Significance: Intraoperative parameters of renal cortical microperfusion (RCM) have been associated with postoperative ischemia/reperfusion injury. Laser speckle contrast imaging (LSCI) could provide valuable information in this regard with the advantage over the current standard of care of being a non-contact and full-field imaging technique.Aim: Our study aims to validate the use of LSCI for the visualization of RCM on ex vivo perfused human-sized porcine kidneys in various models of hemodynamic changes.Approach: A comparison was made between three renal perfusion measures: LSCI, the total arterial renal blood flow (RBF), and sidestream dark-field (SDF) imaging in different settings of ischemia/reperfusion.Results: LSCI showed a good correlation with RBF for the reperfusion experiment (Conclusions: LSCI is capable of imaging RCM with high spatial and temporal resolutions. It can instantaneously detect local perfusion deficits, which is not possible with the current standard of care. Further development of LSCI in transplant surgery could help with clinical decision making. Others have shown the potential of near-surface perfusion imaging for the prediction of postoperative complications, including reduced creatinine clearance, delayed graft function, and even graft rejection.,–,,The value of the common techniques, such as postoperative duplex sonography or an arterial renal blood flow (RBF) probe, to monitor total RBF is limited by the fact that such techniques do not account for local perfusion heterogeneities.To date, an objective, intraoperative imaging tool that can help visualize the RCM during surgery is still missing. In this paper, we report on the use of laser speckle contrast imaging (LSCI), a real-time, non-contact, full-field imaging technique with a large FOV that can visualize blood flow in tissues without the administration of a fluorescent dye,22.1®, Groningen, The Netherlands) at 4°C and perfused for three and a half hours at a mean pressure of 25 mmHg. HMP was oxygenated  were slaughtered for consumption purposes and were handled according to standardized legal procedures. The pigs were stunned by electricity and died from exsanguination. Approximately 2 liters of blood were collected in a beaker with 25,000 IU of heparin  during exsanguination. Kidneys were removed from the cadaver en bloc, the renal artery was dissected free, and surrounding tissue was removed. The left kidney was used for all the experiments since this side displayed a better visible arterial bifurcation. After 30 min of warm ischemia , the kidneys were flushed with 500 ml cold 4°C saline. Next, the kidneys were installed in a kidney holder and placed on the hypothermic machine perfusion (HMP)  controlled by in-house developed software .®, MEDOS Medizintechnik AG, Heilbronn, Germany) was built in the oxygenator. The flow sensor is a clamp-on flow sensor . The pressure sensor is a Truewave® disposable pressure transducer . As perfusion medium, 500 ml of autologous leukocyte depleted blood was used. The blood was diluted with 300 ml Ringers lactate  and supplemented with 10 ml 8.4% bicarbonate , 10 ml 5% glucose , 6 mg mannitol , 0.33 ml dexamethasone , ®, Novo Nordisk, Bagsværd, Denmark), and 3 ml bicarbonate was maintained. Carbogen (95% The setup of the normothermic machine perfusion (NMP) was described in detail elsewhere,2.3®-imaging  analysis software that has previously been demonstrated by our group to qualify gut microvascular blood flow during in-vivo ischemia/reperfusion experiments.®, Jai, Copenhagen, Denmark) was placed on a fixed 3D-printed holder to ensure invariability in distance and incident angle of the camera and laser  was set at an LSCI is based on the principle of coherent laser light that is backscattered from tissue, which forms a speckle pattern at the detector. The phase shift of this backscattered light results in a random interference pattern, the so-called speckles. Due to motion in the tissue, i.e., movement of the RBCs, the interference pattern starts fluctuating, causing a dynamic speckle pattern, which is blurred by the finite exposure time of the detector. The speckle contrast nd laser . The disal setup  was plac2.4We have designed a set of four hemodynamic experiments to study the LSCI as a tool for organ perfusion measurement . During 2.4.1Validating the relation between the RBF and LSCI values is crucial for clinical use. Apart from the resemblance with the reperfusion during a kidney transplant, this experiment could provide us with insights about the correlation between RBF and LSCI values in low- and high-flow values at the beginning and end of the experiment, respectively. After HMP and a cold flush with 500 ml of 0.9% NaCl, the kidney was installed in the NMP organ chamber, warming up for 60 min with a pressure of 85 mmHg and a sinusoidal flow with a frequency of 60 Hz to mimic a physiological situation. During this hour, the kidney warms-up from 4°C to 37°C.2.4.2Similar to the reperfusion experiment Sec. , this ex2.4.3We could evaluate the ability to distinguish between well and non-perfused tissues within the FOV by inducing a local ischemic area. The speed at which the large local perfusion difference can be visualized gives an indication of the added clinical value in case of an unwanted local perfusion deficit during a kidney transplant. A catheter  was inserted in the renal artery before NMP and sutured in the inferior bifurcation of the renal artery. The catheter was inflated inducing local ischemia in one part of the kidney. The ischemia was induced twice. The first time, a short 5-min warm ischemic period was followed by a 10-min recovery time. The second time, a long 15-min warm ischemic period was followed by a 40-min recovery time.2.4.4Although local ischemia Sec.  causes a2.5–® Video Microscope System, MicroScan BV, Amsterdam, The Netherlands) was held in place using a tripod connected to a laboratory table to minimize movement artifacts. The tripod has SDF imaging can track the movement of individual RBCs enabling quantitative blood flow measurements and the detection of subtle microvascular changes. of RBCs . The regThe renal capsule had to be locally removed at the tip of the SDF microscope to be able to image the RCM during this part of the experiments. SDF imaging, RBF measurements, and LSCI were performed simultaneously in only one kidney due to the complex nature of imaging the RCM using SDF imaging. Five consecutive repetitions of the short local ischemia experiments were executed followed three gas injections with oxygen, one with room air, and one with nitrogen, respectively.2.6Data are presented as 3The average weight of the six slaughterhouse kidneys was 3.1The reperfusion experiment was performed five times in five kidneys. During the hour, all kidneys warmed-up to 37°C, which led to an increase in RCM .. This e3.2The flow experiment was performed 10 times in five kidneys. The 3.3The local ischemia experiment was performed on five kidneys. The experiment failed for two kidneys; one due to malfunctioning of the balloon catheter and the other one due to the appearance of the ischemic area on the posterior side of the kidney. Typical images are shown in 3.4The gas-bubble infusion was performed once on five kidneys and characterized by a slow, local return of RCM, as depicted in 3.5The results of the local ischemia, oxygen, room air, and nitrogen injection are shown in 4ex vivo perfused human-sized porcine slaughterhouse kidneys in various models of ischemia/reperfusion. The local reperfusion experiments demonstrated a high correlation between the LSCI and SDF, superior to LSCI correlations with RBF. The good correlation between LSCI and SDF emphasizes the high temporal and spatial resolution of LSCI in its ability to visualize RCM. LSCI not only display a clear distinction between perfused and non-perfused tissues, it also tracks the transient ischemia, induced by injection of gasses with different absorption characteristics, in a real-time manner. Yet, the effect of the monitoring of near-surface cortical microperfusion on clinical decision making should be further investigated in a clinical trial.We report on the visualization of RCM using The reperfusion experiment displayed a high correlation between the RBF and LSCI indicating that the reperfusion after ischemia during renal transplantation could be monitored using LSCI. This has the advantage over conventional intrarenal probes that it can detect local perfusion deficits in an early stage. The slight difference between RBF and LSCI could potentially be explained by the kidney’s redistribution mechanismDuring the flow experiment with a stepwise change in total RBF, LSCI displayed only a moderate correlation with total RBF. We hypothesize that the functional tissue in the cortex is preserved at the expense of a reduction in flow to the medulla in a response to a decrease in RBF. This would result in a redirection of flow to the cortex .The local ischemia was immediately visible on the real-time live feed. The experiment has a good correlation for both the short and long ischemic period, which is comparable to the correlation found during the LSCI-SDF comparison. This can be explained by the fact that there is no blood to redistribute, thus the decrease in flow is proportional to the RBF. It instantly displays a distinction between well- and non-perfused tissues, whereas visible tissue discoloration takes a prolonged amount of time. This fast and accurate assessment of the RCM by LSCI has potential clinical impact. For example, Hoffman et al.As mentioned before, SDF imaging directly visualizes individual RBCs. Comparing LSCI to both SDF and RBF gives us valuable information about the perfusion measured using LSCI. However, SDF imaging of a kidney is tedious and required removal of the renal capsule making it unsuitable for clinical practice. The good correlations between LSCI and SDF thus indicate that LSCI can give valuable information with the advantage of being a non-contact and full-field imaging method.–An important clinical need in which LSCI can easily be implemented is organ transplantation. Since prolonged anastomosis time is detrimental for organ quality,–The use of LSCI has already been described in rat kidneys,in vivo, the kidney will be subject to movement as a result of respiratory movement and pulsations of the heart during transplantation. Others have tried to overcome this using fiducial markers.,,One of the main challenges that has to be overcome before LSCI can be implemented into clinical practice is movement artifacts.5ex vivo machine perfused human-sized porcine kidneys, LSCI was capable of detecting local changes in RCM with a high spatial and temporal resolution. In various settings of local ischemia, LSCI correlated well with SDF imaging. However, LSCI does not always fully correlate with the total RBF due heterogeneity of blood flow between the medullar and cortical microcirculation underlining the added value over conventional arterial flow sensors. The implementation of LSCI during transplant surgery could help with the early establishment of an appropriate treatment plan directly after reperfusion of the organ.In the setting with Click here for additional data file.Click here for additional data file.Click here for additional data file.


The mammalian ULK kinase complex is the most upstream component in the macroautophagy/autophagy signaling pathway. ULK1 and homolog ULK2, the sole serine/threonine kinases in autophagy, transduce an array of autophagy-inducing stimuli to downstream autophagic machinery, regulating autophagy from autophagosome initiation to fusion of autophagosomes with lysosomes. ULK signaling is also implicated in a diverse array of non-canonical processes from necroptosis to ER-Golgi trafficking to stress granule clearance. However, the exact mechanisms by which ULK regulates these diverse processes remain largely unknown. Most notably, the number of validated ULK substrates is surprisingly low. Our study identifies new ULK substrates from a wide array of protein families and signaling pathways and supports an expanded range of physiological roles for the ULKs. We further characterize several new substrates, including the PIK3C3/VPS34-containing complex subunit PIK3R4/VPS15 and the AMPK component PRKAG2. Finally, by analyzing PIK3R4/VPS15-deficient models we discover novel aspects of ULK signaling with potential relevance in selective autophagy. Furthermore, bioinformatics analysis provided insight into the range of ULK-dependent and -independent phosphoproteomic changes that occur when the kinases are removed. Our datasets give unprecedented insights into ULK signaling and provide a useful resource to advance our understanding of these substrates and their relevant pathways.Using an array of mass spectrometry techniques to study the phosphoproteome of in vitro, indicating that any differences in roles between the paralogs are not due to varying target specificity.It is a fascinating question as to how a handful of kinase complexes facilitate the dramatic rearrangements of membranes and proteins required for autophagosome formation. The ULK complex plays an indispensable role in the process. It contains the regulatory components ATG13, ATG101 and dimeric RB1CC1/FIP200 along with either ULK1 or ULK2. While ULK1 and ULK2 share functions in autophagy, independent roles have been reported. Interestingly, by comparing the ability of ULK1 and ULK2 complexes to phosphorylate a diverse range of substrates in peptide array format, we showed that their substrate repertoires are virtually indistinguishable The ULK complex coordinates nutrient and energy signals from the MTORC1 and AMPK complexes respectively. However, the interplay between ULK and AMPK signaling is controversial. ULK negatively regulates AMPK activity via an unknown mechanism, and AMPK has independently been shown to positively and negatively regulate ULK-dependent autophagy. We discovered that the nucleotide-sensing AMPK component PRKAG2, mutated in the cardiac glycogen storage disease PRKAG2 syndrome, is phosphorylated by both ULK and AMPK at serine 124, with phosphorylation sensitive to both serum and energy status. Based on previous PRKAG2 characterization studies, we speculate that phosphorylation might modulate AMPK complex localization. Our data therefore add an exciting new piece to the puzzle of ULK-AMPK regulation. Notably, as glycogen storage diseases are often associated with the impairment of autophagy, it is possible that further study of ULK-dependent PRKAG2 phosphorylation may yield disease-relevant insights.Immediately downstream of the ULK complex is the class III phosphatidylinositol 3-kinase (PtdIns3K) PIK3C3/VPS34-containing complex. This is comprised of lipid kinase PIK3C3/VPS34 and regulatory subunits BECN1/Beclin1, PIK3R4/VPS15 and both NRBF2 and ATG14 (complex I), or UVRAG (complex II). Both generate the signaling phospholipid phosphatidylinositol-3-phosphate, but in different cellular contexts. Typically, complex I activity is associated with autophagy initiation and complex II activity with endolysosomal trafficking, as well as the later stages of autophagy.in vitro and in vivo ULK substrate. Notably we also showed that VPS26B, part of the retromer complex which acts immediately downstream of PtdIns3K complex II in endolysosomal sorting, is phosphorylated by ULK at serine 302/304. We also present the first evidence of ULK-dependent PIK3C3/VPS34 serine 249 phosphorylation without kinase or substrate overexpression, and show that both nutrient and iron deprivation are upstream stimuli, giving physiological context to recent phenotypic data identifying serine 249 as a phosphorylation-activated LC3-interacting region.The regulation of PtdIns3K complex activity by ULK is an exciting and evolving area of study. ULK positively regulates PtdIns3K complex I upon autophagy initiation; however, a more comprehensive understanding of ULK-dependent regulation of both complexes is crucial. We identified PtdIns3K complex II component UVRAG as an Importantly, we identified the pseudokinase PIK3R4/VPS15, as a novel ULK substrate. Previously thought to be an inert scaffold, recent structural insights indicate that PIK3R4/VPS15 may dynamically transmit activating signals to PIK3C3/VPS34’s lipid kinase domain. However, no functional post-translational modifications have been identified in PIK3R4/VPS15 to date. We identified 6 novel ULK substrate residues in PIK3R4/VPS15 , mutatioPIK3R4/VPS15 knockout model. Unexpectedly, we discovered novel ULK-dependent phenotypes with potential relevance for the regulation of autophagy. We showed that upon PIK3R4/VPS15 ablation ULK phospho-substrates accumulate along with aberrant bodies positive for a range of autophagy proteins in PtdIns3K complex I-dependent manner. Our data revise previous findings identifying these bodies as stalled autolysosomes, instead favoring SQSTM1/p62-ubiquitin-positive aggregates, which sequester autophagic signaling proteins upon chronic inhibition of autophagic flux. The study of ubiquitin-induced SQSTM1 aggregation and phase separation has provided valuable insight into the mechanisms of redox homeostasis and selective autophagy. Our data suggest that these aggregates are sites of high local ULK activity, which actively remodels their outer surface by controlling the recruitment and distribution of autophagic machinery. Together, these data support a cargo-centric model for the initiation of selective autophagy, indicating that autophagy initiation machinery are subject to tight spatial regulation on cargo mediated by ULK kinase activity. Alongside recent data showing that multiple ULK complex translocations to distinct surface regions are required for the capture of mitochondria by autophagosomes, these data support the importance of spatial regulation of autophagic machinery in selective autophagy.To study the ULK-PIK3R4/VPS15 signaling axis, we generated the first human


PHF6 mutations frequently coexist with JAK3 mutations in T-ALL patients. However, the role(s) of PHF6 mutations in JAK3-driven leukemia remain unclear. Here, the cooperation between JAK3 activation and PHF6 inactivation is examined in leukemia patients and in mice models. We found that the average survival time is shorter in patients with JAK/STAT and PHF6 comutation than that in other patients, suggesting a potential role of PHF6 in leukemia progression. We subsequently found that Phf6 deficiency promotes JAK3M511I-induced T-ALL progression in mice by inhibiting the Bai1-Mdm2-P53 signaling pathway, which is independent of the JAK3/STAT5 signaling pathway. Furthermore, combination therapy with a JAK3 inhibitor (tofacitinib) and a MDM2 inhibitor (idasanutlin) reduces the Phf6 KO and JAK3M511I leukemia burden in vivo. Taken together, our study suggests that combined treatment with JAK3 and MDM2 inhibitors may potentially increase the drug benefit for T-ALL patients with PHF6 and JAK3 comutation.T-cell acute lymphoblastic leukemia (T-ALL) is a malignant hematologic disease caused by gene mutations in T-cell progenitors. As an important epigenetic regulator,  NOTCH1 or JAK-STAT, super-enhancer generating mutations of TAL1, deep deletion of cell cycle-related genes , and inactivating mutations of WT1, LEF1, GATA3, and PHF6 ). The mice received medication daily until death. For the in vitro experiment, MOLT-4 cells were treated with tofacitinib (2.0 μM), idasanutlin (0.5 μM) or 0.2% DMSO in culture medium for 48 h and then proliferation and apoptosis were examined.Phf6 WT/KO + JAK3M511I cells, 1 µg RNA per sample was used for RNA sample preparations. Transcriptome sequencing was performed on Illumina NovaSeq 6000 platform  to a total target depth of 10 million 150 bp paired end reads. Differential expression analysis was performed by DESeq2 R package (1.16.1).For RNA-Seq analysis of The ChIP assay was performed with the ChIP assay kit  according to manufacturer’s recommendations. Chromatin from cross-linked HA-PHF6-overexpressed K562 or control cells was sheared using an ultrasonicator  to obtain DNA (100–400 bp). Immunoprecipitation was conducted with ChIP-grade HA-tag antibody , PHF6 antibody  or normal IgG antibody .RNA-Seq and ChIP-Seq data are available at GEO under accession number GSE159444 and GSE159549.JAK/STAT mutations occurred in 98 samples (21.83%) . Ho. HoJAK3Mlls Fig. . This suPHF6 and JAK3 mutations cooperatively drive T-ALL progression probably via inhibiting the BAI1-MDM2-P53 signaling pathway, in addition to activating the JAK3/STAT5 signaling pathway. We further demonstrated that combination therapy with tofacitinib and idasanutlin reduced the Phf6 KO + JAK3M511I leukemia burden in vivo. Our study suggested that the combined usage of JAK3 and MDM2 inhibitors should increase the drug benefit for T-ALL patients with PHF6 and JAK3 comutation.In conclusion, we revealed that Supplementary-PHF6


Rheumatoid Arthritis (RA) is an incurable autoimmune disease that promotes the chronic impairment of patients’ mobility. For this reason, it is vital to develop therapies that target early inflammatory symptoms and act before permanent articular damage. The present study offers two novel therapies based in advanced drug delivery systems for RA treatment: encapsulated chondroitin sulfate modified poly(amidoamine) dendrimer nanoparticles (NPs) covalently bonded to monoclonal anti-TNF α antibody in both Tyramine-Gellan Gum and Tyramine-Gellan Gum/Silk Fibroin hydrogels. Using pro-inflammatory THP-1 , the therapy was tested in an inflammation in vitro model under both static and dynamic conditions. Firstly, we demonstrated effective NP-antibody functionalization and TNF-α capture. Upon encapsulation, the NPs were released steadily over 21 days. Moreover, in static conditions, the approaches presented good anti-inflammatory activity over time, enabling the retainment of a high percentage of TNF α. To mimic the physiological conditions of the human body, the hydrogels were evaluated in a dual-chamber bioreactor. Dynamic in vitro studies showed absent cytotoxicity in THP-1 cells and a significant reduction of TNF-α in suspension over 14 days for both hydrogels. Thus, the developed approach showed potential for use as personalized medicine to obtain better therapeutic outcomes and decreased adverse effects. Inflammatory arthritis is a term used to classify a group of conditions such as rheumatoid arthritis (RA), which affects the immune system and causes pain, stiffness, and joint damage ,2. DiffeTNF α has an important role in the pathogenesis of RA, since it is overexpressed in the synovial joints of patients . TherefoNanotechnology, as nanoparticle-based delivery systems, provides a unique series of advantages for the development of delivery systems including high loading capacity, controlled release, prolonged circulation, and target delivery . Even soDespite being relatively simple and effective, is has been challenging to translate such approaches into clinical settings. One of the reasons for this may be a reliance on the settings selected for their study. Current in vitro cellular methods, applied in several areas of drug discovery, comprise the use of static conditions which do not emulate the dynamic natural physiological environment found in the human body, thereby failing to produce reliable data ,18. ThusIn previous works, Tyramine-Gellan Gum (Ty-GG) hydrogels  and Tyra−1)  was linked to CS/PAMAM dendrimer NPs previously produced by the authors  . For that, anti-TNF α Ab-CS/PAMAM dendrimer NPs and CS/PAMAM dendrimer NPs were allowed to react with a secondary antibody Alexa Fluor 488 rabbit anti-mouse lgG (H + L)  in PBS solution (1:1000) for 1 h. Then, the solutions were analyzed in the fluorescence spectrometer.−1 of TNF α  for 4 h at room temperature. As controls, non-modified dendrimers were also incubated. After the time of reaction, the solutions were centrifuged, and the supernatants were kept at −80 °C for further analysis. The concentration of free TNF-α in the supernatant samples was determined using a Human TNF-alpha DuoSET ELISA kit  and a DuoSet Ancillary Reagent Kit 2 , following the manufacturer’s instructions. The samples were measured at an optical density of 450 nm using a microplate reader . Absorbance values were converted into concentrations using a standard curve of TNF-α in the range of 0 to 1000 pg mL−1.The capacity of anti-TNF α Ab-CS/PAMAM dendrimer NPs to capture TNF α was also evaluated. For the evaluation, modified dendrimers were incubated with 1000 pg mLTy-GG previously produced by the authors  was used−1)  in PBS and a solution of hydrogen peroxide (H2O2) (0.36% (v/v))  in distilled water were prepared. Then, 1% Ty-GG solution (w/v) was prepared in distilled water and used for the preparation of Ty-GG hydrogels and Ty-GG/SF hydrogels. To obtain the Ty-GG/SF hydrogels, 1% (w/v) of Ty-GG solution was mixed with 2% (w/v) SF (1:1) solution. At this point, anti-TNF α Ab-CS/PAMAM dendrimer NPs were mixed in the Ty-GG and Ty-GG/SF solutions at a final concentration of 0.5 mg mL−1. Then, two different crosslinking levels were tested by adding different amounts of HRP and H2O2 solutions to Ty-GG and Ty-GG/SF solutions, hereafter denominated C1, C2, C3, and C4, as described in To prepare the hydrogels, first a solution of horseradish peroxidase (HRP) (0.84 mg mL−1 of CS/PAMAM dendrimer NPs solution was prepared in a carbonate-bicarbonate coupled buffer (pH 9.2). Then, 50 µL of 10 mg mL−1 of FITC in anhydrous dimethyl sulfoxide (DMSO)  solution was added per each mL of CS/PAMAM dendrimer NPs solution under agitation and kept in the dark at 4 °C for 8 h. In the end, the FITC-labelled CS/PAMAM dendrimer NPs were dialyzed in ultrapure water for 48 h and freeze-dried. Then, FITC-CS/PAMAM dendrimer NPs, at a final concentration of 0.1 mg mL−1, were mixed with Ty-GG and Ty-GG/SF solutions and HRP and H2O2 solutions  . Initially, 10 mg mL−1 to 0.5 mg mL−1. The UV absorbance of dendrimers NPs was read at 280 nm in a microplate reader to quantify the anti-TNF α Ab-CS/PAMAM dendrimer release . Three samples per condition were evaluated at each time point. The absorbance values were converted into concentrations using the calibration curve.To evaluate the release profile of anti-TNF α Ab-CS/PAMAM dendrimer NPs, each release system developed, C1, C2, C3, and C4 (more details in v/v) of penicillin and streptomycin , under standard culture conditions . When ≈80% confluence was reached, cells were trypsinized and seeded in a well of a 24-well plate at a density of 5 × 105 cells per well. For induction of THP-1 cells’ differentiation into macrophages, cells were cultured under RPMI with 100 nM phorbol 12-myristate-13-acetate (PMA) , hereafter designated as healthy cells. After 24 h, the medium was replaced with a RPMI medium without PMA and incubated for another 48 h. At this point, to develop an inflammation in vitro model, cells were incubated with 100 ng mL−1 of Lipopolysaccharide (LPS)  in an RPMI medium and incubated for 5 h to induce an inflammatory response.The Human monocytic cell line (THP-1)  was expanded in RPMI 1640 Medium, GlutaMAX™ Supplement, HEPES , supplemented with 10% fetal bovine serum  and 1% (−1 anti-TNF α Ab-CS/PAMAM dendrimer NPs. Only cells under LPS stimulation were studied (hereafter designated as LPS stimulation). Cells’ metabolic activity and proliferation were monitored along seven days of culture. The TNF α neutralization was assessed along 14 days of culture.Cells under LPS stimulation were incubated with each release system developed, C1, C2, C3, and C4 (more details in v/v) of Alamar Blue®  was added to the different conditions.Cells’ metabolic activity was evaluated on days one, three, and seven of culture with Alamar Blue at each time point. For this, an RPMI culture medium containing 10% .The culture plates were kept in the dark, at 37 °C in the CO−1.The proliferation of the THP-1 cells at days one, three, and seven of culture was analyzed by dsDNA quantification. At each timepoint, cells were washed with PBS solution and lysed with ultrapure water. The cells’ lysate solution was placed into 1.5 mL microtubes and then stored at −80 °C for further analysis. The Quanti-IT PicoGreen dsDNA Assay Kit  was used to quantify dsDNA, accordingly with manufacturers’ instructions. Briefly, 28.7 µL of each sample was mixed with 71.3 µL of PicoGreen solution and 100 µL 1X TE buffer in a well of a 96-well white microplate. Then, the plate was incubated in the dark for 10 min and the fluorescence was read using an excitation of 480/20 nm and an emission of 528/20 nm in a microplate reader. DNA concentration was determined using a standard curve in the range of 2 to 0 µL mL−1.The neutralization of TNF α as evaluated at one, three, seven and fourteen days of culture. At these time-points, the medium of each condition was recovered and stored at −80 °C. To determine the levels of TNF α captured, the TNF-α concentration in the samples was measured using the Human TNF-alpha DuoSET ELISA kit and the DuoSet Ancillary Reagent Kit 2, following manufacturers’ instruction. The optical density was measured at 450 nm using the microplate reader and the values were converted into concentration using a TNF-α calibration curve ranging from 1000 to 0 pg mL5 cells/well in TCP coverslips in 24-well plates. As conducted under static conditions, for induction of THP-1 cell differentiation, cells were cultured under RPMI with 100 nM PMA. After 24 h, the medium was replaced with RPMI medium without PMA and incubated for another 48 h. Then, the cells were incubated with 100 ng mL−1 of LPS in an RPMI medium and incubated for 5 h to induce an inflammatory response. After LPS stimulation, the coverslips were transferred to the lower chamber of a dual-chamber bioreactor [THP-1 cells were seeded at a density of 5 × 10oreactor  with an 2 atmosphere and the compartment containing the cells and hydrogels was perfused at a rate of 12.5 µL h−1. After one, three, seven, and fourteen days of perfusion, the culture medium contained in the collection tubes was removed and stored at −80 °C to analyze of the amount of TNF α present through an ELISA assay, as described above. Delivery systems cultured in THP-1 cell-based inflammation in vitro models under standard static conditions were used as control.To evaluate the therapeutic effect of the developed approaches under dynamic conditions, one condition of each delivery system was selected and added to the cells cultured on the lower chamber of the bioreactor. Then, the entire piping system was assembled and the syringes were filled with medium and connected to the syringe pump. The dual-chamber bioreactor was kept at 37 °C in a humidified 5% COp < 0.05. All assays were triplicated and the results were presented as mean ± standard deviation.Statistical analysis was performed by GraphPad Prism 8 version,  where a Shapiro–Wilk normality test was previously made to evaluate the data normality. Statistical significances were obtained as * The present work is focused on the development of anti-TNF α Ab-CS/PAMAM dendrimer NPs loaded into Ty-GG and Ty-GG/ SF hydrogels as a promising delivery system. This way, hydrogels would increase the controlled and sustained release of anti-TNF α Ab-CS/PAMAM dendrimer NPs at the target site, improving their therapeutic efficacy while requiring less frequent administrations . With thFree antibodies have been reported to present the capacity to neutralize the target agent, TNF α . Herein,To further assess the anti-TNF α Ab-CS/PAMAM dendrimer NPs’ effectiveness in TNF α sequestering, the difference between the initial and the final amount of free TNF α in solution was quantified following the dendrimer NPs’ addition b. The daAt this point, anti-TNF α Ab-CS/PAMAM dendrimer NPs were loaded into Ty-GG and Ty-GG/SF disc hydrogels . The gelConsidering the success of the hydrogel-based NPs’ encapsulation, it was critical to optimize Ty-GG and Ty-GG/SF hydrogels’ crosslinking levels to maximize anti-TNF α Ab-CS/PAMAM dendrimer NPs . Thus, aIn RA, there is a disproportion between pro-inflammatory and anti-inflammatory cytokines, resulting in a more pronounced pro-inflammatory phenotype ,31,32,33DNA quantification was used to assess THP-1 cells’ proliferation under different conditions. At day one, cell proliferation was decreased in C1, C2, C3, and C4 CTRL as compared to the LPS stimulated cells. However, this effect dissipated over seven days, with no final differences among the conditions. The lack of influence of the GG hydrogels and dendrimer nanoparticles over cell viability has been previously known for over a decade ,36.Overall, the results of metabolic activity and cell proliferation analysis provided evidence regarding the safety of these materials for biomedical applications.In order to evaluate the capturing efficacy of TNF α by C1, C2, C3, and C4, the level of free TNF α in the culture medium along 14 days of culture was quantified . Again, A dual-chamber bioreactor was used to mimic the dynamic physiological environment found in the human cartilage and obtain more reliable data concerning the challenges faced by the drug delivery system. Conditions that led to faster and slower NPs release were chosen for this setup. C3 was the condition that presented higher NPs release levels and a better ability to decrease free TNF α. Since C3 is a Ty-GG/SF hydrogel-based strategy and C2 and C4 were conditions with similarly slow-release profiles, C2 was chosen (considering it is based on a Ty-GG hydrogel). Therefore, anti-TNF α Ab-CS/ PAMAM dendrimer NPs loaded-Ty-GG and Ty-GG/SF hydrogels were tested for 14 days using a THP-1 cell-based inflammation in vitro model under dynamic conditions . The conIt was possible to verify that the TNF α concentration present in the supernatant medium significantly decreased along the 14 days of culture under both dynamic and control conditions. However, under static conditions, C2 was the only one presenting no differences to the control, in accordance with the previously release profile evaluation. Firstly, analyzing the results obtained for static and dynamic conditions, it was observed that the concentration of TNF α was always lower in dynamic conditions. However, in dynamic conditions, on day 14, the levels of free TNF α decreased abruptly, indicating that all anti-TNF α Ab-CS/ PAMAM dendrimer NPs were released from the hydrogels. Under static conditions, a less accentuated decrease of TNF α was observed, indicating a slower release of dendrimer NPs over time. Noteworthy, no significant differences were observed between the dendrimer NP-loaded hydrogels tested.In this study, an improved system based on anti-TNF α Ab-CS/PAMAM dendrimer NPs loaded into Ty-GG and Ty-GG/SF hydrogels to tackle inflammatory diseases such as RA was developed. The produced anti-TNF α Ab- CS/PAMAM dendrimer NPs demonstrated a capacity to maintain Ab biological activity, enabling a high immobilization degree of TNF α. Furthermore, it was possible to fine-tune anti-TNF α Ab-CS/PAMAM dendrimer NP-loaded Ty-GG and Ty-GG/SF hydrogels by altering crosslinking levels, thereby enabling the control of the dendrimer NPs release profile over time. The in vitro studies conducted using the THP-1 cell-based inflammation model under static conditions demonstrated that cells cultured with the developed dendrimer NPs-loaded hydrogels were metabolically active and proliferative along the culture time. When their capacity to capture TNF α was evaluated, it was observed that developed systems retained their capacity to neutralize TNF α even after 14 days. Finally, dynamic studies performed with the THP-1 cell-based inflammation model showed that the dendrimer NP-loaded hydrogels maintained an even higher therapeutic effect over time as compared with the static studies, demonstrating the importance of using a physiologically mimetic environment. These relevant results support the successful use of these drug delivery systems in several inflammatory diseases, including RA, in in vivo settings. Overall, the developed approaches which make use of bioengineered nanoparticle-loaded hydrogels can fill the current gaps concerning traditional therapies, providing a more targeted and thus more effective treatment while enabling less frequent administration and, thus, reducing the side effects caused by an overdosing of drugs.The developed approach showed potential to be used as personalized medicine to obtain better therapeutic outcomes and decreased adverse effects. Thus, in the near future, it is expected that the knowledge acquired in the tissue engineering field can be an important tool in the development of clinical medicine.


This study aims to analyze the 100 most cited papers and research trends on stereotactic body radiotherapy (SBRT).We used Web of Science to identify the 100 most frequently cited papers on SBRT on September 29, 2021 and extracted the following data: publication year, source title, country/region, organization, total citations, and average number of citations per year. The research type and research domain were classified independently by the authors. Then we carried out a bibliometric analysis to determine the trends in research on SBRT.These 100 papers were cited a total of 26,540 times, and the median number of citations was 190 . “Stereotactic body radiation therapy for inoperable early stage lung cancer” by Timmerman et al. had the highest number of total citations (1688 times). International Journal of Radiation Oncology, Biology, Physics published the largest number of papers (37 papers), followed by Journal of Clinical Oncology (13 papers). The USA contributed the most papers (67 papers), followed by Canada (18 papers). Primary lung cancer  and oligometastases  were the most cited research areas.To the best of our knowledge, this is the first bibliometric analysis of the most frequently cited papers on SBRT. Our results provide insight into the historical development of SBRT and important advances in its application to cancer treatment. Early-stage non–small-cell lung cancer and oligometastases were the most cited research areas in the top 100 publications on SBRT, and SBRT combined with immunotherapy was a hot topic in the past few years. This study is helpful for researchers to identify the most influential papers and current research hotspots on SBRT. Bibliometric analysis is a method of analyzing data and citation trends for a large body of literature that can help researchers to determine the state of a research area.In the present study, we identified the 100 most frequently cited publications on SBRT and carried out a bibliometric analysis to determine the trends in research on this topic.We used Web of Science to identify the most heavily cited papers on SBRT. We selected the Science Citation Indexing Expanded database and conducted a literature search on September 29, 2021 without restrictions on publication time, language, or type. The search string was as follows: (Title = [stereotactic body OR stereotactic ablative] AND Title = [radiotherapy OR radiation OR radiation-therapy OR irradiation]) OR Title = (SBRT OR SABR). The search results were ranked by the number of times the papers were cited so as to identify the top 100 publications. We then used Web of Science to extract and analyze the following data: publication year, source title, country/region, organization, total citations, and average number of citations per year (12 times the number of citations per month).https://bibliometric.com) and VOSviewer 1.6.14 software were applied to construct the bibliographic coupling network based on journals, countries, co-authorship relations, and keywords so as to implement network visualization analysis.Microsoft Excel software was used for descriptive statistical analysis and visual illustrations. The online platform .The Journal of the American Medical Association (JAMA) in 2010, had the highest number of total citations (1688 times) and second highest average number of citations per year (146.78 times)  and the sixth highest number of total citations (588 times)  published the largest number of papers (37 papers), followed by Journal of Clinical Oncology . These two journals were at the core of the citation network. JAMA had the largest average number of citations per paper . Among the journals that published at least two papers, JCO had the largest average number of citations per paper (390.54 citations).A citation network of journals was constructed based on the average published year Figure 2The authors of the 100 most cited papers were from 17 countries or regions Figure 3A network was constructed of the coauthors of the 100 most heavily cited papers Figure 4The research domains of the 100 most cited papers are shown in The keyword co-occurrence network of the 100 most frequently cited papers is shown in Since radiation therapy has been applied to the treatment of carcinomas, radiation oncologists have attempted to deliver higher radiation doses to target tissues while minimizing toxicity. In the 1970s, after several iterations of radiotherapy technology, SRT was applied to treat brain tumors , Nagata Of the 100 most frequently cited papers on SBRT, 32 focused on early-stage NSCLC. In 2005, Nagata et al. reported the clinical outcomes of a phase 1/2 trial of SBRT in which 45 patients with early-stage NSCLC were treated with a total dose of 48 Gy in four fractions. All tumors showed local response and no adverse events higher than grade 3 were noted  . HoweverThe abovementioned studies demonstrate that SABR is not inferior to surgery for stage I NSCLC, with comparable efficacy and safety for central and peripheral lesions when the doses delivered to normal tissue are strictly limited.Of the top 100 papers on SBRT, 29 focused on oligometastases, which has always been an important SBRT research area . Prior tThe optimal mode of SBRT for oligometastases has yet to be determined. A phase 3 randomized trial showed that the local recurrence rate of single-dose 24 Gy radiotherapy was lower than that 27 Gy SBRT in three fractions, with no significant difference in toxicity . HoweverThe efficacy and safety of SBRT for multiple metastases is unsubstantiated. The phase 1 NRG-BR001 trial yielded preliminary evidence for the safety of SBRT for 3 to 4 metastases or 2 close metastases . HoweverPeter et al. reported that the most common failure after SBRT was distant metastasis . This noTo date, SBRT has been safely used for extracranial oligometastases in a variety of sites including liver, lung, and bone . Phase 3Among the 100 most frequently cited papers on SBRT, nine were focused on primary liver metastases, five on pancreatic metastases, and eight each on prostate cancer and spinal metastases. Following reports of SBRT for the treatment of liver, pancreatic, and prostate carcinoma and spinal metastases in the 2000s, clinical trials have demonstrated the efficacy and safety of SBRT in these malignancies –33.Daniel et al. reported that SBRT was superior to radiofrequency ablation for hepatocellular carcinomas ≥2 cm , while ZThe use of SBRT in pancreatic carcinoma has not been well studied, with most trials focused on optimal dose selection . A recenZhao et al. confirmed the efficacy and safety of SBRT for prostate cancer based on 5-year outcomes . Tsang eConventional EBRT is a standard palliative treatment for spinal metastases; however, complete response rates for pain were as low as 10%–20% . A phaseFurther studies are needed to clarify the applicability of SBRT to the treatment of liver, pancreatic, and prostate carcinoma and spinal metastases and establish the optimal regimens.Only 11 of the 100 most frequently cited papers were published after 2015, of which four were on I-SABR. In 2016, Bernstein et al. summarized the preclinical and clinical evidence for I-SABR in promoting the host antitumor immune response . A phaseResearch on I-SABR has progressed rapidly. A recent phase 2 randomized trial reported that SBRT plus pembrolizumab and trametinib was effective and safe in patients with locally recurrent pancreatic cancer ; and anoI-SABR is an important subject for future research. Larger trials are necessary to investigate the molecular mechanisms underlying the effects of I-SABR, the influence of SABR on the tumor microenvironment, optimal treatment regimen, and criteria for patient selection.IJROBP (37 papers) and JCO (13 papers) published half of the 100 most frequently cited papers. Fewer articles were published in JAMA, The Lancet, and Lancet Oncology, but these often had a high impact. Institutions in the United States made the greatest contribution to SBRT research.R Timmerman, who was the most influential scholar on SBRT, contributed 15 of the top 100 papers. These were published between 2004 to 2017 and were mainly focused on early-stage NSCLC and oligometastases. JY Chang has been the most prolific scholar in recent years, with most papers from his group published after 2013 and pertaining to SBRT vs. surgery for early-stage NSCLC and I-SABR. S Suresh is another new scholar who has published articles on early-stage NSCLC.Early-stage NSCLC and oligometastases are the major focus of research on SBRT. A series of studies have established the standard of SBRT for early-stage NSCLC, and demonstrated that SBRT is not inferior to surgery. Recent studies have provided evidence for the efficacy and safety of SBRT for oligometastases, and larger clinical trials are underway. In the future, research in these two areas will be dominated by large-scale clinical trials.I-SABR has been the most popular research domain in recent years. Immunotherapy combined with SBRT was shown to produce synergistic effects, but many unanswered questions need to be addressed in the future by basic or clinical studies.Based on the most popular keywords, the main interests of researchers are toxicity, SBRT combined with other treatments, and evidence from clinical trials.The number of citations was influenced by many factors such as publication time, research domain, and author. For example, early publications tended to have more citations. Therefore, the number of citations is not a useful metric for identifying the most influential papers. Most of the 100 papers included in our analysis were published before 2015, making it likely that some important new publications were overlooked. We used the number of citations per year to offset the impact of publication time on the most frequently cited papers, and searched important papers published in the last few years and discussed their findings in order to stay abreast of the latest advances in SBRT research.Most of the selected papers focused on early-stage NSCLC or oligometastases, but other domains, while less popular, are also important. As we included different subdivisions in a single bibliometric analysis, it was inevitable that smaller research domains were excluded. However, we discussed the development of each domain in order to determine the research status.We used only the Web of Science search engine to identify publications; therefore, papers in other databases or that were not in English may have been missed, which could have led to bias in citation statistics and the omission of important work.Finally, although we tried to identify the domains and study design of each article, it was difficult to analyze these articles in more detail in a bibliometric analysis.To the best of our knowledge, this is the first bibliometric analysis of the most frequently cited papers on SBRT. Our results provide insight into the historical development of SBRT and important advances in its application to cancer treatment. Early-stage NSCLC and oligometastases were the most cited research areas in the top 100 publications on SBRT, and I-SABR was a hot topic in the past few years. This study is helpful for researchers to identify the most influential papers and current research hotspots on SBRT.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.XZ and YL contributed to the conception of the study. YL analyzed the data. YL, JL, and XC contributed to the review of literatures. YL wrote the manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The therapy of autoimmune rheumatological conditions has undergone significant changes with the introduction of biologic antiinflammatory agents including cytokine antagonists and agents that interfere with the function of T and B cells or those that inhibitintracellular enzymes such as Janus kinase (JAK). Although useful to control inflammation, these agents may be associated with druginduced lung disease, which may be difficult to differentiate from pulmonary disorders caused by the underlying autoimmune diseases.This review aims to provide a description of lung disease, both infectious and non-infectious, that may be induced by the administration ofbiologic anti-inflammatory agents with emphasis on inhibitors of tumour necrosis factor, interleukin-1, interleukin-6 and JAK. The recent introduction of an array of biologic anti-inflammatory agentshas transformed the treatment of numerous rheumatological conditionsand other autoimmune disorders such as inflammatory bowel diseaseand psoriasis. However, despite important clinical benefits, thesebiologic agents may be associated with adverse effects involving multiplesystems, including the respiratory tract. There is increasing evidencethat these biologics may paradoxically induce an autoimmune processand this can be as part of a systemic process such as the inductionof systemic lupus erythematosus, vasculitis, sarcoidosis and antiphospholipid syndrome. There is also an association between thesedrugs and organ-specific auto-immune processes such as interstitiallung disease, uveitis, optic neuritis, peripheral neuropathies, multiplesclerosis and auto-immune hepatitis.The wider use of biological agents also requires constant awarenessof the risks associated with these agents by the treating physicians asexperience is gained by using these drugsLung involvement in patients with rheumatological disordersis common and a wide spectrum of abnormalities may occur.Respiratory physicians need to differentiate lung disease associatedwith the underlying rheumatological condition from adverse effectssecondary to various forms of therapy. It is important to recognisepotential side-effects of therapeutic agents to facilitate the diagnosticprocess. This review will focus on the adverse effects of the mostcommonly used anti-inflammatory biologic disease-modifyingagents on the respiratory system. These agents have been selected andclassified according to their predominant mode of action. Biologicagents are categorised into four classes based on their specific targets:(i) inhibitors of cytokines; (ii) agents targeting signal transductionpathways; (iii) interference with intercellular interactions; and(iv) depletion of inflammatory cells.The classes of biologic agents as well as examples of each are shownin [1] Withnovel applications of highly specific cytokine antagonism, considerableknowledge is being gained about the biology of these molecules, aswell as the impact these targeted therapies may have as treatmentoptions for a variety of inflammatory diseases.[2]Cytokines are small, short-lived protein molecules, which function asimportant mediators of intercellular communication required for anintegrated response of immune and inflammatory processes.The adverse effects of the well-established classes of cytokineinhibitor therapies focusing on tumour necrosis factor (TNF)inhibitors, as well as the interleukin 1 (IL-1) and interleukin 6 (IL6) inhibitors, are described below.[3] TNF-α has a broad spectrum of biological activityand there is considerable evidence showing that either overproductionor inadequate production plays an important role in the pathogenesisof various chronic inflammatory diseases.[3]Tumour necrosis factor-alpha (TNF-α) is produced primarily bymacrophages in response to inflammatory stimuli, and binds to itsreceptors that are found on almost all cells in the body, resulting insignalling required for normal development and functioning of theimmune system.[4] Both forms of TNF-α interact with two types ofreceptors, which are expressed on almost all nucleated cells: TNFreceptor 1 (TNFRI), a 55 kDa receptor also known as the p55 receptorand TNF receptor 2 (TNFRII), a 75 kDa receptor also known as thep75 receptor.[3] Binding of TNF-α to these receptors culminates in avariety of downstream effects including the stimulation of apoptosis,various anti-tumour responses and multiple effects on the immunesystem development and functioning.6]TNF-α exists in two forms: a soluble form and as a precursortransmembrane form, which is also biologically active in cell-to-cell interactions.[4] Five TNF-α inhibitors are available and have beenextensively studied: etanercept, infliximab, adalimumab, certolizumaband golimumab.The preferential receptor for transmembrane TNF-α appears to beTNFRII, which has both shared and opposing effects compared withthose of TNFRI. The differential effect of the various TNF-α inhibitors,particularly on the transmembrane form of TNF-α, may account forthe differences in clinical effect, as well as adverse risk profiles of thedifferent agents.7] In circulation,etanercept will bind sTNF-α and prevent it from interacting with theTNF receptors, resulting in TNF-α antagonism.Etanercept is a soluble recombinant human TNF-α dimeric fusionprotein comprising the extracellular portion of two p75 TNF receptorsfused to the Fc portion of immunoglobulin G (IgG).7]In contrast, adalimumab was the firstfully human mAb against TNF-α to enter clinical trials and as suchis less prone to the risk of the development of antidrug antibodies,enabling more long-term administration.8]Infliximab is a monoclonal antibody (mAb) that neutralises thebiological activity of TNF-α. It is a chimeric antibody comprising bothmurine and human portions.11]Both certolizumab  and golimumab (an IgG1 kappamAb specific for human TNF-α) were developed later and have shownefficacy in neutralising both transmembrane and solubleTNF-α.The adverse effects of TNF-α antagonism involving the respiratorysystem are numerous and may be of a serious nature. These includeboth infectious and non-infectious complications.[12]The role of TNF-α in the normal immune response to infection iswell described and infectious complications resulting from inhibitorsof TNF are therefore to be expected. Evaluating the risk of infectionfrom these agents is difficult, given that they are used in patients whoare already immunocompromised from the underlying disease itselfor the effects of prior or concomitant immunosuppressive therapy.[13] The infection rates appear higher in patients receivingmAbs compared with etanercept and there appears to be a higher rate of infection in patients receiving higher doses.[12] Both common andopportunistic infections have been described.15] The infection riskappears to decrease over time and is highest in the first 6 monthsafter initiation of therapy.[12] Data appear to be conflicting regardingthe rate of serious infections associated with TNF-α inhibitors, withsome studies reporting no increased serious infection rate comparedwith controls, while others show a tendency for serious infections tooccur in line with what is known about the biological role of TNF-αin infection control.16]Infections, especially those involving the respiratory tract, arehowever frequent problems encountered in patients using TNF-αinhibitors.18] TNF-α inhibitorshave been associated with increased susceptibility to tuberculosisinfection, poor granuloma formation, greater rates of tuberculosisreactivation and higher mortality from tuberculosis.18] Screeningfor latent tuberculosis is recommended prior to initiating theseagents. However, the possibility of cutaneous anergy in patients withunderlying autoimmune disease must also be considered.18] A higherproportion of patients receiving TNF-α inhibitors experience extrapulmonary or disseminated tuberculosis compared with infections inthe general population, suggesting that this may be a class effect.18]Owing to the critical role TNF-α plays in the control of tuberculosis,the impact of its inhibition, particularly with the mAbs, on reactivationof tuberculosis is an important consideration.20] Etanercept has been shown to be less effective in themanagement of these conditions. The risk for tuberculosis appearsto be lower with etanercept compared with the use of the mAb formsof TNF inhibition, suggesting that the greater the antigranulomaforming effect, the greater the risk for granulomatous infections.[4]The differential effects of these agents are likely mediatedthrough varying efficacy in eliminating immune cells bearingtransmembrane TNF-α.[4]Infliximab and adalimumab are effective in the managementof various non-infectious granulomatous diseases includingsarcoidosis, granulomatosis with polyangiitis and Crohn’sdisease.22]TNF inhibitors have been shown to be effective in treating both theunderlying inflammatory disease for which they are being used, as wellas providing benefit for any associated interstitial lung disease (ILD).Paradoxically however, these agents themselves may be associatedwith new-onset ILD, as well as exacerbations of pre-existing ILD.[23] Other patterns that have been described includeorganising pneumonia, nonspecific interstitial pneumonia (NSIP),diffuse alveolar haemorrhage (DAH) and lymphocytic interstitialpneumonitis (LIP).23] Studies describing these findings havehowever been criticised for not conclusively demonstrating anabsence of ILD prior to initiation of TNF inhibitors and many patientsusing these agents did so in conjunction with other disease-modifyingantirheumatic drugs (DMARDs) known to produce similar effects inthe lung.[23]The predominant pattern of ILD involvement is a usual interstitialpneumonia (UIP) pattern, which has a high mortality in patientswith prior UIP.[22] A recentreview by Huang et al.[24] highlighted that the use of TNF-α inhibitorsin patients known to have RA-associated ILD may predispose themto more severe symptoms and even death.[24]According to data obtained from the British Society ofRheumatology Biologics Register, the mortality rate from ILD inpatients with rheumatoid arthritis (RA) treated with TNF-α inhibitorswas similar to those treated with conventional DMARDs.25] In almost all instances, the diseaseresolves on withdrawal of therapy, providing compelling evidenceof causality.25] The presence of pulmonary granulomas could alsobe explained by the development of sarcoidosis, hypersensitivitypneumonitis or an unidentified infectious agent, although the latteris unlikely given the continued resolution of disease despite initiationof replacement immunosuppressive regimens after withdrawal ofTNF-α inhibitors.[13]While TNF-α inhibition has been used successfully to treat noninfectious granulomatous diseases, paradoxically TNF-α antagonismhas also been associated with the development of a granulomatous,sarcoid-like disease, which may have both pulmonary andextra-pulmonary effects.[26]This results in an imbalance of the TNF-α-IFN-γ immunoregulatorypathway, which may be responsible for the subsequent development ofgranulomatous change.[23] This could be explained by the differencesin their pharmacokinetic and pharmacodynamic properties, as well astheir effect on transmembrane TNF-α bearing cells.27]The proposed mechanisms for these granulomatous changesinclude an enhanced pro-inflammatory and pro-fibrotic role ofinterferon gamma (IFN-γ) and IL-1 in the absence of TNF-α.23]Accelerated pulmonary rheumatoid nodule formation hasbeen described in patients receiving etanercept. These patientssimultaneously experienced reduced joint symptoms, and resolutionor stabilisation was demonstrated on withdrawal of etanercept, againsuggesting causality.[15] TNF-α is also known to modulate tumourdevelopment and spread; however, cancers arising during therapy withTNF-α inhibitors were not shown to differ by stage at presentation orpost-cancer survival rates. [28]It has been suggested that the use of TNF-α inhibitors may beassociated with an increased risk of lymphoproliferative disorders,especially non-Hodgkin’s lymphoma in patients with RA. It hasbeen shown that the rate of lymphoma is increased in patients withsevere and active RA, the very group of patients that are more likelyto receive these agents.23] Studies have also demonstratedinduction of antiphospholipid antibodies, but thrombotic events havenot been reported in these patients.[23]There is a growing body of literature suggesting that TNF-αantagonists may induce autoimmune diseases, although theappearance of new autoantibodies during therapy is not clearlyunderstood. Most patients who develop autoantibodies remainasymptomatic. However, autoimmune disease may manifest clinicallywith vasculitis, the antisynthetase syndrome, and lupus-like syndromeinvolving the lung and pleura.29]It should be noted that in addition to the adverse respiratoryeffects associated with antagonism of TNF-α, these agents have alsobeen used in clinical trials for treating various respiratory diseasesincluding asthma, chronic obstructive pulmonary disease, sarcoidosisand pulmonary fibrosis. Studies have thus far yielded inconsistentresults.30]IL-1 cytokines are known to function as potent inflammatorymediators, and play a role in normal immune system function, and immune-mediated disease, including cardiovascular, metabolic,neuro-degenerative and neoplastic diseases.[30] Of particularimportance is IL-1-beta (IL-1β), which is an important target forIL-1 antagonism.[31]IL-1 does not refer to a single cytokine, but rather encompasses afamily of molecules, which have an important role in mediatingthe inflammatory response, including the induction of IL-6.The three commercially available IL-1 inhibitors are anakinra,canakinumab and rilonacept.[7] It is structurally similar to endogenous IL-1 receptorantagonist (IL-1Ra), which is a naturally occurring competitiveIL-1R1 antagonist. Apart from its role in the management ofauto-inflammatory diseases, it has also been investigated for themanagement of COVID-19-associated pulmonary complications.[32]Anakinra is a recombinant IL-1 receptor antagonist that binds toIL-1 receptor type 1 (IL-1R1), which is the target receptor for IL-1αand IL-1β.[33]Canakinumab is a human mAb targeting IL-1β, and it was developedfor the treatment of immune disorders including various forms ofinflammatory arthritis such as adult-onset Still’s disease and RA.[34] It is currently used for the treatmentof cryopyrin-associated periodic syndromes (CAPS) in adults andchildren 12 years and older.Rilonacept is a synthetic protein containing the extracellulardomains of IL-1R1 that behaves as a soluble decoy receptor for IL-1β,and binds with high affinity.[7] The risk is surprisingly modest andis likely influenced by additional therapies the patient may be using,the dose used, as well as other underlying co-morbid disease.37]Most notably, the presence of asthma is associated with a greaterrisk of pneumonia and anakinra should be used with caution inasthmatics.35] Reversible neutropenia has also been described andmay contribute to the risk of infection.38]The most serious adverse effect of the IL-1 inhibitors appears to bean increased risk of infection.[39]The Canakinumab Anti-inflammatory Thrombosis Outcome Study(CANTOS) trial,[40] which examined the role of anti-inflammatorytherapy during atherosclerosis, found a reduction in the risk ofcardiovascular events and this was countered by an increase in deathdue to infections (incidence rate in pooled canakinumab group was0.31 events per 100 person-years v. 0.18 events per 100 person-yearsin the placebo group). In addition, the increased risk of infection waslargely found in older patients or those with diabetes.A meta-analysis of anakinra conducted in 2009 showed that therewas an insignificant increase of infections in patients using anakinrav. placebo, including opportunistic infections such as tuberculosis.41]Anakinra should not be initiated in patients with active infectionand should be discontinued in patients who develop severe infection.Caution should also be exercised prior to initiating therapy in patientswith a history of chronic or recurrent infection. The concomitant useof TNF-α antagonists may increase the risk of infection and shouldbe avoided.[42] A severeinflammatory ILD associated with a high mortality has emergedin patients taking IL-1 and IL-6 inhibitors for the management ofsystemic juvenile idiopathic arthritis, suggesting a causal link withthese agents, although the exact relationship remains unknown.45]Anakinra has been associated with drug-induced ILD andpulmonary fibrosis when used in patients with RA.[46] Tocilizumab is an anti-human IL-6 receptor antibody, whichbinds to the IL-6 receptor (IL-6R), thereby interfering with theeffects of cytokine binding. It has been shown to be effective in thetreatment of RA.48] It is also used in the management of juvenileidiopathic arthritis, giant cell arteritis and the cytokine releasesyndrome. In addition to anakinra, tocilizumab is currently beinginvestigated for the management of COVID-19-associated pulmonarycomplications.[49]IL-6 is a pleiotropic cytokine with multiple biological effects, and playsan important role in many immune and inflammatory processes.51] but variousadverse effects have been attributed to it including infections,cytopenia and neutropenia, hyperlipidaemia and abnormal liverenzymes.54] The neutropenia associated with IL-6R inhibitiondoes not appear to be associated with a risk of severe infection.52]Tocilizumab has a good long-term safety profile,[50] The USA[55] has issued blackbox warnings regarding infection risk associated with the use oftocilizumab, noting an increased risk of serious and potentially fatalinfections, as well as an increased risk of tuberculosis (pulmonary andextra-pulmonary). Tocilizumab may in addition mask the symptomsof infection and care should be taken not to overlook a patientpresenting with mild symptoms.[52]The respiratory effects of tocilizumab are mainly mild in naturewith upper respiratory tract infections, bronchitis, cough, dyspnoeaand nasopharyngitis described.56] Most of these occurred in patients on concomitantDMARDs therapy. Conditions described include new-onset ILD,idiopathic pulmonary fibrosis and allergic pneumonitis. Severeexacerbation of RA-associated ILD has also been described.56] Casereports have been published describing organising pneumonia, acutepneumonitis, as well as exacerbation of combined pulmonary fibrosisand emphysema syndrome associated with use of tocilizumab.59]Randomised control trials evaluating the use of tocilizumab in RAhave revealed non-infectious pulmonary adverse effects in a minorityof patients.61] They are classified as targeted synthetic DMARDs.[62]Tyrosine kinases are intracellular enzymes that transmit signalsby transferring phosphate groups to tyrosine residues oncytoplasmic proteins or the intracellular domains of transmembranereceptors.[63] There are four JAK isoformsin humans: JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2), whichfunction in pairs and whose activity is related to different cytokinereceptors.[64]The mechanism of action of Janus kinase (JAK) inhibitors is basedon inhibition of activity of one or more JAK isoforms, thus impedingcertain pathways, particularly the JAK-signal transducer and activatorof transcription (JAK-STAT) pathway.65]The first JAK inhibitor approved by the FDA for RA was tofacitinib,which inhibits JAK1 and JAK3 resulting in the inhibition of IFN-γand IL- 6.Tofacitinib is indicated for the treatment of adult patients withmoderate to severe active RA, who have had an inadequate responseor intolerance to methotrexate. It may be used as monotherapy or incombination with methotrexate or other non-biologic DMARDs. Usein combination with biologic DMARDs or potent immunosuppressantssuch as azathioprine or cyclosporine is not recommended.[66] The most frequentlyreported adverse events that led to temporary discontinuation or dosereduction of tofacitinib were nasopharyngitis and herpes zoster.68]The reactivation of varicella-zoster virus is a concern with the useof tofacitinib as disseminated disease may be fatal and vaccinationshould be considered prior to the initiation of biologic therapy inpatients with RA older than 60 years.[69]During the ORAL sequel long-term extension study that evaluatedthe efficacy and safety of tofacitinib 5 mg and 10 mg twice daily, the observed incidence rates for serious infections, deep vein thrombosisand pulmonary embolism were similar to those seen with TNFinhibitors and other biologic DMARDs.[68] The risk of tuberculosis in patients receiving tofacitinibtreatment varies depending on the prevalence of tuberculosis indifferent geographic areas. Incidence rates are comparable with datafor biologic DMARDs[66] and screening for latent tuberculosis isadvised prior to starting treatment with tofacitinib.Patients with RA are at greater risk of serious infections, includingtuberculosis.[70] with tuberculosisand other mycobacterial infections, cryptococcosis, histoplasmosis,pneumocystis, cytomegalovirus, BK virus infection and listeriosis havealso been reported.The FDA issued a warning regarding serious infections leading tohospitalisation or death with the use of tofacitinib. The most commonserious respiratory infection was pneumonia[71] that shares the sameinfection risks as tofacitinib. One particular safety concern was thepossible increased risk of thromboembolic events related to the useof baricitinib.[72] The European Medicine Agency (EMA) has issuedwarnings to use baricitinib and tofacitinib with caution in patientswith risk factors for thromboembolic disease.[73]Baricitinib is a selective JAK1/JAK2 inhibitor[74] and has demonstrateda favourable risk-benefit ratio in patients with an inadequate responseto biologic DMARDs at a dose of 15 mg daily.[74] The FDA issueda warning for upadacitinib similar to that for baricitinib and venousthromboembolic events occurred in patients with additional riskfactors.[75]Upadacitinib is a selective JAK1 inhibitor,[76] Abatacept, a homologue of CD28,competitively binds to CD80/CD86 resulting in suppression of T cellactivation.[76] Pulmonary hypertension and acute respiratory failurehave been reported in patients receiving abatacept.78]Antigen presenting cells (APCs) such as dendritic cells andmacrophages present antigens to T cells, priming their activation.Optimal activation of T cells requires a co-stimulatory signalprovided by the interaction between CD28 on the surface of T cellsand CD80/CD86 on APCs.[79]Patients receiving rituximab rarely develop an organising pneumonia.Another rare, but potentially fatal, complication that has been reportedwith rituximab is the new onset of an ILD.[80] This complication appearsto be more common in middle-aged males, presents as bilateralinfiltrates on computed tomography (CT) imaging of the lungs, andis associated with hypoxaemia and a restrictive deficit on pulmonaryfunction testing.[80] A summary of the adverse effects of biologic agentson the respiratory system are shown in Rituximab is an anti-CD20 monoclonal antibody that targetsB cells, causing their depletion via multiple mechanisms includingcomplement-mediated and antibody-dependent cytotoxicity.[81]A chest CT study of 332 patients with RA on long-term biologictherapy showed the following forms of respiratory involvement:ILD in 29 patientsairway disease in 76 patients which included bronchial wallthickening, bronchiectasis, bronchiolitis, air trapping and atelectasisco-existing interstitial disease with airway disease in 6 patientsno CT abnormalities of the lungs in 221 patients.[82] This usually requires pulmonary function testing, chestradiographs and high-resolution chest CT. [82] If the diagnosis is stillin doubt, a bronchoscopy with lavage and/or transbronchial biopsiesshould be performed.When evaluating patients with possible drug-induced lung disease,it is important to obtain a thorough history relating to the timingof initiation of medications and the onset of symptoms. Clinicalexamination may yield signs supporting an allergic reaction, but thisis not always the case. Importantly, the respiratory physician mayneed to differentiate the underlying lung disease from other causessuch as infections, malignancy, cardiac failure and adverse effects ofdrugs used.Biologic anti-inflammatory agents used for the treatment ofrheumatological disorders may cause respiratory infections andnon-infectious pulmonary disease. Respiratory physicians need tobe cognisant of the adverse effects of biologic agents when evaluatingrheumatology patients with pulmonary abnormalities.


Scientific research has an essential role in building up the basics of public health and clinical care. As a developing country, Syria has a limited contribution to scientific research. Besides, the current Syrian crisis has inflicted severe damage to the infrastructure of the country, which, in turn, negatively affected the scientific sphere. This study examines the scientific publishing in Syria from 2011 until 2020. It aims to define the real and deep reasons for the slow-down in publication to get over them, push Syria to keep track of the latest updates, and take its place in scientific research.We conducted a bibliometric analysis of articles published in (PubMed and Scopus) Databases from 1/1/2011 until 26/12/2020, using the following search terms ((“Syrian Arab Republic”) OR (Syria) OR (Syrian)) limiting the search to (Affiliation) fields.Syrian medical, dental and pharmaceutical institutions published 1403 papers from 2011 until 2020. There were only 55 papers in 2011, and a peak with 180 papers in 2018. Besides, publications in the last 4 years were 1.135 times more than publications in the last 6 years.. Accordingly, we recommend enhancing research skills, paying more attention to the quality of researches, and holding research workshops and Evidence Based conferences to enhance the scientific endeavor.We noticed a peak in quantity of Syrian medical, dental and pharmaceutical publications in the last decade •We conducted a novel bibliometric analysis to evaluate the research productivity in the current crisis.•Our Results show that papers in Medical, Dental and Pharmaceutical field have been increasing every year.•Otherwise, there is a paucity in publishing compared with equivalent countries.•It takes Syria as an example of poor countries' research interest so it may inspire those have similar circumstances. Publications and researches have an essential role in clinical care, teaching, and training .Nowadays, the scientific field is witnessing a massive acceleration in publishing, with increased quality and quantity of published papers. This can be explained by the well-known role of scientific research in expanding knowledge, supporting job progression, and providing professional education as a form of continuous medical education (CME) ,2.There are many acceptable methods for evaluating the scientific level of individuals and countries. One of them – which was published by South Korean plastic surgeons-considers the number of published papers as an evidence of quantitative evaluation of researches . TherefoIn recent years, Syria become the spot of attention after the devastating war has caused severe damage to the infrastructures, and serious threats to the functioning of educational, economic, and social institutions . Even beDespite obstacles and challenges that faced the research process, Syrian students-medical students in particular-are attempting to keep up with the latest medical research findings and EBM .We conducted a novel bibliometric analysis to evaluate the research productivity in the current crisis which takes a comprehensive look of the scientific publishing in Syria from 2011 until 2020. It aims to define the real reasons behind the current situation in order to get over them, push Syria to keep looking for the latest updates, and take place in scientific research as a major contributor.22.1In this study, we conducted a systematic electronic database search for relevant studies from 1/1/2011 until 26/12/2020 in PubMed and Scopus data bases, using the following search terms: ((“Syrian Arab Republic”) OR (Syria) OR (Syrian)) limiting the search to (Affiliation) field.2.2We included papers that at least one of their authors was affiliated to one of Syrian medical, dental, or pharmaceutical institutions .We excluded all other fields and articles that their authors did not affiliate to medical, dental, or pharmaceutical institutions. There were no restrictions on study design or language.2.3One author (MZBA) developed the data extraction using Endnote X8 and formatted it using Microsoft Excel. The extracted data included year, date, title, abstract, and authors' names and their affiliations. We removed duplicated data for the combined database, and six authors  scanned the papers according to our inclusion and exclusion criteria to select appropriate articles. We included 1403 articles for bibliometric analysis, and manually defined: city , field  depending on article's field, research type  and specialties for medical articles. Every marked phase was done twice by two different authors.We did descriptive statistics (number of publication and percentage) using Microsoft Excel 2019 to present our Results in tables and figures.3From initial search on two databases depending on our search terms, we found 3686 papers, and after removing the duplicated studies and manual search exclusions, we ended up with only 1403 papers that met our inclusion criteria and then we included them in the analysis .Fig. 1FlWe found that Syrian medical, dental and pharmaceutical institutions published 1403 papers in total, in the period from 2011 until 26/12/2020.The number of published papers has been increasing by years. It started in 2011 with only 55 published papers and then reached 279 in 26/12/2020 .Table 1NThe significant peak was in 2018 with 180 papers. We noticed that the last 4 years' publications counted 1.135 times more than the last 6 years' publications .Fig. 2FrOut of 1403 papers; 828 (59.0%) were in the medical field, while 282 (20.1%) were in dentistry, and 293 (20.9%) papers were in pharmacy .Fig. 3BaDamascus University and its affiliated institutions published 58% of total papers, which is the highest number of publications with 863 papers. Aleppo University and its affiliated institutions came next with 349 papers, while Latakia University and its affiliated institutions took the third place with 132 papers. Hama, Homs and their affiliated institutions participated in 34 and 31 papers, respectively. Dara'a University and its affiliated institutions took the last place with only 19 papers .Table 2NOut of 1403 papers, 728 were carried out in partnership with external institutions.The prevailing study type was primary observational with 795 (56.7%) papers, followed by the secondary type with 277 (19.7%) papers. While primary interventional type counted 131 (9.3%) papers, basic scientific researches included 200 papers: 151(10.8%) of them were conducted in vitro and the other 49 (3.5%) researches were conducted on animals .Fig. 4PiWe have also classified the studies that were in medical field into their specialties .Fig. 5PiWe found out that epidemiology achieved the highest percentage with 88 papers, oncology was in the second with 81 papers, and gastroenterology was in the third place with 71 papers. General surgery published the highest number of surgical papers with 57 papers; in contrast, both thoracic and plastic surgery had the lowest number of surgical papers, only 6 papers for each one. There were 32 publications in psychology, and only 8 papers were about COVID-19 pandemic. Forensic medicine had the lowest number of papers in general, with only one published paper. We also found out that 31 papers interested in general medical education .Fig. 6Nu4This study provides a comprehensive review of the research productivity and health care practitioners’ situation in Syria during the last decade.The Results showed paucity of publishing -in comparison with equivalent countries-, with ascending increase since 2011 and a slight peak in the last three years.Syria, in total , takes the 15th position of 22 Arab countries, and the 14th position out of 16 Middle Eastern countries, with 113 H-index and 8010 documents. While Jordan takes the 7th position of both Arab and Middle Eastern Countries in documents’ number with 176 H-index and 45884 documents, and Tunisia takes the third position of Arab Countries with 193 H-index and 94962 documents according to Scimago Journal & Country Rank [Paucity can be explained by many reasons such as: lack of mentors, being a low-income country, and having the current crisis. The crisis -which started in 2011- has been accompanied with economic and scientific sanctions, devastating social and demographic situations that constrained connecting with trials' participants, less financial resources, and immense stress on medical institutions ,9. At thMore reasons can be mentioned: using Arabic as the official language in education -while English is the most widely used in research-, poor internet connection, limited participation and lack of training, important gap in medical knowledge in Syria, and insufficient motivation for medical students ,9,13,14.As for the peak, it was mainly caused by the increased awareness among physicians and medical students , which gAccording to our data: Damascus affiliations reached the highest number of publications, because of having more experienced seniors and mentors, and more encouraging scientific atmosphere . On the Medicine colleges published almost 828 papers, whereas dentistry and pharmacy colleges together published only 575 papers. This can be explained by the orientation of medical students toward evidence-based medicine (EBM) practice and continuous medical education, and updating educational curricula by adding EBM subject to the medical curricula.The dominant proportion of these studies were observational (56.7%), which need less time, less funding and fewer experiments . SecondaThe leading specialties were: epidemiology, oncology, and gastroenterology respectively. The priority of these three specialties is caused by having more active mentors, and many external contributions, especially by Tobacco Center affiliated researchers . Psychia4.1We did not include papers that their authors affiliated to institutions out of service due to Syrian war. We also did not do manual search for papers published locally in libraries. Moreover, included studies were evaluated depending on number only, neither citations nor quality of journals was considered.4.2As previously mentioned, there is an apparent difference in publication between governorates and faculties.Alhamid et al. (2017) proposed solutions to overcome the obstacles in research productivity among medical students such as: holding research workshops, improving research skills by expert members, and adding new opportunities in school curriculum .So, we recommend holding annual evidence based medical, dental, and pharmaceutical conferences to keep up with the latest updates, and to enhance internal collaborations to share the available expertise equally among governorates.To increase the general scientific outcome, seniors and mentors should give students the chance to collaborate in their latest researches, as the recent studies have shown that the early young researchers are involved in publication, the more they achieve in future .We advise Syrian students to improve their academic English language because it has a higher number of citations, readings and it is accessible to a larger audience ,24. Like5We noticed a peak in quantity of Syrian medical, dental and pharmaceutical publications in the last decade. Depending on our data, we expect this number of publications to increase in the next few years. Accordingly, we recommend enhancing research skills, paying more attention to the quality of researches, and holding research workshops and Evidence Based conferences to enhance the scientific endeavor.There are no funding sources.There is no need.There is no source of funding.MZBA: contributed to project administration, literature search, study design, data analysis, data interpretation, writing, reviewing, and editing. Both HS and RB: contributed to data analysis, data interpretation, literature search, writing, reviewing, and editing. AG: contributed to data analysis, data interpretation, software, study design, and creating figures. MNS: contributed to data analysis, data interpretation, and validation. LHK: contributed to data analysis, data interpretation, literature search, and writing. AG: contributed to supervision, data interpretation, planning, reviewing, and editing. All authors read and approved the final manuscript.This study does not have patients.1.Name of the registry: not applicable2.Unique Identifying number or registration ID: not applicable3.Hyperlink to your specific registration (must be publicly accessible and will be checked): not applicableMuhamad Zakaria Brimo Alsaman.Not applicable.All data generated or analyzed during this study are included in this article.The authors declare that they have no competing interests.


Knockdown of PMCA4 in human umbilical vein endothelial cells (HUVECs) leads to an increase in tubule formation and upregulation of regulator of Calcineurin 1 (RCAN1.4) signaling, a downstream effector of the calcineurin/nuclear factor of T-cells (NFAT) pathway i i  which ha [Ca2+]i . Recentl [Ca2+]i . HoweverATP2B1 (si-PMCA1), ATP2B4 (si-PMCA4), or non-targeting (si-NT) scrambled control (Dharmacon) in HUVECs (TCS Cellworks) was achieved using published methods  and The 10.13039/501100000770University of Manchester.This work was supported by the None.