Moreover, U0126, a specific inhibitor of ERK, exhibited the same effects with ALR-S on a thrombotic process in FeCl3-injured HAECs, suggesting the thrombotic role of the ERK/NF-κB pathway and the antithrombotic role of blocking the ERK/NF-κB pathway by ALR-S. In conclusion, our study revealed that the ERK/NF-κB pathway is a potential therapeutic target in arterial thrombosis and that ALR-S has good characteristics for the cure of arterial thrombosis via regulating the ERK/NF-κB signaling pathway.Saccharides from Arctium lappa. L. root (ALR-S) is a high-purity fructosaccharide separated from the medicinal plant Arctium lappa. L. root. These compounds showed many pharmacological effects in previous studies. In the present study, the antithrombotic effects of ALR-S in arterial thrombosis via inhibiting platelet adhesion and rebalancing thrombotic and antithrombotic factor expression and secretion were found in rats and human aortic endothelial cells (HAECs). This study also showed that inhibition of oxidative stress (OS), which is closely involved in the expression of coagulation- and thrombosis-related proteins, was involved in the antithrombotic effects of ALR-S. Furthermore, studies using FeCl The endothelium is responsible for many functions, such as the control of vascular permeability and tone, maintenance of vessel integrity, regulation of vascular inflammation, and prevention of thrombosis , 2. Endorombosis , 4.2 can relax blood vessels and inhibit platelet aggregation. The FXa-TFPI-FVIIa-TF tetramer was combined by TFPI with tissue factor (TF), then coagulated factors VII(FVII) and X(FX), thus inactivating tissue factor pathway and inhibiting coagulation reaction. TM can reversibly bind to the endothelial cell protein C receptor and reduce the level of free thrombin, thus preventing thrombin from cleaving fibrinogen or activating platelets. After endothelial cell injury, TM on the endothelial cell surface is released into the blood, which reduces the thrombin binding rate and the antithrombotic effect of endothelial cells [PGIal cells . Therefoal cells .2, ADP, and other substances to further promote platelet adhesion, aggregation, and thrombosis.When VECs are damaged, they slough off, leading to exposure of subendothelial tissue and potentially the release of a large amount of TF. The TF complex with FVII in the blood activates the exogenous coagulation pathway to produce thrombin. Thrombin activates XIII and causes fibrin to form blood clots. In addition, when VECs are injured, a large amount of vWF is released to stimulate platelet activation and produce platelet aggregation. At the same time, activated platelets release TXAκB, AP-1, and/or other signaling pathways through extracellular regulated protein kinase 1/2 (ERK1/2). These activation events cause lipid peroxidation, which injures VECs and promotes the overexpression of thrombosis factors, inflammatory factors, and adhesion factors. These factors can further injure VECs, cause thrombosis, and induce cardiovascular disease [After vascular injury, VECs, vascular smooth muscle cells, leukocytes, fibroblasts, and platelets are important sources of ROS, which can regulate platelet activation , 8, trig disease , 16.In addition to treating thrombosis with antithrombotic agents, there is increasing interest in the use of natural food products and biologically active ingredients for the prevention and treatment of thrombosis. ALR-S contain fructosaccharides that can be separated at high purity. These polysaccharides have many pharmacological effects, such as anti-inflammatory and immune regulatory activity , 18; ant3-induced rat arterial thrombosis model and a human aortic endothelial cell (HAEC) injury model to explore whether ALR-S can inhibit excessive OS and regulate the expression of thrombosis-associated factors through the ERK/NF-κB signaling pathway, leading to antithrombotic effects.In this study, we used a FeClALR-S was extracted and purified from Arctium lappa L. root in our laboratory. The dry ALR powder was macerated in 80% alcohol at 60°C for 6 h and refluxed in aether petrolei at 45°C for 1.5 h to remove soluble contaminants. The residues were vacuum-dried and were macerated in distilled water at 95°C for 3 h; then, the filtrate was concentrated. The Sevag method was used to remove proteins . The depMale Sprague-Dawley (SD) rats weighing 180 g to 220 g were purchased from the Animal Center of Dalian Medical University . The rats were housed under 12 h light/12 h dark cycles at 21 ± 3°C and 55 ± 5% relative humidity.n = 10): (1) control group , (2) FeCl3-induced arterial thrombosis model group , (3) low-dose ALR-S group , (4) middle-dose ALR-S group , (5) high-dose ALR-S group , and (5) heparin group .Sixty male SD rats were randomly divided into six groups . Different concentrations of ALR-S, heparin, or normal saline were injected, respectively, into the right femoral vein at 2 ml/kg for each group. Twenty minutes later, the common carotid artery thrombosis model was prepared according to Kurz et al.'s method , 26. In After 10 minutes, blood samples were collected from the rats via the abdominal aorta. The blood was anticoagulated by 3.8% sodium citrate and then centrifuged  to obtain plasma. The supernatant (plasma) was stored at -80°C.The same length of the right common carotid artery segment containing the thrombus of all rats was dissected and weighed. Then, the arterial thrombus were carefully removed and the blood vessels were stored at -80°C.v/v) fetal bovine serum , 4.5 g/l glucose, 4.0 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in an incubator with 5% CO2.Human aortic endothelial cells (HAECs) were purchased from Shanghai Institute of Biochemistry and Cell Biology  and cultured in DMEM  with 10% (3), (3) low-dose ALR-S group (0.1 mg/ml ALR-S), (4) middle-dose ALR-S group (0.2 mg/ml ALR-S), and (5) high-dose ALR-S group (0.4 mg/ml ALR-S). HAECs (1 × 105) were seeded on six-well plates and pretreated at 90% confluence with different concentrations of ALR-S or vehicle for 4 h and then exposed to 0.4 mmol/l FeCl3 for 2 h.HAECs were divided into five groups: (1) control group (serum-free DMEM), (2) model group (0.4 mmol/l FeCl3), (3) ALR-S (0.4 mg/ml ALR-S), and (4) U0126 (10 μmol/l U0126). HAECs (1 × 105) were seeded on six-well plates and pretreated at 90% confluence with ALR-S or U0126 for 4 h and then stimulated with 0.4 mmol/l FeCl3 for 2 h.For ERK1/2 inhibition study, HAECs were divided into five groups: (1) control group (serum-free DMEM), (2) model group (0.4 mmol/l FeCl2O2), and cellular SOD, LDH, glutathione (GSH), and nitric oxide (NO) in culture medium in HAECs were detected with kits .Plasma malondialdehyde (MDA), superoxide dismutase (SOD), lactate dehydrogenase (LDH), hydrogen peroxide (HPlatelet-rich plasma (PRP), platelet-poor plasma (PPP), and washed platelets were prepared as described previously , 28. BriμM) for 30 min at 37°C in the dark. The relative fluorescence units of the platelets were measured by a fluorescence microplate reader  using excitation at 488 nm and emission at 525 nm.Platelets ROS were detected using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe according to the instructions in the detection kit . Briefly, the platelets were incubated with the probe (5 μM) for 30 min at 37°C in the dark. Fluorescence in each group was measured by flow cytometry  using excitation at 488 nm and emission at 525 nm. Cell fluorescence was also observed by inverted fluorescence microscopy.Intracellular ROS in HAECs were detected with the same detection kit as μg) of protein were separated by 8%~15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the target proteins were transferred to polyvinylidene difluoride (PVDF) membranes . The membranes were blocked with 5% milk for 60~90 min at room temperature and were incubated at 4°C overnight accordingly with the primary antibodies specific for TM , TFPI , TF, FPA , PECAM-1, p-ERK , ERK, NOX4, P65, lamin B, and GAPDH .A commercial protein isolation kit  was used for harvesting and extracting the nuclear proteins, cytoplasmic proteins, and total protein in tissues or HAECs. Equal loading quantities . Emitted light was measured by a multispectral imaging system , and a Gel-Pro Analyzer  was used for analyzing bands of each group.2, 6-keto-PGF1α, vWF, and PECAM-1 in the supernatant were detected according to the instructions of the ELISA kits . The OD values were measured at 450 nm using a microplate reader . The contents of TXB2, 6-keto-PGF1α, vWF, and PECAM-1 were calculated according to the standard curve.The supernatant was obtained from centrifuged rat plasma  or from cell culture medium . The contents of TXBHAECs were washed with PBS, collected by centrifugation , and placed in 2.5% glutaraldehyde overnight at 4°C. The cells were rinsed three times with 0.1 M phosphate buffer for 15 min each, fixed for 1 hour with 1% osmium acid, and rinsed again as before. The cells were blocked with 1% uranium acetate for 2 hours and then dehydrated with an acetone gradient  for 15 minutes at each concentration and twice with 100% acetone for 10 minutes each. The cells were immersed consecutively in the following solutions: acetone : embedding solution (1 : 1) for 2 hours at 37°C; acetone : embedding solution (1 : 4) overnight at 37°C; and pure embedding solution for 2 hours at 45°C. Finally, the blocks of cells were sectioned and observed.8 platelets/ml.PRP, PPP, and washed platelets were prepared as described in μM BCECF probe  for 1 h. The fluorescence microplate reader  was used for detecting fluorescence in each group.Platelet adhesion test was performed as described previously . HAECs wT-test, one-way ANOVA, and the Student-Newman-Keuls (SNK) method were used for data analysis. p < 0.05 referred that the difference was statistically significant.Statistical analysis was performed by IBM SPSS 21.0 . All values are presented as the mean ± SD. Compared with the control group, the model group showed a significant increase in thrombus weight . The ALR2, 6-keto-prostaglandin Fα1 (6-keto-PGFα1), vWF, PECAM-1, TM, TFPI, TF, and FPA, was significantly improved by different doses of ALR-S  and ischemic heart disease (IHD), even acute myocardial infarction, which are often life-threatening. Polysaccharides, one of the most important active ingredients of traditional Chinese medicine decoction, have attracted more and more attention. ALR-S, extracted from a traditional medicinal plant, showed multiple pharmacological activities. However, the effects and mechanism of ALR-S on thrombosis have not been reported. The present study demonstrated that ALR-S effectively alleviated arterial thrombosis by rebalancing thrombotic/antithrombotic factors and inhibiting platelet adhesion. The underling mechanism is related to the inhibition of the OS and ERK/NF-3 is widely used to establish a thrombus model for scientific researches in vivo, and previous reports indicated that FeCl3-mediated thrombosis involves inducing VEC oxidative injury and platelet adhesion [3-induced arterial thrombosis model and FeCl3-induced HAEC injury model were used, respectively, in in vivo and in vitro experiments to investigate the effects and mechanism of ALR-S on arterial thrombosis.VECs are very important to maintain blood homeostasis. VEC dysfunction caused by various stimuli promotes thrombosis in the body. Oxidative stress is a common and important cause of endothelial dysfunction. FeCladhesion , 36. The3-induced thrombus formation [2 and PECAM-1 are secreted on stimuli to inhibit platelet aggregation. This study showed that ALR-S significantly inhibited FeCl3-induced arterial thrombosis in rats by rebalancing the abovementioned thrombotic/antithrombotic factors, including vWF, TF, TXB2 (the metabolite of TXA2), 6-keto-PGF1α (the metabolite of PGI2), TM, TFPI, and PECAM-1. In vitro studies also showed that ALR-S inhibited platelet adhesion to HAECs and improved thrombosis-relative imbalance  induced by FeCl3 injury in HAECs. These results suggest that ALR-S exert the antithrombotic effects mainly by protecting VECs and improving VEC dysfunction.VECs are extensively involved in blood homeostasis by synthesis and secretion of many active substances to regulate platelet activation, coagulation, and fibrinolysis. VEC damage is an initial stage of FeClormation . TF is rormation , 38. TFPormation . vWF, stormation . TM inhi2O2, LDH, and MDA and the expression of endothelial NOX4 and upregulated SOD activities in FeCl3-induced artery thrombosis in rats. These results indicated that ALR-S inhibited OS via regulating the relative enzymes, as evidenced by decreased ROS production and lipid peroxidation. In vitro studies showed that ALR-S inhibited OS in HAECs by reversing the FeCl3-induced changes in the production of ROS, the activities of GSH and SOD, and the expression of endothelial NOX4. These results in in vivo and in vitro investigations suggest that ALR-S exert the role of protecting VECs and improving VEC dysfunction mainly by inhibiting endothelial OS.Endothelial OS state was examined further to investigate the mechanism of ALR-S on arterial thrombosis. OS plays an important role in the early regulation of thrombosis by activating VECs. Activated VECs then produce a large amount of ROS, leading to a vicious circle of EC dysfunction . The preκB is then activated by p-ERK and translocates into the nucleus to bind to specific DNA fragments and regulate gene and protein expression [κB pathway. U0126, the accepted specific inhibitor of ERK, is frequently used to block the ERK/NF-κB pathway. Here, U0126 was used for further clarifying the role of the ERK/NF-κB pathway in mediating ALR-S action. As expected, U0126 demonstrated the same effects with ALR-S on FeCl3-injured HAECs, including inhibiting platelet adhesion and rebalancing the factors of TF, TFPI, PECAM-1, and TM. Moreover, both U0126 and ALR-S inhibited the nuclear translocation of NF-κB, as evidenced by the decreased expression of nuclear P65. These results suggest that ALR-S may inhibit the ERK/NF-κB pathway and then the NF-κB translocation into the nucleus, leading to the downregulated expression of thrombotic factors and upregulated expression of antithrombotic factors.As previously reported, iron can induce cell oxidative damage and produce higher ROS levels to activate ERK1/2 phosphorylation. NF-pression –44, leadκB pathway in VECs, which may lead to the VEC protection and the alleviation of arterial thrombosis. The study also indicated that the ERK/NF-κB pathway may be a potential therapeutic target in arterial thrombosis and ALR-S could have a positive role in arterial thrombosis and related cardiovascular diseases.In conclusion, our research revealed the effects and mechanism of ALR-S against arterial thrombosis in vitro and in vivo. ALR-S inhibited platelet adhesion to VECs and rebalanced thrombotic/antithrombotic factor expression and secretion by inhibiting the oxidative stress and ERK/NF-Further studies are needed to clarify the regulatory mechanism of ALR-S on platelet adhesion and aggregation via effects on platelets and effects on the interaction of VECs and platelets in arterial thrombosis.


Several studies have confirmed that mobilizing bone marrow-derived stem cells (BMSCs) ameliorates renal function loss following cisplatin-induced acute kidney injury (AKI). The aim of this study was to explore whether the combination of granulocyte-colony stimulating factor (G-CSF) and plerixafor (AMD3100) exerts beneficial effects on renal function recovery in a model of cisplatin-induced nephrotoxicity.C57BL/6J mice received intraperitoneal injections of G-CSF (200 μg/kg/day) for 5 consecutive days. On the day of the last injection, the mice received a single subcutaneous dose of AMD3100 (5 mg/kg) 1 h before cisplatin 20 mg/kg injection. Ninety-six hours after cisplatin injection, the mice were euthanized, and blood and tissue samples were collected to assess renal function and tissue damage. Cell mobilization was assessed by flow cytometry (FCM).P < 0.05).Mice pretreated with G-CSF/AMD3100 exhibited longer survival and lower serum creatinine and blood urea nitrogen (BUN) levels than mice treated with only G-CSF or saline. Combinatorial G-CSF/AMD3100 treatment attenuated tissue injury and cell death, enhanced cell regeneration, and mobilized a higher number of stem cells in the peripheral blood than G-CSF or saline treatment. Furthermore, the mRNA expression of proinflammatory factors was lower, whereas that of anti-inflammatory factors was higher, in the G-CSF/AMD3100 group than in the G-CSF or saline group (all These results suggest that combinatorial G-CSF/AMD3100 therapy mobilizes BMSCs to accelerate improvements in renal functions and prevent cisplatin-induced renal tubular injury. This combinatorial therapy may represent a new therapeutic option for the treatment of AKI and should be further investigated in the future. The two most common causes of acute kidney injury (AKI) are ischaemia/reperfusion (I/R) injury and nephrotoxic agent exposure . Cisplat+ stem cells from the bone marrow into the peripheral blood [Stem cell therapy has attracted great interest as a treatment for AKI. Numerous studies have demonstrated that stem cells can repair damaged tubular cells and attenuate cisplatin-induced AKI , 4. Boneal blood . It exeral blood  and myocal blood . Howeveral blood , 13. Theal blood . Howeveral blood . Other sal blood , 17. TheBased on the above findings, we speculated that combinatorial therapy with G-CSF/AMD3100 may be more effective in recruiting BMSCs into renal tissue to repair and ameliorate cisplatin-induced AKI than either therapy alone. In addition, the mechanisms underlying the renoprotective effects of BMSCs require further elucidation. In the present study, we first demonstrated that G-CSF/AMD3100 mobilizes BMSCs into the peripheral blood and prevents cisplatin-induced AKI in mice. Second, we demonstrated that mobilization of BMSCs by G-CSF/AMD3100 decreases tubular cell apoptosis and promotes proliferation. The protective effects of this combination therapy are associated with a reduction in the levels of putative biomarkers of renal injury and inflammation.All experiments were performed using C57BL/6J mice and were conducted according to the Huazhong University of Science and Technology Guide for the Care and Use of Laboratory Animals. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology, Wuhan, China.Experiments were performed with 7- to 8-week-old male C57BL/6 mice (weighing 20–25 g) purchased from Hua Fukang Company . The animals were housed according to the institutional animal research guidelines and were maintained under constant temperature, a 12-h light/dark cycle, and 50 ± 5% humidity with standard mouse chow and water available ad libitum.n = 12), cisplatin  + saline group (n = 12), Cis + G-CSF  group (n = 12), and Cis + G-CSF + AMD3100  group (n = 12). G-CSF treatment was administered over five consecutive days, and AMD3100 was administered 60 min before AKI induction. At 96 h after the last cisplatin injection, the mice were euthanized. Blood and tissue samples were collected to assess renal function and tissue damage.The experimental design is shown in Fig. n = 6 per group). The mice were euthanized 96 h after treatment, and peripheral blood samples were collected for subsequent flow cytometry (FCM) analyses.To confirm bone marrow stem cell mobilization following G-CSF/AMD3100 administration, mice were irradiated with 2 separate doses of 4.5 Gy of whole-body γ-radiation at a 2-h interval to induce bone marrow ablation (BMA). Mice were randomly assigned to the following groups: the irradiation + saline group, irradiation + G-CSF/AMD3100 group, non-irradiation + saline group, and non-irradiation + G-CSF/AMD3100 group. Beginning 1 day after irradiation, G-CSF/AMD3100 was administered as described in the above-mentioned treatment protocol. Control mice that were not subjected to irradiation were injected with an equivalent dose of saline . The remaining cells were labelled with FITC-labelled rat anti-mouse CD34 , APC-labelled rat anti-mouse CD133 , FITC-labelled rat anti-mouse CD44 , PE-labelled rat anti-mouse CXCR4 , and PE-labelled rat anti-mouse C-kit .Serum samples were obtained from mice 96 h after cisplatin injection. BUN and creatinine levels were subsequently measured using an autoanalyser .After the mice were euthanized, kidney specimens were fixed immediately in 10% buffered formalin for 24 h and then embedded in paraffin, and some kidney specimens were embedded in OCT for freezing. The kidney tissues were cut into 3-μm-thick sections for periodic acid-Schiff staining (PAS) and immunohistochemistry (IHC). Tubular injury was diagnosed based on the presence of tubular epithelial necrosis, cast formation, tubular dilatation, and brush border loss. Renal injury severity was scored in a blinded fashion as described in a previous study based on the percentage of tubule lesions in ten randomly selected  non-overIHC was performed to identify Ki-67-positive cells, which were counted in 20 randomly selected cortical and outer medullary (OSOM) fields at a magnification of × 400 by a blinded investigator. In addition, kidney tissue sections were subjected to immunofluorescence staining for BrdU using mouse anti-BrdU monoclonal antibodies (Roche) and DyLight 594-conjugated secondary antibodies (Amyjet) to evaluate tubular epithelial cell proliferation. The number of BrdU-positive cells was determined by counting the number of positive nuclei in 20 randomly selected non-overlapping cortical and OSOM fields at a magnification of × 400.Apoptosis was evaluated using a terminal deoxynucleotidyl transferase dUTP nick end-labelling (TUNEL) assay kit . Briefly, C57BL/6J mouse kidney sections were deparaffinized, rehydrated, digested with proteinase K, and labelled with TUNEL reaction mixture for 60 min at 37 °C. The TUNEL-positive cells, which corresponded to apoptotic tubular epithelial cells, were counted in 20 randomly selected cortical fields at high-power magnification (× 400). All tissue sections were viewed and labelled by a blinded examiner.A total of 50 μg of total renal cell lysate was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes . Western blotting was performed as described in our previous study . The fol−ΔΔCt method, where △△CT = Target group − reference group [Total RNA was extracted from tissues with TRIzol Reagent (TaKaRa) according to the manufacturer’s instructions. RNA reverse transcription was conducted using a PrimeScript™ RT Reagent Kit (TaKaRa). PCR enzymes and master mixes (DBI Bioscience), along with primers specific for mouse GAPDH, TNF-α, IL-6, IL-10, kidney injury molecule-1 (Kim1), and neutrophil gelatinase-associated lipocalin , were used for real-time PCR, Kim1, and Ngal. Relative expression levels were normalized to the levels of GAPDH and calculated using the 2ce group . The priIL-6 forward (F): 5′-TCCAGTTGCCTTCTTGGGAC-3′; reverse (R): 5′-TGCACAACTCTTTTCTCATTTCCAC-3′IL-10 forward (F): 5′-ATCAGCAGGGGCCAGTAC-3′; reverse (R): 5′-AAGGCTTGGCAACCCAAGT-3′Kim1 forward (F): 5′-TACCTGGAGTAATCACACTGAAGCA-3′; reverse (R): 5′-TTCAATCTTAGAGACACGGAAGGC-3′Ngal forward (F): 5′-GGCAGCTTTACGATGTACAGCA-3′; reverse (R): 5′-TCTGATCCAGTAGCGACAGCC-3′TNF-α forward (F): 5′-TCACAAAACTTGAGAGTCGTGGTG-3′; reverse (R): 5′-AAAGTGGCTCTACGTTATATTCTGCC-3′GAPDH forward (F): 5′-GCCAGCCTCGTCTCATAGACA-3′; reverse (R): 5′-AGAGAAGGCAGCCCTGGTAAC-3′P value < 0.05 was considered significant.The results are presented as the mean ± SEM. Multiple group comparisons were performed by ANOVA followed by Bonferroni multiple-comparison post hoc test. For survival analysis, the Kaplan-Meier method was used, and the groups were compared using the log-rank test. Statistical analysis was carried out using the SAS statistical software for Windows version 8.2 . All tests were two-tailed, and a P < 0.01). Furthermore, the G-CSF/AMD3100 group had a better survival rate than the G-CSF group (P < 0.05). These results suggest that combinatorial G-CSF/AMD3100 therapy has the ability to protect against cisplatin-induced renal toxicity.BMSCs can repair injured renal tubules, and G-CSF can mobilize BMSCs into the peripheral blood. Therefore, we examined the ability of pretreatment with G-CSF/AMD3100 to prolong the survival of mice injected with cisplatin. As shown in Fig. P < 0.01). The mice that received G-CSF or G-CSF/AMD3100 exhibited significantly lower serum creatinine and BUN levels than mice that were treated with saline. Moreover, the levels of BUN and creatinine were notably lower in the G-CSF/AMD3100 pretreatment group than in the G-CSF-treated alone group (P < 0.05). These results suggest that pretreatment with G-CSF/AMD3100 significantly attenuates the deterioration of renal function after cisplatin-induced AKI and that the combination of G-CSF/AMD3100 is more effective than pretreatment with G-CSF alone.We measured BUN and serum creatinine levels to assess the severity of renal dysfunction in each group after cisplatin injection. As shown in Fig. P < 0.05; Fig. We next examined the histological changes in the kidney after treatment with cisplatin. Renal tissues were subjected to PAS staining Fig. a–h. Acco+ and CD133+ cells are widely recognized as haematopoietic stem cell (HSC) and endothelial progenitor cell (EPC) biomarkers, respectively. In this study, BMSCs in peripheral blood samples from C57BL/6 male mice were analysed by FCM analysis. As expected, compared with saline treatment, G-CSF and G-CSF/AMD3100 treatment significantly enhanced circulating CXCR4+ cells, as shown in Fig. P < 0.05 and **P < 0.01, respectively). In addition, there were more CXCR4+ cells in the G-CSF/AMD3100 group than in the G-CSF-treated group (P < 0.01) ; however, the percentages of these cells were significantly higher in the non-irradiation + G-CSF/AMD3100 group than in the non-irradiation + saline and irradiation + G-CSF/AMD3100 groups (P < 0.01). These results further prove that BMSCs are mobilized into the peripheral blood by G-CSF/AMD3100.To further determine whether G-CSF/AMD3100 administration mobilizes BMSCs into the peripheral blood, we induced BMA via irradiation to prevent BMSC development and mobilization. As shown in Supplemental Fig. P < 0.05). G-CSF/AMD3100-treated mice exhibited higher protein levels of PCNA than G-CSF-treated mice  and a 1.5-fold increase compared with G-CSF treatment . Interestingly, G-CSF/AMD3100-treated mice exhibited significantly fewer TUNEL+ cells than G-CSF-treated mice   and CXCR4+/CD44+ cells in the circulating blood was performed flow cytometry analysis after induction of AKI in control cisplatin, G-CSF and G-CSF/AMD treated mice. Results are expressed as the means ± SD (n=5 per group),*P<0.05, **P<0.01, vs. the cisplatin group, and the indicated test group. Supplemental Figure 2. Effect of bone marrow ablation (BMA) and G-CSF/AMD3100 treatment in C57BL/6J mice on mobilization of stem cells in the peripheral blood. Irradiated C57BL/6J mice received treatment with G-CSF/AMD3100 or saline, as described in the Materials and Methods section. 96 hours after the last injection of cytokines, collected of peripheral blood. The percentage of CXCR4+CD34+ cells (A), CXCR4+CD133+ cells (B) in the circulating blood was performed flow cytometry analysis. Results are expressed as the means ± SD (n=5 per group),*P<0.05, **P<0.01, NS : no significance. Supplemental Figure 3. Effects of G-CSF/AMD3100 on the Kim-1, Ngal mRNA expression. (A)The expression of Kim-1 mRNA was detected by RT-PCR. (B) The expression of Ngal mRNA was detected by RT-PCR. Results are expressed as the means ± SD (n=6), # P<0.001, vs. the control group; **P<0.01, vs. the cisplatin group and the indicated test group. Supplemental Figure 4. Schematic diagrams illustrating the mechanism of G-CSF/AMD3100 mobilizing bone marrow–derived stem cells rescues mice from Cisplatin-induced acute renal failure. Exogenous AMD3100 specifically blocks CXCR4 mediated SDF-1/CXCR4 interactions in bone marrow microenvironment, resulting in BMSCs snap out the bone marrow niche, mobilize to the peripheral blood, and homing into injured kidney. The BMSCs promote renal repair via improving renal tubular cells proliferation and regeneration, regulating apoptosis and inflammatory cytokines.


The cerebrospinal fluid (CSF)/serum quotient of albumin (QAlb) is the most used biomarker for the evaluation of blood–cerebrospinal fluid barrier (B-CSF-B) permeability. For years QAlb was considered only as an age-related parameter but recently it has also been associated to sex. The aim of the present study was to explore the impact of sex in the determination of B-CSF-B dysfunction.The analysis was retrospectively conducted on subjects consecutively admitted to the neurological ward. CSF and serum albumin levels were measured by immunonephelometry and pathological QAlb thresholds were considered: 6.5 under 40 years, 8.0 in the age 40–60 and 9.0 over 60 years.1209 subjects were included in the study. 718 females and 491 males (age: 15–88 years): 24.6% of patients had a diagnosis of multiple sclerosis, 23.2% suffered from other inflammatory neurological diseases, 24.6% were affected by non-inflammatory neurological diseases, and for 27.6% of patients the final neurological diagnosis could not be traced. Dysfunctional B-CSF-B was detected more frequently  and median QAlb value were higher  in males than in females in the overall study population and in all disease subgroups. QAlb and age were positively correlated both in female (p < 0.0001) and male (p < 0.0001) patients, however the slopes of the two regression lines were not significantly different (p = 0.7149), while the difference between the elevations was extremely significant (p < 0.0001) with a gap of 2.2 units between the two sexes. Finally, in a multivariable linear regression analysis increased age and male sex were independently associated with higher QAlb in the overall study population (both p < 0.001) and after stratification by age and disease group.Accordingly, identification and validation of sex-targeted QAlb thresholds should be considered as a novel tool in an effort to achieve more precision in the medical approach. Brain homeostasis is maintained by means of two different barriers: the blood–cerebrospinal fluid (CSF) barrier (B-CSF-B) formed by the choroid plexus epithelial cell, and the blood–brain barrier (BBB) formed by the cerebral blood vessel endothelium , 2. SincWithin threshold values QAlb indicates a “normal” B-CSF-B permeability, while values beyond the threshold reflect B-CSF-B dysfunction . The useQAlb values were recently reported to be higher in men than women regardless of age in a large population of patients with unspecified disorders and in healthy volunteers . The impOwing to sex-related differences in brain development, structure, and neurotransmission , exploriOur aim was to investigate whether sex has an impact on B-CSF-B permeability by analysing QAlb in a cohort of neurological patients.Clinical and laboratory anonymized data were retrospectively collected from patients hospitalized from 2000 to 2018 in the ‘S. Anna’ University Hospital , in Ferrara, northern Italy. The study was approved by the local Committee for Medical Ethics in Research, “Comitato Etico di Area Vasta Emilia Centro della Regione Emilia-Romagna”  and written informed consent was obtained.All patients included in the study had undergone lumbar puncture for diagnostic purposes. Blood and CSF samples had been withdrawn at the same time. CSF and serum samples were analysed at room temperature immediately after centrifugation or stored in aliquots at − 80 °C until assay. All analyses were performed on the 2nd–4th ml of CSF after lumbar puncture. For every patient albumin levels were measured as part of the diagnostic work-up in cell-free CSF and paired serum samples by immunochemical nephelometry with the Beckman Array Protein System or IMMAGE 800 Immunochemistry System  according to the procedure of Salden . AlbuminExclusion criteria were (i) age less than 16 years (ii) lack of demographic data (sex and/or age) and (iii) the presence of xanthochromia and/or high levels of CSF red blood cells (> 2000/µl) as suggestive of traumatic lumbar puncture or subarachnoid haemorrhage.These subjects were subsequently diagnosed with multiple sclerosis (MS), other inflammatory neurological diseases (OIND), including inflammatory diseases of both central and peripheral nervous systems, non-inflammatory neurological diseases (NIND) and patients with untraceable definite neurological diagnosis (UNK). For all subjects, sex, age, and QAlb values were collected.®) version 21.0 for Windows, OSX  and Prism® 8 (GraphPad Software Inc.) were used for the statistical analysis.Continuous variables with non-normal distribution at the Kolmogorov–Smirnov test were reported as median  and comparisons were made with Mann–Whitney test. Categorical variables were reported as count (percentage) and Chi square test used for comparison. Correlations between age and QAlb were investigated with Spearman test. In regression analysis F-test was used to compare the fits of linear models. Independent predictors of Log-transformed QAlb were explored using multivariable linear regression, and age and sex were used as reciprocal covariates in the model. Two tailed p-values < 0.05 were considered statistically significant. The Statistical Package for the Social Sciences  patients had a diagnosis of MS, 281 (23.2%) suffered from OIND, 297 (24.6%) were affected by NIND, and for 334 (27.6%) the definite neurological diagnosis could not be traced (UNK). The main clinical-demographic features of the study population are reported in Table The women:men ratio was 1.46:1 in the overall study population, 2.19:1 in MS, 0.89:1 in OIND, 0.97:1 in NIND and 2.41:1 in UNK group. In the overall study population age at admission was higher in men (48.0 years) than in women (44.0 years) and in the UNK subgroup it was 51.0 years versus 42.4 years, respectively. No further significant difference in age distribution was observed across groups.In our overall study population, based on QAlb, men were reported with a B-CSF-B dysfunction more frequently than women (44.0% vs. 20.1%). After stratification by age, 40.8% men vs. 11.8% women under 40 years of age were reported with B-CSF-B dysfunction, 44.3% vs. 23.4% between 41 and 60 years, and 47.1% vs. 33.3% over 60 years , and in all age subgroups: 5.74 vs. 4.19 in the under 40 s, 7.33 vs. 5.03 in the group aged between 41 and 60 years, and 8.87 vs. 6.52 in the over 60 s  and in men  ; 92 OIND ; 94 NIND ; 334 UNK . As reported in Fig. At the time of sample collection disease severity was scored using Kurtzke’s Expanded Disability Status Scale (EDSS)  in 286 MIn our study we have shown that both older age and male sex are independently associated with higher QAlb in a large population of neurological patients. Because QAlb reflects B-CSF-B dysfunction , 5, thisOur findings are in line with those from a study by Parrado-Fernández et al. which was recently conducted in a population of over 20,000 patients with unspecified diseases as well as in control subjects . They obSince it was introduced in 1977 , QAlb haQAlb is reported to be normal or only rarely increased in MS . In the Elevated QAlb values have been reported above all in OIND and also, although less frequently, in NIND and in patients without neurological disorders , 27. AccAs has also been observed in other studies , in our Sex-specific differences in QAlb values may be secondary to a number of mechanisms. The female hormone, 17β-estradiol , may driAlso, a difference in the CSF flow rate between sexes may explain the sex-specific differences in QAlb. For decades QAlb has been regarded as increasing with age , 13, 34,Other factors such as the use of oral contraception in females  or postuFrom another perspective, the different distribution of QAlb defined B-CSF-B dysfunction by sex indeed opens up a discussion on the relevant potential implications for clinical practice. If this difference is genetically determined , 33, we Even in the absence of B-CSF-B alteration, however, the sex-specific difference of QAlb may still reflect corresponding differences in B-CSF-B permeability and/or CSF flow/turnover also in normal subjects. In this perspective, differential drug delivery to the CNS should be hypothesized, and sex-specific pharmacological strategies may be required to ensure the most effective drug concentration crossing the BBB.Finally, it is worth reporting that it has recently been shown that glial cells in the CNS can produce albumin , 38–40, Our study has some limitations. The analysis did not include a healthy donor population so a comparison between neurological patients and a healthy population could not be made. Thus, it is possible that the male/female and age differences might be due to altered responses to disease. However, we did include groups with different neurological pathologies, i.e., MS, OIND and NIND, which ultimately showed similar QAlb distribution, and were in line with existing evidence on healthy subjects reported elsewhere . FurtherOur study reveals that, regardless of age, male neurological patients show B-CSF-B dysfunction more frequently than females, based on QAlb values. This evidence opens up discussion on whether the current QAlb threshold needs to be revised e.g., by introducing a corrective factor, in consideration of sex-genetically background differences, or whether B-CSF-B permeability truly differs between sexes, making men more prone to B-CSF-B dysfunction than women. In any case, future studies are required to evaluate whether the gap of 2.2 units we found between male and female neurological patients might be applied as a sex-related corrective factor to the thresholds for the evaluation of B-CSF-B permeability through the QAlb.In an era of cost-effective personalised medicine, these considerations could have practical implications on sex-specific prognosis of neurological disorders as well as on the choice of the best strategy for drug delivery into the CNS.


Surface electromyography (sEMG) can be used to assess the integrity of the neuromuscular system and its impairment in neurological disorders. Here we will consider several issues related to the current clinical applications, difficulties and limited usage of sEMG for the assessment and rehabilitation of children with cerebral palsy. The uniqueness of this methodology is that it can determine hyperactivity or inactivity of selected muscles, which cannot be assessed by other methods. In addition, it can assist for intervention or muscle/tendon surgery acts, and it can evaluate integrated functioning of the nervous system based on multi-muscle sEMG recordings and assess motor pool activation. The latter aspect is especially important for understanding impairments of the mechanisms of neural controllers rather than malfunction of individual muscles. Although sEMG study is an important tool in both clinical research and neurorehabilitation, the results of a survey on the clinical relevance of sEMG in a typical department of pediatric rehabilitation highlighted its limited clinical usage. We believe that this is due to limited knowledge of the sEMG and its neuromuscular underpinnings by many physiotherapists, as a result of lack of emphasis on this important methodology in the courses taught in physical therapy schools. The lack of reference databases or benchmarking software for sEMG analysis may also contribute to the limited clinical usage. Despite the existence of educational and technical barriers to a widespread use of, sEMG does provide important tools for planning and assessment of rehabilitation treatments for children with cerebral palsy. Cerebral palsy (CP) is the most common form of motor disability in childhood. It describes a group of permanent disorders of movement and posture, caused by disturbances in the fetal or infant brain . The cliIn recent decades, significant developments have occurred in many electrodiagnostic studies that provide powerful quantitative approaches to instrumentation-based assessments in cardiology (electrocardiology), neurology , skeletal muscle functioning . In this work we will specifically focus on sEMG. It can be used for evaluation of the integrity of the nervous system in neurological disorders with motor deficit, playing an important role in neurorehabilitation and predicting the outcome of neuromuscular disorders –17. MultThis article specifically reports the potential clinical value of techniques based on sEMG in neurorehabilitation medicine of children with CP and addresses the barriers limiting a widespread clinical usage of sEMG for this patient population. In the first section, we will briefly consider the motor impairments that result from a lesion occurring in the developing brain and describe sEMG applications for the assessment of neurological impairments and for performing interventions/treatment in children with CP. In the second section, we will present the results of a survey directed to the physiotherapists, neuro-developmental disorders therapists and medical doctors of a department of pediatric rehabilitation related to the barriers limiting a widespread clinical use of sEMG techniques in clinical assessment and neurorehabilitation of children with CP.One of the most widespread and used classifications of CP is that proposed by Surveillance of Cerebral Palsy in Europe. According to their criteria, all CP subtypes have an abnormal pattern of movement and posture and classification is applied in a hierarchical manner using the predominant type of muscle tone and movement abnormality, resulting in the following categories: spastic, dyskinetic and ataxic . The mosComprehensive descriptions of motor impairments in children with CP have been reported in numerous studies both for lower and upper limbs. Children with CP may develop various motor dysfunctions, including dystonia, contractures, hyperreflexia, muscle weakness, lack of coordination , 24, incGait impairments in CP are also typical . In partSince any reflection on functional disability in CP should consider the mechanisms and methods of their assessment, sEMG monitoring may be useful for assessing and treatment of motor impairments and various examples will be considered below.Since the discovery of sEMG in 1912, myoelectric activity measurements provided many examples of normal and pathological skeletal muscle function, improved our knowledge about the neural control of movement and contributed to the development of clinical applications . sEMG reStandard measurements and processing procedures of the sEMG signals are in great demand for a better understanding of neuromuscular control and are important for various biomedical applications and clinical diagnosis. In clinical practice, real-time sEMG can be used by physiotherapists as control if the movement requested is performed by the proper target muscle or by means of compensatory mechanisms, as a direct measurement of variations consequent to mobilization, verticalization, trunk fixation, or to assess the effect of different orthoses on muscle activation, which can vary toward or away from the normal pattern. Below we consider examples of using sEMG in children with CP for assessing neurological impairments and performing interventions.Walking is typically considered one of the most essential activities of daily living , 54. CliIt is worth noting that the muscles are often weak and atrophic in children with CP, resulting in significantly reduced volumes in leg muscles and in bone changes , 61, 62.sEMG is commonly used to assess muscular coordination in clinical gait analysis but could also be used in functional diagnosis or in the monitoring of therapeutic outcomes. In particular sEMG could be useful for the assessment of the “paretic component,” i.e., defective activation of peripheral muscle effectors on most affected side of hemiplegic children. Reduced and insufficient speed dependent modulation of the ankle dorsiflexors' activity  around foot contact at end swing (and to a lesser degree at end stance) can significantly affect the ankle dorsiflexion torque and consequently the foot trajectory in children with CP  64). Th. Th64). Functional corticospinal connectivity in CP can be assessed by estimating the oscillatory drive of the motor cortex to the spinal cord using coherence analysis of sEMG signals within and between muscles. Indeed, in children with CP, there is a frequent problem of foot drop associated with impaired control of the ankle dorsiflexors  and reflHyperreflexia is a frequent feature in neurological disorders characterized by a velocity-dependent increase in tonic stretch reflexes (muscle tone) with exaggerated tendon jerks, resulting from hyper-excitability of the stretch reflex . sEMG exClinical analysis of muscle activity is thus necessary for deciding whether to intervene or not and in particular for determining the degree of post-treatment reduction of the spastic component. Experiments and data analysis using muscle models confirmed a tight coupling between kinematics and motor output in children with spastic diplegia, for instance, during the phases of lower limb muscle lengthening in the gait cycle . In partChildren with CP might have higher levels of activation in specific muscles and/or large amounts of coactivation of agonist and antagonist muscles in the same joint, which could increase muscle fatigue. Mechanical manifestations of muscle fatigue are defined as a reduction in the force-generating capacity of the neuromuscular system, which occurs during sustained activity . Muscle Typically a decrease in frequency and an increase in root mean square of sEMG signals are interpreted as myoelectric manifestations of muscle fatigue , 77. FatNeuromuscular or functional electrical stimulation (FES) is an example of the appropriate usage of temporal characteristics of sEMG recordings for determining the application of multi-channel electrical stimuli to superficial skeletal muscles in order to control, compensate and/or correct their contractions –85. In aSome studies used electromyographic biofeedback as a non-invasive, safe, and effective treatment for children with CP –95. ChilTo perform a movement, the central nervous system (CNS) should engage many muscles and control corresponding forces exerted around joints involved. Furthermore, muscles differ in the fiber composition  and structure , may be divided into compartments and comprise quite different number (thousands) of motor units. During locomotion or other movements, tens of muscles are active and need to be coordinated simultaneously. The idea that the CNS can control the complexity of interactions to promote a certain motor act by adopting a modular decomposition and therefore a limited number of primitives has recently received a lot of attention . In the This approach has had a large impact on the analysis of motor control in the field of neurorehabilitation since it implies that the CNS generates forces and movements by optimizing the control strategy of either individual muscles or (more likely) muscle synergies . ConcernThe final neural output of spinal locomotor circuitry is represented by the spatiotemporal activation of α-motoneurons (MN). It can be evaluated indirectly by using sEMG recordings from a large number of lower limb muscles and mapping their activity patterns onto the approximate rostrocaudal location of the motor pools of the corresponding muscles in the lumbosacral enlargement , 128. ThA number of “barriers” exist limiting the widespread application of sEMG techniques in clinical assessment and neurorehabilitation of children with CP. We aimed at examining these barriers by gathering information from the clinicians involved in pediatric rehabilitation to generate opinion on the current use of sEMG and its clinical utility. We think that getting some background views and perspectives will help our understanding of the current applications of sEMG in neurorehabilitation of children with CP as well as of the potential obstacles to its use in the clinical setting in this particular area. To this end, we conducted a survey directed to the physiotherapists, neuro-developmental disorders therapists and medical doctors related to the barriers limiting a use of sEMG techniques in clinical assessment and neurorehabilitation of children with CP.An online survey involving the personnel of the Department of Pediatric Neurorehabilitation of the IRCCS Santa Lucia Foundation was conducted. Of the 36 invitations sent, 28 invitees completed survey questionnaires. Professional background was varied, with 16 (57%) physical therapists, 7 (25%) medical doctors, and 5 (18%) neuro-developmental disorders therapists (Google Forms) was used to collect the answers electronically. They were requested to respond to the questionnaire by selecting one of the answers to each statement  contributors totally agree with the statement that sEMG is rarely used in clinical neurorehabilitation , evaluate muscular fatigue, evaluate the appropriateness of muscle activation in specific motor acts, identify pathological patterns of motor unit behavior, evaluate maximal voluntary activation, characterize involuntary muscle activations , and characterize muscle fiber conduction velocity , we described motor impairments resulting from a lesion occurring in the developing brain and corresponding sEMG applications. The use of sEMG signals through state-of-the-art and advanced methodologies is becoming essential in rehabilitation engineering and in clinical neurophysiology. Many publications in peer-reviewed journals provide various arguments and examples related to the current clinical applications of sEMG showing the information available for neurorehabilitation of children with CP , 132–136Despite these successes and evidences, clinical applications remain very limited because of many barriers acknowledged by the end-users . In addition, publication of clinical results in the experimental papers or state-of-the-art reviews is necessary but far from being sufficient pointing to the important issue of disseminating rehabilitation innovations and evidence-based practice . The resn = 28) and included experts in pediatric rehabilitation: physical therapists, neuro-developmental disorders therapists, and medical doctors  may have an impact on increasing the use of sEMG in clinical practice. However, given the primary usage of this information by clinicians, we suggest specific theoretical-practical training to be carried out both during university courses for health rehabilitation professions and during medical specialization courses with outlets in neurorehabilitation. This implies recruitment of specialized professionals as teachers, availability of medical technology in university hospitals and therefore allocation of state or university funds.These perceived reasons for the potential barriers  do suggeDespite the uniqueness of the sEMG technology and the successes in clinical applications for planning and assessment of treatment of children with cerebral palsy, clinical application and practice in rehabilitation departments remain very limited because of many barriers. Various educational and technical barriers to a widespread use of sEMG were acknowledged by the end-users . Overcoming these barriers requires a highly interdisciplinary educational approach. Rehabilitators and engineers should have overlapping education and this would lead to overcoming the existing communication gap developing a common language and promoting the use of sEMG systems.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Human neutrophils exert a well-known role as efficient effector cells to kill pathogenic micro-organisms. Apart from their role in innate immunity, neutrophils also have the capacity to suppress T cell-mediated immune responses as so-called granulocyte-myeloid-derived suppressor cells (g-MDSCs), impacting the clinical outcome of various disease settings such as cancer. Patients undergoing chemotherapy because of an underlying malignancy can develop prolonged bone marrow suppression and are prone to serious infections because of severe neutropenia. Concentrates of granulocytes for transfusion (GTX) constitute a therapeutic tool and rescue treatment to fight off these serious bacterial and fungal infections when antimicrobial therapy is ineffective. GTX neutrophils are mobilized by overnight G-CSF and/or Dexamethasone stimulation of healthy donors. Although the phenotype of these mobilized neutrophils differs from the circulating neutrophils under normal conditions, their anti-microbial function is still intact. In contrast to the unaltered antimicrobial effector functions, G-CSF/Dexamethasone-mobilized neutrophils were found to lack suppression of the T cell proliferation, whereas G-CSF-mobilized or Dexamethasone-mobilized neutrophils could still suppress the T cell proliferation upon cell activation equally well as control neutrophils. Although the mechanism of how G-CSF/Dex mobilization may silence the g-MDSC activity of neutrophils without downregulating the antimicrobial activity is presently unclear, their combined use in patients in the treatment of underlying malignancies may be beneficial—irrespective of the number of circulating neutrophils. These findings also indicate that MDSC activity does not fully overlap with the antimicrobial activity of human neutrophils and offers the opportunity to elucidate the feature(s) unique to their T-cell suppressive activity. Patients who undergo chemotherapy are prone to develop neutropenia and are thereby susceptible to serious bacterial and fungal infections . In addiNext to their role of efficient innate immunity killers of micro-organisms, neutrophils are also recognized to be involved in modulation of adaptive immune responses in various disease settings including cancer –9. ImmatIn this study we investigated whether neutrophils obtained upon overnight mobilization of neutrophils into the bloodstream in healthy GTX donors may have a potentially relevant impact as MDSCs in the treatment of oncology patients.Heparinized peripheral blood samples were collected from healthy granulocyte transfusion donors 1 day after combined G-CSF  and dexamethasone  treatment (G-CSF/Dex), or upon their preference with G-CSF or dexamethasone alone, as described previously . Blood sNeutrophils and peripheral blood mononuclear cells (PBMC) were isolated from whole blood by gradient centrifugation using isotonic Percoll  with a specific density of 1.076 g/mL. T cells were isolated from the PBMC fraction by magnetic-activated cell sorting with the Pan T cell isolation kit of Miltenyi-Biotec  according to the manufacturer's instructions. Neutrophils were obtained from the pellet fraction after erythrocyte lysis with hypotonic ammonium chloride solution at 4°C as previously described .4 U/mL penicillin, 10 ng/mL streptomycin, 200 mM glutamine, and 0.00035% (v/v) β-mercaptoethanol . To induce proliferation, the T cells were stimulated by anti-CD3  and anti-CD28  monoclonal antibodies . Neutrophils from blood, collected from the pellet fraction after density centrifugation, were added in a 1:3 ratio , in the presence or absence of neutrophil-activating stimuli: fMLF , TNFα  or LPS .Purified T cells were labeled with CFSE  and cultured in 96-well flat bottom plates  for 4–6 days at 37°C in IMDM medium , supplemented with 10% (v/v) fetal calf serum , 10+ and CD8+ T cells via flow cytometry.After 4–6 days, the cells were harvested from the culture plates and stained with APC-labeled anti-CD4  and PerCPCy5.5-labeled anti-CD8  antibodies. The T cell proliferation was assessed by measuring the CFSE dilution of CD46/mL) stimulated with: fMLF (1 μM), TNFα (10 ng/mL), LPS (20 ng/mL) + LPS-binding protein (LBP)  or PMA  in the presence of Amplex Red (0.5 μM) and horseradish peroxidase (1 U/mL). Fluorescence was measured at 30-s intervals for 4 h with the HTS7000+ plate reader . Maximal slope of hydrogen peroxide release was assessed over a 2-min interval.NADPH oxidase activity was assessed as the release of hydrogen peroxide, determined by the Amplex Red method  by neutrophils  and PECy7-labeled anti-CD16 .Flow cytometry data were acquired using Canto II flow cytometer (BD Biosciences) and analyzed using FlowJo software .t-test. The results are presented as the mean ± SEM. Data were considered significant when p < 0.05.Statistical analysis was performed with GraphPad Prism version 8 for Windows . Data were evaluated by one-way ANOVA or unpaired two-tailed student's We received blood from healthy granulocyte transfusion donors routinely treated with the combination of G-CSF and dexamethasone to test whether the mobilization of neutrophils into the bloodstream resulted in a change of MDSC activity.One day after G-CSF/Dex administration, the absolute neutrophil count in the peripheral blood was ~30 times increased compared to the neutrophil count before administration . The rapTo investigate the MDSC activity  of these G-CSF/Dex-mobilized neutrophils, we now performed additional T cell proliferation assays. In our previous study , where wNeutrophils from G-CSF/Dex donors or from healthy controls were cultured simultaneously for 5 days in the presence of isolated CFSE-labeled T cells from an unrelated healthy donor and were left unstimulated or activated with either fMLF, TNFα, or LPS. Just as previously described, T cell proliferation was induced by the strong and uniform activation by the combination of monoclonal anti-CD3 and anti-CD28 antibodies and quantified as relative “recursor frequency”: i.e., percentage of naïve cells in the initial population that underwent one or more divisions upon anti-CD3/anti-CD28 antibodies . The pre+ or CD8+ T cells, neither under resting conditions nor upon their activation , whereas the Dex-mobilized neutrophil fraction only comprised phenotypically mature neutrophils  patients, unable to generate ROS, do not show MDSC activity while the extent of trogocytosis was indistinguishable from that of control neutrophils . The facin-vivo, for instance, in the ovarian cancer microenvironment  as novel forms of effective immunotherapy to treat cancer –32. In cIn this study, we explored whether g-MDSC activity of neutrophils can be selectively inhibited when treating cancer, while leaving the effector mechanisms of neutrophils against microbial pathogens unaffected, and show that mature G-CSF/Dex-mobilized neutrophils indeed meet such conditions . AlthougThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by Sanquin Research Institutional Ethical Committee. The patients/participants provided their written informed consent to participate in this study.TK and CA are the principle investigators, who conceived, and designed the study. CA, IH, and CF performed the experiments. RB contributed to the design of the study. CA and IH initiated many of the experiments and performed the analysis. CA wrote the manuscript together with TK. 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.


CSF induced transcriptomic changes in pathways promoting cell malignancy, such as apoptosis, survival, cell motility, angiogenesis, inflammation, and glucose metabolism. A genetic signature extracted from the identified transcriptional changes in response to CSF proved to be predictive of GBM patient survival using the TCGA database. Furthermore, CSF induced an increase in viability, proliferation rate, and self-renewing capacity, as well as the migratory capabilities of GBM cells in vitro. In vivo, GBM cells co-injected with human CSF generated larger and more proliferative tumors compared to controls. Taken together, these results provide direct evidence that CSF is a key player in determining tumor growth and invasion through the activation of complex gene expression patterns characteristic of a malignant phenotype. These findings have diagnostic and therapeutic implications for GBM patients. The changes induced by CSF contact might play a role in the increased malignancy of SVZ-proximal GBM.Glioblastoma (GBM) is the most common and devastating primary cancer of the central nervous system in adults. High grade gliomas are able to modify and respond to the brain microenvironment. When GBM tumors infiltrate the Subventricular zone (SVZ) they have a more aggressive clinical presentation than SVZ-distal tumors. We suggest that cerebrospinal fluid (CSF) contact contributes to enhance GBM malignant characteristics in these tumors. We evaluated the impact of human CSF on GBM, performing a transcriptome analysis on human primary GBM cells exposed to CSF to measure changes in gene expression profile and their clinical relevance on disease outcome. In addition we evaluated the proliferation and migration changes of CSF-exposed GBM cells  Glioblastoma (GBM) is the most common and aggressive type of primary brain cancer in adults, accounting for 54% of new gliomas and 45% of primary malignant tumors . In the Tumor location greatly influences the prognosis of GBM patients. More than half of all patients with GBM have tumors that touch the lateral ventricle or even reach into an important brain neurogenic region known as the subventricular zone (SVZ) –15. ThesThe cause for worse outcome for patients suffering from SVZ-infiltrating GBMs is not known. Although it is tempting to speculate that the neurogenic characteristics of the SVZ are the underlying causes of this clinically observed phenomenon. The SVZ is the largest neurogenic niche in adults and is highly regulated by the flow of cerebrospinal fluid (CSF). The CSF milieu has the properties to sustain the neurogenic niche environment, regulating neural stem cells proliferation, differentiation, and migration , 16–18. in vitro and in vivo. We observed that CSF is an important contributor to tumor growth and invasion through the activation of gene expression patterns characteristic of a malignant phenotype.The role of CSF in modulating the aggressiveness of GBM, or other gliomas for that matter, is largely understudied in the field of neuro-oncology. In this work we have taken on the hypothesis that CSF contact enhances GBM malignant characteristics and we have resolved to study the effects of CSF exposure on human GBM-derived BTICs. Our study evaluated CSF impact on GBM gene expression profile as well as cell proliferation and migration Under Mayo Clinic institutionally-approved protocol we established primary BTIC cultures from tumor tissue from patients undergoing surgical resection for newly diagnosed GBM without prior treatment as described previously by our group , 19. Cliin vitro. Tumor initiation potential was determined in vivo by orthotopic implantation in immunosuppressed mice .Primary-cultured BTICs derivation and culture protocols were performed as previously described , 21. BriFor CSF collection and processing, samples were obtained intra-operatively upon opening of the dura mater. CSF samples were spun at 200 × g for 5 min at 4°C, filtered by 0.45 μm to eliminate cells and debris, and immediately aliquoted and stored at −80°C until use.For CSF stimulation, BTICs were cultured on laminin-coated plates. Twenty-four hours prior to experimentation, cells were maintained in base media (without EGF and FGF) to avoid confounding effects. Protein concentration in CSF was measured and CSF was utilized at a 1:100 dilution in base media, unless otherwise stated. CSF samples were matched based on gender and age, specifically cCSF73 was matched to ncCSF12 or ncCSF1276 and cCSF-37 to ncCSF25. Cells were maintained under CSF stimulation in incubation conditions for 24 h or as required for analysis.2 flasks until 80% confluency was reached, then treated with cancer or non-cancer CSF (CSF was pooled from 3 cases each). Cells were incubated in CSF or control conditions for 24 hrs. Upon incubation, cells were centrifuged and RNA was extracted. Samples were assessed for RNA quality and quantity using a 2100 Bioanalyzer (Agilent) with a RNA 6000 Nano Chip (Agilent) and diluted to a final concentration of 50 ng/μl. All samples used in this study had RIN values >9.2 μg of RNA were submitted for microarray using the Illumina Human HT-12 v4 chip. The scanned images of the microarrays (.dat files) were processed using Illumina's GenomeStudio software. The probe level intensities were quantile normalized across samples with background subtraction. Probes with detection p-values > 0.05 in all samples were excluded. All samples had >40% probes expressed and passed this step of QC. In addition, all samples passed the Illumina's internal QC threshold of signal intensity ratios between 5′ vs. 3′ probes targeting house-keeping genes. The bimodal distributions of the normalized and log2 transformed probe intensities were plotted to determine the threshold of expression as 6.6 (data not shown). Genes with average expression values in all experimental groups below the threshold of expression were filtered out. The principle component analyses (PCA) as well as the unsupervised hierarchical clustering analyses of all samples using the remaining 21,023 probes identified two outlier samples which were consequently removed for the following analysis of variance (ANOVA) and weighted gene co-expression network analysis (WGCNA). Source of variance analyses identified CSF treatment, cell line, and CSF treatment/patient interactions as main contributors to variance, and were included in the ANOVA model. Pair-wise group comparisons were also performed to obtain fold change and p-values between any two groups.Cells were grown in 25 cmhttp://software.broadinstitute.org/gsea/msigdb).WGCNA  was perfp-value < 0.05. Canonical pathways were assessed for significant enrichment and directionality of effect utilizing a z score >±2 and p-value < 0.05 (right-tailed Fisher's exact-test) on IPA. Network Analysis was also performed on Metacore, using Analyze Networks (AN) algorithm (default settings) to generate a list of biological sub-networks highly enriched and unique for the uploaded data. In this workflow the networks are prioritized based on the number of fragments of canonical pathways on the network, ranked by p-value, G-score and interpreted in GO terms. Transcriptional regulators of DEG genes were also identified with MetaCore Interactome. Transcription factors (TF) are ranked according to their Z-score (the level of connectivity of the TF to the DEG list). Larger Z-scores represent higher levels of connectivity between the transcription factor and the DEG list.Gene ontology (GO) and pathway analysis were performed on differential gene expression (DEG) results using Metacore  and Ingenuity Pathway Analysis . DEGs were defined as genes with a Fold change (FC) higher than 1.2 or lower than −1.2 and adjusted http://gliovis.bioinfo.cnio.es/) (mRNA expression from the Affimetrix HT Human Genome U133 array). Unsupervised hierarchical clustering of both genes and samples was performed using Partek Genomics Suite . The quantile normalized and log2 transformed expression values were used for unsupervised hierarchical clustering of both samples and probes. Pearson Correlation Coefficient was used as the distance metric between samples and genes. The distance between two clusters is defined as the average of distances between all pairs of objects using the average linkage method. The expression values of each gene across samples were standardized to mean of 0 and scaled to standard deviation of 1. Clinical data collection for overall survival and disease/progression free was obtained from the GBM provisional cohort in the cBioPortal for Cancer genomics .Pre-processed and normalized gene expression data was retrieved from GlioVis portal (n > 4 per condition) and incubated overnight to allow for attachment in base media. A 10 μL aliquot of alamarBlue reagent was added to the wells containing 100 μL of base growth media with or without treatments, and cells were incubated for at least 4 h at 37°C. Fluorescence was measured using a plate reader  at 24, 48, 72 h after CSF stimulation.Cell culture growth and viability was evaluated by alamarBlue (Invitrogen). GBM cells were seeded at 2.000 cells/well in a laminin-coated 96-well plate  using ImagePro Software (Media Cybernetics).For gradient migration assay, cells were plated on a glass bottom multiwell plate (ibidi™) coated with poly-L-ornithine solution  and laminin. A 2 × 10http://bioinf.wehi.edu.au/software/elda/.The assay was performed as previously described . BrieflyNuclear Ki67 expression was evaluated to determine cells in active proliferative phases. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, and blocked for 1 h in PBS containing 0.1% Triton X-100 (Sigma-Aldrich) with 10% goat serum prior to overnight incubation with anti-Ki67 . Alexa 594-labeled secondary antibody  was used for visualization, and DAPI was used to counterstain cell nuclei. Slides were visualized and recorded with an inverted fluorescence microscope and the number of Ki67+/DAPI cells was counted in at least 8 randomly selected fields at 20x per slide. For mouse tissue, after fixation in 4% paraformaldehyde and paraffin embedding, brains were sliced at 5 μm, deparaffinazed and treated for antigen retrieval in citrate buffer for 30 min, followed by primary antibody staining as described above.Cyclin D1 expression was measured by real-time quantitative PCR, after 24 h exposure to CSF. Total RNA was isolated using TRIzol (Invitrogen) and the RNeasy mini kit (Qiagen) following the manufacturer's instructions. Reverse transcription was performed using Superscript III (Invitrogen) and real time quantitative PCR was performed on a Quant Studio 3 (Applied Biosystems) with Power SYBR Green PCR Master Mix. Relative quantification of mRNA expression was calculated by the ΔΔCT method after adjusting the levels to the corresponding internal GAPDH control for each sample. Primers sequences were as follows: human GAPDH sense: 5′-AGGTCGGTGTGAACGGATTTG-3′, antisense 5′-TGTAGACCTGTAGTTGAGGTCA-3′; human Cyclin D1 sense 5′-CAATGACCCCGCACGATTTC-3′, antisense 5′-CATGGAGGGCGGATTGGAA-3′.ad libitum access to food and water with a 12 h light/dark cycle. All animals were used for study between 8 and 15 weeks of age.All animal procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines. Athymic nude mice  were housed three to five per cage and maintained on in vivo studies  proteins . One week post-transduction, cells were sorted by flow cytometry to select GFP-expressing cells. Cells were washed in PBS and resuspended in PBS before injection to remove any trace of growing media.Glioblastoma cells GBM1A (previously known as GBM 0913) originally established by Vescovi et al.  and prev studies , 24–26. 5 GBM1A GFP-Luc cells were resuspended in 3 μl of CSF (or PBS for controls) plus 2 μl 0.2% Pura Matrix (Corning) and injected intracranially into the right hemisphere . Injection was performed with an automatic system at a 0.5 μl/min rate and injection needle was left in place for an extra minute to reduce backflow. Injection needle was then retrieved and skin incision was closed with surgical glue. Isoflurane was discontinued, and the animal placed on a heating pad. All mice completely recovered within 5 min. Buprenorphine (10 mg/kg) was used as pre- and post-operative analgesic.We performed intracranial injection as described previously by our group , 19, 27.Mice were randomized into 3 experimental groups of 8 animals each: (1) control GBM1A coinjected with PBS and Pura Matrix, (2) GBM1A coinjected with cancer CSF and Pura Matrix, and (3) GBM1A coinjected with non-cancer CSF and Pura Matrix. Tumor formation and growth were followed by bioluminescence (BLI) every week. Mice were euthanized 4 weeks after injection. Brains were fixed using transcardiac perfusion, and post-fixed overnight at 4°C in 4% paraformaldehyde.A) of a number (n) of parallel sections spaced at a constant distance (t), using the following equation: est (V) = t*(A1 + A2 + A3 +& An). Three 5 μm brain slides per tumor sectioned at 50 μm intervals were used for this calculation.Tumor volume was calculated performing a morphometric analysis of brain sections that presented tumor. The morphometric volume (MFV) determination was done using the Cavalieri principle , which aIn vivo bioluminescence images of tumor-implanted mice were obtained using the IVIS Spectrum System (Perkin Elmer) that has a cooled CCD camera to capture images of animals and tissues in a light-tight box. D-luciferin  was injected intraperitoneally at a dose of 10 mg/kg and allowed to distribute for 5 min. Mice were then anesthetized using isoflurane and imaged in prone position. Imaging times ranged from 5 s to 5 min, depending on the total tumor burden as a function of light emission from tumor cells. Region of interest analysis was performed using Living Image Software.The mean ± SD light emission over the time was plotted for each brain tumor engrafted mice. Bioluminescent signal was normalized and analyzed as the absolute total flux (photons/steradian/cm2). Mice were imaged at 1, 2, and 3 weeks after tumor engrafting.post-hoc analysis. Results represent the mean ± SEM of four replicates in three independent experiments unless stated otherwise. Statistical significance is represented by *p < 0.05, **p < 0.01, ***p < 0.001.Statistical analysis was performed using GraphPad Prism 8. Multiple comparisons analysis was determined by a one-way ANOVA, followed by Tukey In order to establish the extend of the effects of CSF contact on GBM, we performed a transcriptome analysis in CSF-treated GBM cells. To the best of our knowledge, there is not data to date on CSF induced transcriptomic changes in GBM cells. To identify common properties of CSF derived from brain cancer compared with non-cancer patients, we have pooled in this study CSF derived from 3 brain cancer patients (high grade gliomas) and CSF from 3 control patients  .p-values between any two groups. We then analyzed the expression fold change of the GBM-CSF treated samples compared to controls , and we found 1,252 differentially expressed genes (DEGs) in the GBM612 sample  and 561 DEGS in the GBM276 sample  (p < 0.001)  , among tgulated) . Most im< 0.001) , all proGlobal analysis of the entire dataset by WGCNA highlighted the relevance of one module  containing the highest number of differentially expressed transcripts common to both cell lines , 2B. WGCThe results of this analysis summarize the complexity of functions regulated by the CSF in the contest of GBM pathophysiology.in vitro, we have attempted to identify patient subgroups in the TCGA database that carry this particular gene expression profile. We have selected DEGs from p-value < 0.05 and a FC > 2 in either cell line analyzed and generated a list of 35 upregulated genes to represent the CSF-induced gene signature in GBM. Gene expression measurements for the entire dataset in the TCGA GBM provisional cohort were retrieved from GlioVis portal (http://gliovis.bioinfo.cnio.es/). We then performed independent hierarchical clustering of both genes and patient samples and we generated 11 patient clusters with different transcriptomic profiles (p < 0.0001), NNMT (p < 0.0001), CCL2 (p = 0.002), GBP1 (p = 0.0004), RCAN1 (p = 0.0044), SERPINA3 (p = 0.0075), ZFP36 (p = 0.018), CEBPD (p = 0.0423), and TNC (p = 0.0462).In order to establish the clinical relevance of the gene signature induced by CSF in GBM cells profiles . We nextprofiles . Clusterprofiles . The genp < 0.01). This response was proved to be dose-dependent (data not shown). Interestingly, GBM1A and GBM965 showed maximum response at different concentrations, with the GBM1A line being the most susceptible to respond to treatment   . Taken tp < 0.05, p < 0.05, p < 0.0001, in vitro.Here we evaluated the cell migration response of BTICs to human CSF by transwell assay and video-microscopy on a 2D surface during 24 h as described previously , 29. GBMin vivo. In order to determine whether the in vitro effects of CSF on BTIC proliferation and migration persist when cells are in the brain microenvironment, we tested using a human orthotopic GBM model in mice . PuraMatrix is a non-toxic, biodegradable, and biocompatible hydrogel composed by 16 amino acids . This hydrogel mimics several properties of the natural extracellular matrix in which cells can easily proliferate, differentiate, and migrate . Interestingly, female mice developed larger tumors than their male counterparts   . No diffin vitro and in vivo confirming that CSF exposure to GBM-derived BTICs affects their malignant phenotype. Our functional experiments showed that CSF modulates the most critical features of aggressive behavior in cancer cells: proliferation, invasiveness and tumor initiation.In this work we first demonstrated by transcriptomic analysis that CSF induces changes in pathways regulating apoptosis, survival, cell mobility, angiogenesis, response to inflammatory stimuli and metabolism. Based on these results, we identified a brain cancer CSF-induced gene signature linked to survival outcome in GBM patients in the TCGA database. We then further validated our findings As briefly mentioned earlier, clinical outcome for GBM tumors is strongly influenced by their location in the brain , 12. It Altogether, these characteristics support the theory that SVZ+ and SVZ– GBMs carry a different biological behavior, concluding that SVZ+ GBM has a much more aggressive profile with a worse overall prognosis for patients. The prognostic difference could theoretically stem from intrinsic characteristics of the tumor, or from its SVZ-contacting or SVZ-distal location. At this time, there is no conclusive evidence that links the clinical features of SVZ+ GBM to a defined molecular subtype or other intrinsic tumor characteristic . AlthougWe have shown here that pathways pivotal for cancer development are indeed modulated by exposure to CSF, particularly when derived from cancer patients (which has obvious implications in the context of tumor recurrence), as regulation of genes involved in cell proliferation, migration, angiogenesis, metabolism and inflammatory responses was observed in our microarray analysis. Most of these pathways converge upstream on the activation of common master regulator genes, such a STAT3, MYC, and FOS which have been proven consistently to be involved in tumorigenesis , althougOne of the most significant pathways identified by our analysis involves the upregulation of STAT3 and SERPINA3, which could both be induced by the presence of cytokines of the IL-6 family in the CSF . These cConsistent with the fact that the SVZ is the largest neurogenic niche in direct contact with the CSF, and with the above clinical features of GBM, our work suggests a pivotal role for CSF in GBM malignancy and progression. As we have shown here, the gene expression changes induced by CSF in GBM cells have important repercussion for the clinical outcome of these patients: the 35 CSF-induced signature genes that we used in our analysis, showed an average lower expression in patients with a significantly better survival. This suggests that modulation of targets of CSF components could contribute significantly to disease outcome. It is crucial for future studies to investigate the nature of the CSF components responsible for the changes observed in GBM cells that we have reported here, and identify the upstream regulators of such tumorigenic effects. Such CSF components could be soluble proteins or ligands present in suspended cells or exosomes. Previous reports have found both populations of shed tumor cells as well as immune cell sets in the CSF of GBM patients. However, it has been described that the lymphocyte population of the CSF in malignant brain tumors has a far fewer percentage of T cells than non-tumoral neurosurgical disorders, implicating depressed cell-mediated immunity of the cells in the CSF . Additioin vivo study was the increased tumor size in female mice compared to males. This is also somewhat in contrast with the clinical observation that GBM is more prevalent and more severe in male patients than females  CSF alone enhances proliferation and tumor initiating properties of GBM cells;2) CSF promotes GBM cells migration and invasiveness, regardless of LV proximity;in vitro and in vivo and it alters GBM gene expression to induce a malignant signature with relevant clinical implications.3) CSF affects GBM cells behavior, That CSF modulates GBM is extremely important in explaining what we observe in clinical settings of increased aggressiveness of those tumors close to the SVZ. Components of the CSF, once identified, should be the target of the next class of drugs used to treat this disease.GSE161528.The data presented in this study are deposited in the GEO repository, accession number The studies involving human participants were reviewed and approved by Mayo Clinic Internal Review Board. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Mayo Clinic Institutional Animal Use and Care Committee.AC, KC, AQ-H, YA, and HG-C: conceptualization. AC, NZ, JP, ML-V, PS-M, KC, AQ-H, and HG-C: methodology. AC, NZ, JP, ML-V, PS-M, EN, YA, and HG-C: data analysis. AQ-H, YA, and HG-C: supervision. AQ-H and HG-C: funding acquisition. All authors have reviewed, edited, read, and agreed to the published version 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.