse monoclonal B3102E8 anti-human IgE . After several washes with TBST, the signal was developed using the ECL-Clarity Western-blotting reagent  and detected with a ChemiDoc XRS. When required, quantifications were performed using Image Lab software tools. For analysis, the signals in intensity/μg protein from at least two independent membranes and two sequential exposition times were referred to that of gmPV1 and averaged.Aliquots containing 15–150 ng of protein were spotted in duplicate on a nitrocellulose membrane and blocked for 1 h in TBST containing 1% BSA. Detection was performed by a 2-h incubation with the anti-amyloid fibril OC antibody , anti-amyloid oligomer A11 antibody  and sera from patients allergic to fish (1:10 dilution) prepared in TBST-1% BSA and a 30-min incubation with horseradish peroxidase-labeled mouse monoclonal B3102E8 anti-human IgE  or goat anti-rabbit IgG . The signal was developed by the ECL-Clarity Western blotting reagent (Bio-Rad) and detected with a ChemiDoc XRS. Quantifications and analysis were performed as before using the signals from gmPV1 as normalization factors.Protein concentrations were determined using a Quick Start™ Bradford assay kit , BSA standards and absorbance readings at 595 nm.GPVi is the fraction of the globular fold of the isoform i, Bi is the coefficient measuring the extent of intrinsic proteolytic degradation, and IgEBGPVi and IgEBAPVi are the average intensities of the IgE binding of the globular and amyloid folds of the isoform i, respectively.Simulation of the IgE binding (IgEB) in muscle extracts was performed using a linear combination of the weighted contributions of the component isoforms in the different structural states as follows:Supplementary Material


Mycobacterium tuberculosis and Toxoplasma gondii. Enhancement of P2X7 receptor responses may be useful in pathogen clearance particularly in individuals with defective microbial killing mechanisms. Ginsenosides from Panax ginseng have been discovered to act as positive allosteric modulators of P2X7. Here we describe a novel modulator binding site identified by computational docking located in the central vestibule of P2X7 involving S60, D318, and L320 in the lower body β-sheets lining the lateral portals. Potentiation of ATP-mediated responses by ginsenosides CK and Rd caused enhanced ionic currents, Ca2+ influx and YOPRO-1 uptake in stably transfected HEK-293 cells (HEK-hP2X7) plus enhanced cell death responses. Potentiation of ATP responses by CK and Rd was markedly reduced by mutations S59A, S60A, D318L and L320A supporting the proposed allosteric modulator binding site. Furthermore, mutation of the conserved residues S60 and D318 led to alterations in P2X7 response and a higher sensitivity to ATP in the absence of modulators suggesting residues in the connecting rods play an important role in regulating P2X7 gating. Identification of this novel binding site location in the central vestibule may also be relevant for structurally similar channels.P2X7 receptors are important in the regulation of inflammatory responses and immune responses to intracellular pathogens such as  P2X7 activation results in the activation of the NLRP3-caspase 1 inflammasome2, release of pro-inflammatory mediators 3, shedding of transmembrane proteins 4, and occurrence of cell death5. Moreover, P2X7 is implicated in immunity to intracellular pathogens such as Mycobacterium tuberculosis and Toxoplasma gondii amongst others making it a potential target for the modulation of the immune response7.P2X7 is an ATP-gated ion channel expressed on various immune cell populations including monocytes and macrophages9. The ginseng root extract contains a complex mixture of bioactive compounds such as polysaccharides, glycolipoproteins, and polyacetylenic alcohols, however the most extensively studied compounds are the ‘steroid-like’ dammarane triterpenoid glycosides termed ginsenosides8. Ginsenosides can be further divided into the protopanaxadiols , and the protopanaxatriols , based upon whether sugar moieties are attached to carbon-3 (diols) or carbon-6 (triols). Metabolism of ginsenosides in the gut by microbial flora species results in the formation of readily absorbable metabolites including the ginsenoside compound K (CK)10. Ginsenosides have been demonstrated to modulate a plethora of immune cell functions such as phagocytosis, the function of inflammatory enzymes, the production of both pro- and anti-inflammatory cytokines, the modulation of intracellular signalling pathways, and regulation of the inflammasome9. Moreover, there is increasing evidence of the potential antimicrobial properties of ginseng, with ginsenosides providing protection against viruses , bacteria  and fungi 12.Ginseng has been utilised throughout Asia for centuries where it is considered a ‘panacea’, promoting longevity and treating many diseases, although the exact mechanisms by which ginseng can exert these biological effects are not fully understood13. These chemicals could enhance the action of the physiological agonist ATP, increasing both the sensitivity and the maximum response of P2X7 to agonist, thus defining the ginsenosides as positive allosteric modulators13. To date, there have been few positive allosteric modulators (PAMs) identified for P2X7 including clemastine, polymyxin B, ivermectin and tenidap16 and currently nothing is known about their molecular binding sites. Much more is known about negative allosteric modulator binding to P2X7 with a detailed allosteric binding site recently being described for AZ10606120 that may also accommodate other antagonists including JNJ-47965567, A804598, and A74000318. Most of what we know about P2X receptor structure comes from the crystal structures of closed and open state zebrafish P2X4 (zfP2X4), giant panda P2X7 (pdP2X7) and human P2X3 receptors19 plus detailed mutagenesis studies investigating ectodomain and transmembrane domain movements22. Our aim in this study was to identify the putative molecular binding site for ginsenoside PAMs on the P2X7 receptor using a molecular modelling approach combined with site-directed mutagenesis. We describe a novel modulator binding site located in the central vestibule region of P2X7 involving two β-sheets in the lower body region enclosing this cavity. We demonstrate that mutations of two key residues, D318 and L320, abolished potentiation of P2X7 responses by the ginsenosides CK and Rd.We recently identified that the activity of P2X7 receptors could be modulated by protopanaxadiol ginsenosides such as CK, Rd, Rb1 and Rh2in vivo metabolite CK, act as positive allosteric modulators of human P2X7 receptors requiring the presence of agonist (ATP) for their action. This, together with data suggesting pre-treatment with P2X7 antagonists such as AZ10606120 could prevent P2X7 opening even in the presence of CK13 led to the hypothesis that ginsenosides may bind to the open ATP-bound conformation of P2X7. To investigate this we generated homology models of human P2X7 (hP2X7) based upon the closed and open states of zfP2X4 (pdb 3H9V and 4DW1)23 and the closed state of pdP2X7 (pdb 5U1L)17. Model building and preliminary docking of the ginsenosides CK and Rd to human P2X7 was carried out using Swissmodel and Autodock Vina respectively, as previously described for P2X424. Subsequent analysis to further define the binding pocket was performed using consecutive runs of Sitemap software and docking calculations with Glide. The best predicted binding site for ginsenosides is located within the central vestibule of hP2X7 in a large hydrophobic cleft lined by β-sheets that define the lower body wall of the trimeric channel  thus conel Fig.  and connInduced fit docking calculations using Glide revealed two elements to the putative ginsenoside binding site; a glucose-specific subsite located close to the top of the central vestibule cavity and a hydrophobic cleft co-ordinating the steroid backbone of the ginsenosides Figs  and 2C. 13. These two ginsenosides have highly similar structures with a single glucopyranose ring attached to carbon-20  and the mutant hP2X7 receptors were stably expressed in HEK-293 cells. The G99EE mutant was generated to mimic the sequence found in human P2X4 receptors (QEENS) vs human P2X7 (QGNS) Fig.  since P22X7 Fig. .5. Stimulation with ATP resulted in uptake of YOPRO-1 by all hP2X7 mutants. N100A, N100W, S101A and G99EE mutations in hP2X7 resulted in poorer dye uptake responses to ATP  to enter the cells, bind to DNA and fluoresceATP Fig. . ConversATP Fig. . FollowiATP Fig. . CalculaATP Fig. . NotablyATP Fig. . S59A anATP Fig. . We confATP Fig. . FurtherATP Fig.  and respATP Fig. .Figure 32+ responses using fura-2AM loaded cells. Stimulation with either ATP or BzATP resulted in a transient peak response followed by a sustained increase in intracellular calcium  of the edges of the lateral portals, widening the central vestibule19. We propose that upon opening of the P2X7 channel, a PAM binding site is revealed within the central vestibule and co-ordination of a chemical containing a glucopyranose moiety culminates in potentiation of the agonist-triggered response. Binding of the ginsenoside chemicals may stabilise the open state or may provide an enhanced movement of the connecting rods (β-sheets linking the ATP binding to TM movement). Roberts et al. demonstrated that locking of the β-sheets across the lateral portal by forcing a disulphide bridge between 60 C and 320 C in hP2X1 channels, inhibited channel responses to ATP29. Breaking this forced bridge using DTT effectively reversed the lock29. In addition, Samways et al. have demonstrated that certain residues in the central vestibule β-sheet region of hP2X4 were amenable to modification with MTSET+ 27. S59 and S62 (hP2X4 numbering) were mutated to cysteine residues and application of MTSET+ caused potentiation of ATP-responses at these mutant P2X4 channels. This suggests that residues in the lower body β2 sheet are accessible via lateral portals and that modifications can positively affect channel gating27.Since the crystal structure of zfP2X4 in the apo and ATP bound states were published30. W46 and W50 in TM1 and D331 and M336 (rat P2X4 numbering) in TM2 are thought to contribute to the ivermectin binding site31 although residues E56 and D58 (hP2X4 numbering) may also contribute to the allosteric binding site for ivermectin27. Ivermectin is also thought to potentiate human P2X7 receptors32 but there is no evidence as yet whether this is due to interaction with a similar transmembrane binding site to P2X4. The possibility exists that ivermectin may bind to a site overlapping the ginsenoside site in the central vestibule however the following pieces of data may argue against this; firstly, ivermectin could not potentiate YOPRO-1 responses32 whereas ginsenosides can effectively potentiate YOPRO-1 responses  and removal of the charged residue E51 at the top of TM1 abolished this effect33. Whilst we have not measured Pf% through P2X7 in this study, it is unlikely that ginsenosides affect calcium permeability due to the predicted binding site sitting well away from the transmembrane regions. We have measured sustained calcium levels in P2X7 expressing HEK cells and show that CK and Rd can significantly increase intracellular calcium levels  may contribute to the activation of the NLRP3 inflammasome34.There are few well described positive allosteric modulators of P2X channels. Within the family, ivermectin is best characterised for its actions on P2X4 and there is some evidence for PAM action in the region of the lateral portals. The binding site for ivermectin is thought to be much lower, residing within the transmembrane helicesOur finding that S60A, D318L, and L320A mutations created hP2X7 receptors with larger responses and higher sensitivity to ATP was somewhat unexpected. The overlap with these residues being highlighted as critical interacting residues with ginsenosides CK and Rd is unlikely to be coincidence and is suggestive that these residues make a difference to the ease of opening of the channel in response to agonist binding. Patch clamp data suggests the kinetics of channel opening are similar and there is no apparent difference in deactivation kinetics Fig. . Indeed,2+). Additionally, these experiments exposed a difference between the potentiating effects of CK and Rd on the mutants. Partly this may be due to the co-exposure of the channel to agonist (ATP) plus ginsenoside for only 5 seconds before the agents were washed away. All of the mutants were poorly potentiated by Rd in comparison to CK  enhanced the sensitivity of P2X7 to ATP by showing reduced cell viability at low ATP concentrations Fig. . The S6037. Furthermore, potentiators acting at this site might also be useful in inducing apoptotic cell death in tumour cells. It is also possible that we may be able to design antagonists to bind to this novel binding site potentially generating a series of open channel blockers to increase drug discovery opportunities for this important P2X receptor.In conclusion, the identification of a modulator binding site on P2X7 opens up new potential therapeutic benefits for disorders linked to dysfunctional P2X7 responses such as the defective killing of intracellular pathogens associated with loss-of-function polymorphisms in P2X7−1 penicillin and 10 mg mL−1 streptomycin  with selection under 400 μg mL−1 G418 . All cells were maintained at 37 °C in a 5% CO2 humidified incubator.HEK-293 cells stably transfected with wild-type or mutant human P2X7 (hP2X7) receptors (EE-tagged hP2X7 in pcDNA3.1 plasmid) were maintained in DMEM/F-12 media containing L-glutamine  supplemented with 10% FBS , 10000 U mLIq competent E.coli high efficiency cells  were transformed with 5 µl of digested product and colonies selected following growth at 37 °C for 16 hours. Plasmids were extracted using Qiagen miniprep kit and mutations verified by sequencing (Eurofins Genomics).Point mutations were introduced into the WT hP2X7 plasmid using the Stratagene Quikchange II site-directed mutagenesis kit (Agilent Technologies). Primers were purchased from Sigma Aldrich. PCR was performed for 16–18 cycles using Pfu turbo polymerase and products digested with DpnI for 1 hour at 37 °C. NEB 5-alpha F’5 cells were pelleted prior to resuspension in primary mouse anti-hP2X7 antibody  at a dilution of 1:100 in PBS/0.5% BSA buffer. Cells were incubated for 1 hour on ice. Following a washing step, cells were resuspended in secondary goat anti-mouse IgG AlexaFluor 488 (Fisher Scientific) at a dilution of 1:100 and incubated in the absence of light for 1 h on ice. Cells were washed and fluorescence was measured on a CytoFLEX flow cytometer . Data was analysed using CytExpert software . All washes were conducted in PBS containing 0.5% (w/v) BSA at 300 g for 5 mins.To quantify cell surface expression of hP2X7, 1 × 104 cells per well the day before experiments into poly-D-lysine (Millipore) coated 96-well plates . The membrane impermeant dye YOPRO-1 iodide  was used at a final concentration of 2 μM in low divalent buffer . Data was acquired on a Flexstation 3 plate reader (Molecular Devices) using excitation wavelength 490 nm, emission wavelength 520 nm and 6 reads per well. ATP ± ginsenosides at 10X final concentration was automatically injected at 40 seconds from a 96-well V-bottomed drug plates (Greiner). Pipette height was set to 180 µl and the rate of injection was 4. Data was analysed as AUC between 50 and 300 seconds using SoftMax Pro v5.4 software (Molecular Devices).Cells were plated at 2.5 × 104 cells per well the day before experiments into poly-D-lysine coated 96-well plates. Cells were loaded with 2 μM Fura-2-acetoxymethyl (AM) calcium indicator dye  in standard HBSS buffer containing 250 µM sulfinpyrazone for 40 minutes at 37 °C. This loading buffer solution was removed and replaced with standard low divalent buffer containing 250 µM sulfinpyrazone. A Flexstation 3 plate reader was used to acquire data using dual excitation wavelengths 340 nm and 380 nm with an emission wavelength of 520 nm, 3 reads per well. ATP ± ginsenosides/vehicle at 10X final concentration was injected after 40 seconds. Data was analysed as AUC between 150 seconds and 300 seconds using SoftMax Pro software (Molecular Devices).Cells were plated at 2.5 × 102, 1 mM MgCl2, 13 mM glucose, 10 mM HEPES, pH 7.3) prior to seal formation, and low divalent buffer solution was used for agonist and PAM application in most experiments. Agonists were applied using a computer-controlled fast-flow system  with the perfusion capillaries placed in close proximity to the cell under investigation.Stably expressing HEK-hP2X7 cells were plated onto 13 mm glass coverslips 24 hours prior to use. Membrane currents were recorded in the whole-cell patch-clamp configuration using an EPC10 amplifier (HEKA Elektronik) and borosilicate glass electrodes (TF-150 World Precision Instruments), resistance 3–8 Ω when filled with standard internal solution . Cells were voltage clamped at −60 mV and dialysis of intracellular contents was performed for 1 minute prior to experimental procedures. Cells were continuously perfused by gravity feed with standard divalent buffer solution  24 hours prior to stimulation with increasing concentrations of ATP (1 μM–3 mM), or a combination of ATP and CK  in the presence or absence of AZ10606120 (10 μM), for 24 h. Following incubation, resazurin  was added to cells for 2 h at 37 °C and fluorescence was measured on a Flexstation 3 plate reader .Cells were plated at 5 × 1038. The Schrödinger Prime software was used to construct an energy-based all-atom model with the OPLS3 forcefield, keeping the ATP coordinates from the template. Regions of low sequence homology were refined using extended serial loop sampling in the Prime software.The coordinates of zfP2X4 in the ATP-bound open state (PDB: 4DW1) were used as a template. The sequence of human P2X7 was aligned to the template using the ClustalW algorithm39. Ligands were then re-docked to the new receptor structure using the Glide SP algorithm40 and standard potentials. Structures within 30 kcal mol−1 of the lowest energy structure were retained.Three-dimensional models of the ginsenosides CK and Rd were generated using the LigPrep software within the Schrödinger Maestro suite. The OPLS3 forcefield was used to generate 32 low-energy conformers for each ginsenoside. Induced fit docking was performed using the automated extended sampling protocol in the Schrödinger Maestro suite. This first performed several initial docking runs, in which sidechains were either trimmed, or their van der Waals potentials softened according to their flexibility. Side chains were then rebuilt and those within 5 Å of the ligand were optimised using Prime41, and had a cubic box with dimensions of 30 Å. Each of the 32 conformers were docked using the extended sampling protocol. For each ginsenoside, the resulting poses were clustered by heavy atom RMSD using the average-linkage method, and a representative structure was chosen from the model closest to the centroid of the most populated cluster. For CK and Rd the most populated cluster made up 27% and 59% of all solutions respectively.The receptor grid was centred on the highest-scoring potential binding site, found using SitemapAll graphs were plotted and statistical tests performed using GraphPad Prism v7. Significance was taken as P < 0.05.Supplementary InformationMovie S1


Background: Recent studies of human sera showed that the majority of the respiratory syncytial virus (RSV) neutralizing antibodies are directed against pre-fusion conformation of the fusion (F) protein of RSV and revealed the importance of pre-fusion antigenic site Ø specific antibodies. However, detailed analysis of multiple antigenic site-specific competitive antibody responses to RSV F protein and their contribution to virus clearance in humans are lacking.Methods: We prospectively enrolled a cohort of RSV infected hematopoietic cell transplantation (HCT) adults (n = 40). Serum samples were collected at enrollment  and 14 to 60 days post-enrollment . Antigenic site-specific F protein antibodies were measured against pre-fusion site Ø, post-fusion site I, and sites II and IV present in both the pre-fusion and post-fusion F protein conformations utilizing four different competitive antibody assays developed with biotinylated monoclonal antibodies (mAb) D25, 131-2A, palivizumab, and 101F, respectively. The lower limit of detection were 7.8 and 1.0 μg/mL for the competitive antibody assays that measured site Ø specific response, as well as sites I, II, and IV specific responses, respectively. Neutralizing antibody titers to RSV A and B subgroups was determined by microneutralization assays.Results: The overall findings in RSV infected HCT adults revealed: (1) a significant increase in antigenic site-specific competitive antibodies in convalescent sera except for site Ø competitive antibody (p < 0.01); (2) comparable concentrations in the acute and convalescent serum samples of antigenic site-specific competitive antibodies between RSV/A and RSV/B infected HCT adults (p > 0.05); (3) significantly increased concentrations of the antigenic site-specific competitive antibodies in HCT adults who had genomic RSV detected in the upper respiratory tract for <14 days compared to those for ≥14 days (p < 0.01); and (4) statistically significant correlation between the antigenic site-specific competitive antibody concentrations and neutralizing antibody titers against RSV/A and RSV/B .Conclusions: In RSV infected HCT adults, antigenic site-specific antibody responses were induced against multiple antigenic sites found in both the pre-fusion and post-fusion F conformations, and were associated with a more rapid viral clearance and neutralizing antibody activity. However, the association is not necessarily the cause and the consequence. Pneumoviridae. RSV is transmitted via fomites and large droplet aerosols. RSV, traditionally considered as the most common respiratory pathogen in children <5 years old, has more recently been found to have a high prevalence in immunocompromised individuals  is a negative-sense, single-stranded RNA virus of the family ividuals , 2. RSV ividuals . The hemividuals . A licenividuals . Moreoveividuals –11.RSV fusion (F) surface glycoprotein is a class I protein that mediates viral entry into host cells by transforming from a metastable trimeric pre-fusion conformation (pre-F) to a highly stable post-fusion conformation (post-F). The antigenic topology of RSV F is substantially altered during this transition. Molecules that prevent these structural changes can prevent viral fusion and have potential as therapeutics for treatment of RSV infection. Some groups of epitopes, referred to as antigenic sites, are generally conserved on both the pre-F and post-F conformations, whereas others are found either on the pre-F or post-F conformation . Six antRSV is primarily a mucosally restricted virus causing damage to the upper and lower respiratory tracts during its multiple rounds of replication. It can elicit cellular and humoral immune responses at variable levels in HCT recipients, depending upon the degree to which individuals remain immunocompromised post-transplantation. RSV-specific neutralizing antibody play an important role in preventing severe infection while cellular immune responses are thought to play a critical function in destroying RSV infected cells and thus clearing the infection. Previously RSV naïve but immunocompetent infants can shed RSV from their upper respiratory tract (URT) for up to 21 days; by contrast, immunocompromised children can shed RSV for several months , 20. In HCT recipients with laboratory-confirmed RSV upper respiratory tract infection at enrollment and negative chest radiography findings were enrolled within 72 h of RSV diagnosis and stratified by level of risk for progression to the lower respiratory tract as previously described . From JaViral RNA extraction and RSV/A and RSV/B detection in nasal wash samples by rtRT-PCR in a CLIA certified Respiratory Virus Diagnostic laboratory (CLIA ID# 45D0919666) were performed as previously described .TM Antibody Biotinylation Kit  per manufacturer's instructions. The source of the fusion protein was from sucrose purified RSV/A/Bernett  for sites I, II and IV. One hundred μL of spRSV  was coated onto the Immulon 2HB 96-well plate  for 18 h at 4°C. For site Ø competitive antibody assay, 100 μL of HEp-2 cell suspension (24 x 104 cells /mL) in 10% FBS/MEM were seeded on 96-well Falcon tissue culture plates from Corning . The plates were incubated at 37°C with 5% CO2 and 85% humidity for 24 h to form monolayers. Cells were inoculated with spRSV (MOI = 0.002) and incubated at the same condition for 2 days. The plates from all four assays were blocked for 1 h with 5% milk (Carnation Instant Nonfat Dry Milk) in 1X phosphate-buffered saline. One hundred μL of D25, 131-2A, palivizumab, and 101F at a concentration of 1.25 μg/mL in 5% milk  and 5% milk in 10% Defined Fetal Bovine Serum in Minimum Essential Medium (for D25) were added in duplicate followed by 2-fold serial dilutions  for generating a standard curve on each plate. Next 50 μL of 2-fold serial dilutions of serum samples  in duplicate were added to the coated plates, and then immediately 50 μL of 100 ng/mL of biotinylated mAbs  generated with a PierceTM Antibody Biotinylation Kit  per manufacturer's instructions, were added, followed by 1 h incubation. After washing, HRP-conjugated streptavidin  was added for an additional hour. Wells containing biotinylated mAb without sera served as positive controls representing maximum binding. Wells without biotinylated mAbs containing either 5% milk instead of sera or wells containing sera served as negative controls. A four-parameter logistic (4PL) regression model was used to calculate the competitive antibody concentrations (μg/mL) based on the dynamic range of the standard curve by interpolating the concentration of the standards that corresponds to the absorbance value at which the test serum sample resulted in 50% inhibition. The lower limit of detection (LLoD) was 1.0 μg/mL for site I, II, and IV competitive antibody assays, and 7.8 ug/mL for site Ø competitive antibody assay. Samples with concentration below the LLoD were assigned a value of 0.5 and 3.9 μg /mL, respectively.Four antigenic-site competitive antibody assays were used to measure concentrations of D25-competing antibody (site Ø), 131-2A-competing antibody (site I), palivizumab-competing antibody (site II), and 101F-competing antibody (site IV) in serum that compete with biotinylated mAbs for binding to the respective antigenic site of the RSV fusion protein. D25 and 101F were purchased from Cambridge Biologics, LLC, Brookline, MA, USA. Palivizumab was from MedImmune, LLC, Gaithersburg, MD, USA. 131-2A was from EMD Millipore Corporation, Temecula, CA, USA. mAbs were biotinylated with a Pierce2 for 90 minutes. One hundred μL of HEp 2 cells was added to each well in the 96-well plate and incubated for 6–7 or 7–8 days for RSV/A and RSV/B microneutralization assays, respectively. Then the 96-well plates were fixed and stained with 10% formalin/0.01% crystal violet solution for ~24 h. After the 96-well plates were air-dried, they were read. Neutralizing antibody titers were defined as the final dilution at which there was a 50% reduction in viral cytopathic effect (CPE). Any serum sample resulting in a titer < LLoD (2.5 log2) was assigned a value of 2 log2. Any mAbs resulting in a titer < LLoD was retested using the stock (1.0 mg/mL) and assigned a value of 0.2 log2 if the titer was negative again.Serum samples and the four mAbs  were analyzed for neutralizing antibodies (Nt Ab) against RSV/A/Tracy and RSV/B/18537 in HEp-2 cells using a qualified microneutralization assay as previously described . Serum st-test was used to determine whether the geometric means of neutralizing antibody titer (GMT) (log2) or geometric mean of log transformed competitive antibody concentrations (GMC) differed significantly between acute and convalescent samples. A two-sample t-test was used to determine whether the neutralizing antibody (log2) or GMC of log transformed competitive antibody concentrations differed significantly between RSV/A and RSV/B infected patients, as well as between HCT recipients who shed virus for < 14 and ≥ 14 days. Statistical significance was indicated for p-values < 0.05. Pearson's correlation coefficients were calculated between neutralizing antibody titers and site-specific competitive antibody concentrations. Statistical analyses were performed using the SPSS Statistic 22 .For demographic characteristics and clinical outcomes, continuous variables and categorical variables were analyzed. A paired p < 0.025). The absolute neutrophil count and the absolute lymphocyte count are within the normal ranges for these cohorts. The median time from transplantation to RSV infection is 169 and 100 days for adults shedding RSV from the URT for < 14 and ≥14 days, respectively.Clinical characteristics at enrollment are summarized for all 40 HCT adults in Specificity of the four competitive antibody assays were confirmed using a panel of monoclonal antibody (12.5 μg/mL) to compete with biotinylated monoclonal antibody specific to their respective antigenic sites . Using aNeutralizing activity of the monoclonal antibodies at a concentration of 40 μg/mL was evaluated in the RSV/A and RSV/B microneutralization assays . The mAbA significant increase in RSV antibody levels  in the convalescent sera compared to the acute sera was measured by all four competitive antibody assays and both microneutralization assays . The GMCThe competitive antibody in GMC (μg/mL) and neutralizing antibody in GMT (log2) were compared between HCT adults shedding virus from their URT for <14 and ≥14 days . For theThe proportion of the contribution for each site-specific competitive antibody GMC to the total GMC for acute and convalescent sera from HCT adults shedding virus <14 days and ≥14 days are illustrated in The site-specific competitive antibody GMC and neutralizing antibody GMT were compared between RSV/A and RSV/B infected HCT adults . No signThe proportion of the contribution for each site-specific competitive antibody GMC to the total GMC for RSV/A and B infected HCT adults is illustrated in p < 0.05)  . The higAn in-depth understanding of the human antibody response to RSV infection will aid the development and evaluation of vaccines and therapeutics against RSV disease. Previous studies have reported on the epitopes targeted by RSV-specific neutralizing antibodies in human sera , 36, as In this prospective cohort study, pre-F and post-F competitive antibodies were elicited after RSV infection in HCT adults. The highest site-specific competitive antibody concentration in acute sera was against site Ø on the pre-F conformation, which is consistent with prior published results showing that prefusion F-specific antibodies are prevalent in polyclonal neutralizing antisera . HoweverSite I had the lowest competitive antibody concentration, which is also consistent with the results published by Gilman and others , 37. TheWe used Pearson's correlation to determine the associations between the four site-specific competitive antibodies and between site-specific antibody concentrations to neutralizing antibody titers. The highest correlations to RSV-specific neutralizing antibody levels were observed with sites II and IV competitive antibodies. These results suggest that antibodies against palivizumab and 101F epitopes are responsible for a significant proportion of the virus neutralizing capacity of sera. It also suggest that the site-specific competitive antibody assays measures changes that directly relates to changes in neutralizing antibody levels. The lower correlation detected between antigenic-site Ø competitive antibody concentrations and neutralizing antibody titers might be related to antigenic site Ø being the least conserved region compared to other antigenic sites in the F protein , 38, 39,The effect of RSV infection by viral subgroups (RSV/A and RSV/B) on the humoral immune responses to the four antigenic sites was evaluated in this prospective cohort study. No significant differences were observed in both the neutralizing antibody level and site-specific competitive antibody concentrations measured between HCT recipients infected with RSV/A vs. RSV/B. In addition, the proportion and the total GMC of the four competitive antibodies were comparable in convalescent sera between RSV/A and RSV/B infected HCT adults. This observation is consistent with antigenic site II and site IV being well conserved between RSV/A and RSV/B subgroups, and suggests a monovalent RSV-F vaccine against infection with RSV/A and RSV/B is likely sufficient for protection against severe RSV disease.n = 40) is not representative of adults in the general population. Higher rises in competitive antibody concentration and neutralizing antibody titer after an RSV infection might occur in healthy adults compared to the HCT adults. In addition, we did not measure competitive antibody concentration to site III and site V, the remaining pre-F antigenic sites. Thus, their contribution to the neutralizing antibody activity post-RSV infection was not determined in this cohort of HCT adults infected with RSV.Our study has some limitations. The small number of RSV infected HCT adults  in HCT adults who shed RSV <14 days compared to ≥14 days, comparable concentrations of competitive antibodies in the convalescent sera between RSV/A and RSV/B infected HCT adults, and a significant positive correlation between site-specific competitive antibodies and RSV-specific neutralizing antibody activity. In conclusion, in RSV naturally infected HCT adults, site-specific competitive antibody responses occurred to antigenic sites found in both the pre-fusion and post-fusion F conformations and were associated with viral clearance. The data might suggest but does not demonstrate that these antibody responses are contributing to the observed more rapid viral clearance. The faster clearance in the individuals with higher antibody responses might just reflect the level of immune competence of these individuals. Therefore, the observed relation between higher antibody responses and more rapid clearance is not necessarily the cause and the consequence. In addition, further evaluation of how the T cell–mediated responses assist in RSV viral clearance is needed. Lastly, HCT recipients would likely benefit from monoclonal antibody immunoprophylaxis or RSV-F vaccine when licensed for prevention of severe RSV infection during their period of vulnerability post-transplantation.The datasets generated for this study are available on request to the corresponding author.This study was carried out in accordance with the recommendations of the institutional review boards of the University of Texas MD Anderson Cancer Center and Baylor College of Medicine with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the University of Texas MD Anderson Cancer Center and Baylor College of Medicine.XY, RC, and PP designed the research. XY, OI, VA, LA, TM, and PP performed the research. XY, LF-S, and PP analyzed the data. DS and RC provided the samples. KP processed the samples. F-AP, VA, LA, TM, LF-S, KP, DS, RC, and PP edited the text. XY wrote the first draft of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.