Atrial fibrillation (AF) is associated with adverse events after cardiac surgery. Multiple studies have reported that posterior pericardiotomy (PP) may be effective for preventing AF after coronary artery bypass grafting (CABG), but some conflicting results have been reported and the quality of evidence from previous meta-analyses has been limited. The present study aimed to systematically evaluate the safety and efficacy of PP for preventing AF after CABG in adults.We conducted a quantitative meta-analysis of randomized controlled trials (RCTs) published before May 31, 2021. The primary outcome was AF after CABG under cardiopulmonary bypass. Secondary outcomes included early pericardial effusion, late pericardial effusion, pericardial tamponade, pleural effusion, length of hospital stay, length of intensive care unit (ICU) stay, pulmonary complications, intra-aortic balloon pump use, revision surgery for bleeding, and mortality.P < 0.0001). PP also effectively reduced the post-CABG occurrence of early pericardial effusion , late pericardial effusion , and pericardial tamponade  as well as the length of ICU stay , while increasing the occurrence pleural effusion . No significant differences length of hospital stay , pulmonary complications , revision surgery for bleeding , use of IABP , or death  were observed between the PP and control groups.Ten RCTs with 1829 patients (910 in the PP group and 919 in the control group) were included in the current meta-analysis. The incidence of AF was 10.3% (94/910) in the PP group and 25.7% (236/919) in the control group. A random-effects model indicated that incidence of AF after CABG significantly lower in the PP group than in the control group (risk ratio = 0.45, 95% confidence interval 0.29–0.64, PP may be a safe, effective, and economical method for preventing AF after CABG in adult patients.The online version contains supplementary material available at 10.1186/s13019-021-01611-x. Postoperative atrial fibrillation (POAF) is one of the most common complications after cardiac surgery –3. The tWe performed this meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement . BecauseThe search strategy was based on the Cochrane System Review Manual . Using tThe following inclusion criteria were used: (1) study design of randomized controlled trial (RCT); (2) study population of adult patients (≥ 18 years old) undergoing CABG surgery under cardiopulmonary bypass (CPB); (3) intervention based on random assignment to receive PP or conventional treatment (no PP); (4) comparison of PP group to control group (no PP); and (5) outcome measurement related to occurrence of AF.The exclusion criteria were: treatment with surgery other than CABG, treatment without CPB, a non-randomized design, an animal study, and patients age < 18 years.All data were independently extracted by two researchers (M.Y.F and Z.Y.L) in duplicate. The following data were independently extracted using standardized and pilot data spreadsheets: first author, year of publication, type of surgery, study design, sample size, patient characteristics , intraoperative data, length of stay, length of ICU stay, AF, early pericardial effusion, late pericardial effusion, pleural effusion, pulmonary complications, intra-aortic ballon pump (IABP) use, pericardial tamponade, revision surgery for bleeding, death in the PP group, and number of individuals in the control group. The extracted data were checked by another investigator, and any inconsistencies were resolved through consensus and discussion. The primary outcome was the occurrence of AF.The quality of evidence provided by each included study was evaluated using the recommended Cochrane bias risk assessment tool. The tool evaluates randomization (selection bias), allocation concealment (selection bias), blinding of research participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective reporting (reporting bias), and blinding of non-results information (other bias). Each study was assessed as having a low, unclear, or high risk of bias.The Grades of Recommendations Assessment Development and Evaluation (GRADE) scale was used to assess the level and strength of evidence for recommendations, as follows: high quality: further research is unlikely to change our confidence in the estimation of effects; moderate quality: further research may affect our confidence in the effect estimate and may change the estimate; low quality: further research is likely to have an important impact on our confidence in the effect estimate and may change the estimate; and very low quality: we are very uncertain about this estimate.To avoid the increased risk of Type I errors in this meta-analysis due to the scarcity of data and repeated cumulative data testing, TSA was used to determine whether evidence was reliable and conclusive. When the cumulative z-curve crosses the test sequence monitoring boundary or an invalid area, the level of evidence for intervention is sufficient and no further testing is required. If the z-curve does not cross any boundaries, there is not enough evidence to draw a conclusion, indicating that further research is still needed. In the current study, we used an alpha error of 0.05, a beta error of 0.20, a 20% reduction in the expected risk ratio (RR) of POAF, and the proportion of events from the control group in our meta-analysis to calculate the information sample size required for TSA.We chose the main outcome to be the occurrence of POAF. Secondary outcomes included in-hospital or out-of-hospital mortality, pulmonary complications, early pericardial effusion, late pericardial effusion, pericardial tamponade, pleural effusion, length of stay in the hospital, length of stay in the ICU, use of IABP, and revision surgery for bleeding.2 and χ2 tests to test the heterogeneity between test results. Statistical heterogeneity was determined by evaluating the I2 value [For each basic hypothesis, the statistical significance level of the two-tailed test was 0.05. All statistical analyses were performed using Review Manager version 5.4  and STATA version 14 . The results are expressed with the Mantel–Haenszel RR and 95% confidence interval (CI)  . We applI2 value , accordiI2 value . All metI2 value , 31 wereFigure The characteristics of the 10 included RCTs and their participants are presented in Table 2 = 64%, Q-test P = 0.003, and effect size RR = 0.40 [95% CI 0.32–0.50, P < 0.05) indicated heterogeneity among the selected studies. On sensitivity analysis, further exclusion of any single study did not substantially change the overall combined RR, which ranged from 0.37 (95% CI 0.29–0.48) to 0.47 (95% CI 0.37–0.59). The random effects model combined with RR showed that PP significantly reduced the incidence of AF after CABG . As shown in Fig. A total of 1829 participants were included in the present analysis (910 in the PP group and 919 in the control group). The cumulative incidence of AF was 10.3% in the PP group and 25.7% in the control group –35. Fixe2 = 64%, P = 0.004; Additional file 2 = 64%, P = 0.004; RR = 0.45; 95% CI 0.31–0.67, P < 0.0001), which were statistically significant and provided substantial evidence of heterogeneity. In addition, this analysis may be affected by the use of β-blockers. Four studies in which patients did not take β-blockers before surgery were analyzed and no significant heterogeneity was observed , with statistical significance [2 = 40%, P = 0.14; Additional file P < 0.05) [We next tested the source of heterogeneity via a sensitivity analysis. Upon exclusion of the study with the smallest sample size , the fixificance –21. As m < 0.05) , 34.P < 0.05; Additional file P < 0.05; Additional file P < 0.05; Additional file P < 0.05; Additional file P < 0.05; Additional file P = 0.66; Additional file P = 0.97; Additional file P = 0.60; Additional file P = 1.0; Additional file P = 0.41; Additional file Compared with the control treatment, PP effectively reduced the post-CABG occurrence of early pericardial effusion , which further clarified the efficacy of PP for preventing AF after CABG. At the same time, in this study, because the included studies were all RCTs, clinical heterogeneity was the main source of statistical heterogeneity, with studies in Turkey accounting for 60% of all studies. To further exclude clinical heterogeneity, we performed a meta-analysis of the six prospective RCTs from Turkey, and the results again showed that PP reduced the incidence of AF after CABG . While these findings may indicate that PP can improve the incidence of AF after CABG, we cannot rule out the effects of potential bias in the included studies.While the previous meta-analyses reported that PP significantly reduced the incidence of AF after cardiac surgery , 25, thoP < 0.05) and the incidence of advanced pericardial effusion  were significantly lower in the PP group than in the control group, indicating that PP can significantly reduce the incidence of pericardial effusion in patients after CABG. Previous research has shown that the incidence of supraventricular arrhythmias is higher in patients with pericardial effusion than in those without pericardial effusion [Our analysis of the effects of PP on early pericardial effusion, late pericardial effusion, pleural effusion, pulmonary complications, length of hospital stay, length of ICU stay, IABP use, and mortality produced varying results. With regard to postoperative pericardial effusion, our study showed that the incidence of early pericardial effusion . In addition, the incidence of pleural effusion in the PP group was significantly higher than that in the control group , indicating that through the PP process, fluid can be discharged freely into the left thoracic cavity, thereby significantly reducing the incidence of pericardial tamponade [P = 0.97). Therefore, the present study indicates that PP provides an effective method for chest drainage to reduce the occurrence of cardiac tamponade without increasing the risk of pulmonary complications. More trials are needed to confirm this finding.The present meta-analysis showed that PP effectively reduced the incidence of postoperative cardiac tamponade compared with that in the control group . Because the duration of treatment in the ICU is related to total hospitalization expenses [P = 0.66), IABP use , need for revision surgery for bleeding , or mortality  between the PP and control groups. Although we found no correlation between PP and these outcomes, these events were very infrequent, which precluded a comprehensive safety assessment of these results. While these results may indicate that the PP procedure did not affect hospital stay, lung complications, revision surgery for bleeding, use of IABP, and mortality, this was an observational analysis that could produce misleading results. Therefore, these results should be interpreted with caution.Our analysis of the postoperative ICU stay showed that PP shortened the ICU stay compared with that in the control group  after removal of the study with the smallest sample size. Figure S2. Pooled analysis for the comparison of the risk for postoperative atrial fibrillation (POAF) without preoperative oral β-blockers. Figure S3. Pooled analysis for the comparison of the risk for postoperative atrial fibrillation (POAF) in Turkey. Figure S4. Pooled analysis for the comparison of the risk for postoperative early pericardial effusion. Figure S5. Pooled analysis for the comparison of the risk for postoperative late pericardial effusion. Figure S6. Pooled analysis for the comparison of the risk for postoperative pericardial tamponade. Figure S7. Pooled analysis for the comparison of the risk for postoperative length of stay in intensive care unit (ICU). Figure S8. Pooled analysis for the comparison of the risk for postoperative pleural effusion. Figure S9. Pooled analysis for the comparison of the risk for postoperative length of hospitalization. Figure S10. Pooled analysis for the comparison of the risk for postoperative pulmonary complications. Figure S11. Pooled analysis for the comparison of the risk for postoperative revision for bleeding. Figure S12. Pooled analysis for the comparison of the risk for postoperative intra-aortic balloon pump (IABP) usage. Figure S13. Pooled analysis for the comparison of the risk for postoperative death. Table S1. Main postoperative data from random controlled trials included in the meta-analysis. Table S2. Actual mode of PP and use of posterior pericardial drains.


We sought to investigate the effect of cilengitide on the neovascularization of abdominal aortic plaques in rabbits and explore its underlying antiangiogenic mechanism on human umbilical vein endothelial cells (HUVECs).  Cilengitide is a selective in vivo experiment, the abdominal aortic plaque model of rabbits was established and injected with different doses of cilengitide or saline for 14 consecutive days. Conventional ultrasound (CUS) and contrast-enhanced ultrasound (CEUS) were applied to measure the vascular structure and blood flow parameters. CD31 immunofluorescence staining was performed for examining neovascularization. Relative expressions of vascular endothelial growth factor (VEGF) and integrin of the plaque were determined. For in vitro experiments, HUVECs were tested for proliferation, migration, apoptosis, and tube formation in the presence of different doses of cilengitide. Relative expressions of VEGF, integrin, and Ras/ERK/AKT signaling pathways were determined for the exploration of underlying mechanism.  For the in vitro experiments, cilengitide treatment was found to inhibit the proliferation, migration, and tube formation of HUVECs. However, cilengitide did not induce the apoptosis of HUVECs. A higher dose of cilengitide inhibited the mRNA expression of VEGF-A, β3, and β5, but not αV. Lastly, cilengitide treatment significantly inhibited the Ras/ERK/AKT pathway in the HUVECs. Conclusions. This study showed that cilengitide effectively inhibited the growth of plaque size by inhibiting the angiogenesis of the abdominal aortic plaques and blocking the VEGF-mediated angiogenic effect on HUVECs. CEUS showed modestly increased size and eccentricity index (EI) of plaques in the control group. Different degrees of reduced size and EI of plaques were observed in two cilengitide treatment groups. The expressions of VEGF and integrin in the plaque were inhibited after 14 days of cilengitide treatment. The neovascularization and apoptosis of the abdominal aorta were also significantly alleviated by cilengitide treatment. For  Stabilizing vulnerable plaque to prevent the occurrence and development of acute cardiovascular and cerebrovascular events is an important goal for the medical community . VulneraAngiogenesis is the formation of new capillaries from preexisting blood vessels. It is regulated by proangiogenic and antiangiogenic factors. Among the proangiogenic factors, vascular endothelial growth factor (VEGF) is the most widely understood mediator in angiogenesis . VEGF plIntegrin-mediated signaling is essential for stabilizing cell adhesion and promoting cell migration, proliferation, and survival , 10. Intαvβ3 and inhibits integrin αvβ3 and αvβ5 functions. Previous studies have confirmed that cilengitide can inhibit angiogenesis and migration in various tumors and promote the apoptosis of vascular endothelial cells, thus inhibiting tumor development [Cilengitide EMD121974) is a cyclic arginine-glycine-aspartate- (RGD-) derived peptide that has a high affinity for integrin elopment . Althoug1974 is aTwelve male New Zealand white rabbits weighing 2.6-3.1 kg (2.811 ± 0.142 kg) were provided by the Zhejiang University Experimental Animal Center  2017-0004). The experimental animal protocol was approved by the Institutional Animal Care and Use Committee of Zhejiang University . The disposal of euthanized animals during the experiments conformed to the Laboratory Animal-Guideline for ethical review of animal welfare  and was approved by the Animal Experimentation Ethics Committee of Zhejiang University. The high-fat feed consisted of 10% lard + 3% cholesterol + 87% essential ingredients at ordinary levels and was bought from Beijing Keao Xieli Feed Co., Ltd.   2019-0003).The rabbits were anesthetized with 3% pentobarbital sodium (30 mg/kg) intravenously. The right femoral artery was then freed under sterile operating conditions after local shaving and disinfection. Following a successful puncture of the femoral artery with a 24-gauge indwelling needle, the Choice PT guidewire was fed to the thoracic aorta using a retrograde approach. A 3.5 mm × 15.0 mm balloon catheter was introduced into the descending aorta about 20 cm along the guidewire. After filling the balloon to 8 standard atmospheric pressure, it was pulled back to the iliac artery at a constant speed for about 5 seconds each time. The abdominal intima was injured five times. The right femoral artery was ligated, and the wound was sutured. After the operation, an intramuscular injection of 400,000 IU/day penicillin was administered for three consecutive days to prevent infection. The rabbits were returned to their housing room and fed with a high-fat feed at 200 g/day.After continuous feeding for 8-10 weeks, the abdominal aortas of model animals were examined with conventional ultrasound (CUS) and contrast-enhanced ultrasound (CEUS) before cilengitide treatments. Local intima-media thickness (IMT) of ≥0.5 mm containing contrast medium was used to indicate that the abdominal aortic plaque model had been established. The inner diameter, peak systolic velocity (PSV), and resistance index (RI) of the abdominal aorta were routinely measured. Simultaneously, the number, position, and size of each plaque, as well as the vascular remodeling index (VRI) and the plaque eccentricity index (EI), were measured and calculated by CEUS. These parameters were then evaluated again after the cilengitide treatment for comparison. The amount of contrast agent in the plaque was evaluated from the number of points reflecting the degree of blood perfusion in the plaque. All ultrasound examinations were performed as blind procedures by an individual ultrasound expert.VRI was calculated as VRI = total vessel area (TVA) at the plaque/TVA of the plaque reference sites . The TVAn = 4 each): T0, T1, and T2 groups. Cilengitide was purchased from MedChemExpress  and was dissolved in dimethyl sulfoxide (DMSO) with a concentration of 100 mg/mL for storage. Before injecting, cilengitide was diluted into a final concentration of 100 μg/mL with saline and administered once daily by intravenous injection in the T2 (200 μg/day) and T1 (100 μg/day) groups. The T0 group was injected with saline using the same volume of cilengitide administered to the T1 group. All groups were treated continuously for 14 days. During the treatment, one accidental death occurred in each of the T1 and T0 groups. On the 15th day, the inner diameter, IMT, PSV, RI of the abdominal aorta, plaque size, and EI at the original mark were measured and recorded by CUS. If new plaque formation was found, the location, size, and echo characteristics of the new plaque were measured and recorded. The degree of contrast agent perfusion was simultaneously analyzed by CEUS.Twelve rabbits with abdominal aortic plaques, confirmed by CUS, were randomly divided into three groups  and sacrificed. Their abdominal aortas were separated. Each aorta specimen was cut to a length of 8 cm according to the ultrasound-labeled plaque position. The plaques were then precisely located by calculating their distances from the right kidney. Each plaque was cut into two halves, one for the histological analysis and the other for the molecular experiment. The former ones of the aorta were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Then the samples were serially sectioned. The sections were performed with the haematoxylin-eosin (H&E) staining, Movat-Russell modified pentachrome (Movat) staining, CD31-positive staining , and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The Movat staining was conducted according to the protocol of the commercial kit . The TUNEL staining was performed following the instruction of the In Situ Cell Death Detection Kit . The points merged by green (TUNEL) and blue (DAPI) were considered the TUNEL-positive cell. The ratios of TUNEL-positive cell and DAPI-positive cell were calculated as the TUNEL-positive cell percentage.The aortas containing the plaques were washed with cold normal saline and quickly removed. A part of the aorta was stored in the freezer at -80°C for uniform testing of western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the rest was used for the ELISA assay. The mRNA and protein levels of VEGF-A were determined by RT-qPCR and western blotting, respectively. Meanwhile, the absolute protein contents of VEGF-A and VEGFR2 were examined by the ELISA kit  according to the protocol of the manufacturer.2. Cells between passages 5-15 were then utilized. Before plating cells, the cell plates were added with 2 μg/mL vitronectin  at 4°C overnight in cell experiments except for tube formation assay. For proliferation, apoptosis, and migration assays, cells were cultured in DMEM alone overnight for starvation. Cells treated with 0 μM cilengitide were used as a control group and received DMSO in the same volume as the other groups that received cilengitide.HUVECs were purchased from American Type Culture Collection . We cultivated the cell in low-glucose Dulbecco's modified Eagle medium  supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 100 ng/mL streptomycin . Cells were incubated at 37°C in an atmosphere with 5% CO3 and then cultured for 24 hours without or with cilengitide at different concentrations. Cell viability was monitored by a Cell Counting Kit-8 (CCK-8) . Briefly, CCK-8 reagents (10 μL) were added to each well and incubated for 1 hour at 37°C. The absorbance at 450 nm was measured by a microplate reader to assess the number of viable cells.Cells were seeded in duplicate in 96-well plates at a density of 8 × 104 and cultured at 37°C for 24 hours. Scratch wounds were created by scraping the cell monolayer with a sterile 10 μL pipette tip. HUVECs were starved by culturing in DMEM without FBS overnight, then treated with cilengitide at different concentrations at 37°C for 24 hours. Untreated cells were used as controls. The migrated cells were imaged in five randomly selected fields of view with a phase-contrast microscope . The percentage of migration was analyzed by the ImageJ software .HUVEC migration was measured by a wound-healing migration assay. Cells were seeded in a 6-well tissue culture plate at a density of 3 × 104 per well. After exposure to 0-10 μM cilengitide, the cells were collected and washed twice with phosphate-buffered saline (PBS), then resuspended in binding buffer at 1 × 106 cells/mL. This suspension was incubated in 5 μL Annexin V-FITC and PI solutions for 15 minutes in the dark at room temperature. Flow cytometric analysis of apoptosis was performed within 1 hour.Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining was used to detect cell apoptosis. HUVECs were plated in 6-well tissue culture plates at a density of 3 × 104 cells/well) together with different concentrations (0-10 μM) of cilengitide were plated on polymerized Matrigel (growth factor reduced)  in 96-well plates. Untreated cells were used as controls. After 8 hours of incubation at 37°C in a cell culture incubator, tube-like structure formation was imaged using an inverted microscope . The length of tubes, tube areas, and the number of capillary-like structures were quantified from representative fields using the ImageJ software.HUVECs  (see ΔΔCt− method.Using the TRIzol reagent (Invitrogen), total RNA was isolated from the arterial tissue or cell samples as previously described. The mRNA was converted into cDNA and performed with quantitative PCR using SYBR® Premix Ex Taq™ II (Takara) by the 7500 Fast Real-Time PCR System . ina) see . PCR proμg of each, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 hour at room temperature, the membranes were incubated at 4°C with primary antibodies overnight as follows: VEGF-A , MCP-1 , MMP-2 , MMP-9 , p-ERK , ERK , p-AKT , AKT , Ras , and β-actin . The membranes were then washed with TBST three times and incubated with the secondary antibody  at room temperature for 1 hour. After washing three times with TBST, the membranes were visualized with an enhanced chemiluminescence detection kit . Antibodies against GAPDH, ERK, and AKT were used as internal references. ImageJ was used to analyze the relative quantity of each protein.Frozen abdominal aorta specimens were lysed by RIPA containing phenylmethylsulfonyl fluoride (PMSF) and homogenized with a homogenizer. Then, the lysis buffer was separated by centrifugation at 14,000 g/min for 5 minutes at 4°C, and the supernatant was taken for western blotting. Similarly, the cell samples were lysed by RIPA containing PMSF and collected for detection of the protein expressions. Total protein concentrations were confirmed by a BCA protein assay kit . Protein samples, 20-50 t-test was used to compare differences between two groups, and one-way ANOVA was applied for the comparison among three or more groups. The control group was set up as 1 for each independent experiment; thus, the SEM for controls was zero. All statistical analyses were performed by GraphPad Prism 8.0. Differences with a p value < 0.05 were considered statistically significant.Experiments were repeated at least three times. All experimental data are presented as the mean ± standard error of the mean (SEM). A two-tail unpaired One rabbit died unexpectedly in groups of T1 and T0, while the rest model animals completed the experiment. We totally detected three, five, and seven plaques by CUS and CEUS before cilengitide treatment among the model animals in T0, T1, and T2 groups, respectively. It indicated the successful establishment of the abdominal aortic plaque model. Then, the changes of each plaque were recorded before and after the cilengitide treatment. Both CUS and CEUS examinations provided adequate images of abdominal aortic plaques in model animals. However, the CEUS showed the plaque boundary more clearly see .p value > 0.05 for all comparisons, p value > 0.05 for all comparisons, αvβ3 inhibitor cilengitide could effectively inhibit the growth of abdominal aortic plaques in model rabbits. It appeared that the higher-dose treatment (200 μg/day) might have been more effective.Evaluated by CUS, we found that there were no statistically significant differences in basic parameters of the abdominal aorta , RI, and VRI among the three groups before or after cilengitide treatments  significantly reduced these elevated expression levels. Also, we confirmed the effect of cilengitide on integrin expression. The relative mRNA expressions of αv, β3, and β5 were all inhibited in the T1 and T2 groups compared with those in the T0 group  could significantly reduce the length of the tube. Higher doses of cilengitide (1 μM and 10 μM) also inhibited the length of the tube, tube areas, and the number of capillary-like structures  acted as sensors and effectors of the vessels that secreted various vasoactive substances against multiple stimulations. The changes regulated by ECs could affect vascular structure and function . So we aor cells , 32. HowVEGF is one of the major angiogenic growth factors that regulate angiogenesis. The VEGF gene family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and PGF. Of these, VEGF-A plays a significant role and is highly expressed in atherosclerotic plaques. Recognized by VEGF receptors, primarily VEGFR-2, VEGF-A promotes endothelial cell proliferation, differentiation, and migration. Besides, it participates in neovascularization through different signaling pathways, such as Ras, ERK, and AKT pathways , 34. In β3 and β5, but not αV. The mechanism of this regulation of integrin expression needs further investigation. Besides, we found that cilengitide inhibited the PI3K/AKT and Ras/MAPK pathways, which were classical pathways that regulated cell proliferation, differentiation, and survival. Collectively, integrin αvβ3 is not only a potential target molecule for tumor diagnosis and treatment but is also a specific target for molecular imaging and treatment of vulnerable plaques [We also showed that cilengitide downregulated the expression of integrins  plaques , 36. TheThere are some limitations to this study. First, the number of rabbits was limited, and our use of only two doses groups was insufficient to determine the optimal cilengitide concentration to inhibit plaque growth and reverse remodeling. Second, we just tested a two-week treatment duration, which did not provide information on long-term efficacy. Third, we did not test whether the rabbit's abdominal aortic plaques would stop growing or even become smaller after the high-fat diet is stopped, with or without drug treatment. Besides, the inhibitory role of cilengitide on the Ras/ERK/AKT signaling pathway was only an observational result. Direction evidence was not provided between the reduced VEGF expression and this signaling pathway. Finally, the contrast agents used in this study, micron-level microbubbles, were limited for visualizing contrast in plaque. It is hoped that developing smaller  contrast agent microbubbles might allow more contrast agents to enter into the new blood vessels resulting from angiogenesis in plaque.Cilengitide effectively inhibited neovascularization and plaque growth in the abdominal aortic plaque models in a rabbit model by reducing the expression of VEGF. Cilengitide also inhibited normal endothelial cell proliferation, migration, and tube formation without leading to cell death. One mechanism for the inhibitory effect of cilengitide on HUVECs is the regulation of VEGF expression. Further studies are needed to reveal the mechanism of cilengitide regulation of integrin gene expression.


L. feeleii is one of the most frequent Legionella species isolated from natural pools of the central region of Spain. This study aimed to evaluate its ecology and to identify this Legionella species as a respiratory pathogen. A PCR assay for detecting the L. feeleii mip gene was developed to identify it in clinical and environmental samples. Culture and PCR were performed in environmental samples from four drinking water treatment plants (DWTPs). Free L. feeleii was only detected in raw water samples (3.4%), while L. feeleii as an Acanthamoeba endosymbiont was found in 30.7% of raw water, 11.5% of decanter biofilm, and 32% of finished water samples. Therefore, Acanthamoeba spp. plays an essential role in the multiplication, persistence, and spread of Legionella species in the environment. The first case of Legionnaires’ disease caused by L. feeleii in Spain is described in this study. The case was diagnosed in an older woman through PCR and sequencing from urine and sputum samples. A respiratory infection could be linked with health care procedures, and the patient presented several risk factors . The detection of non-L. pneumophila, such as L. feeleii, is a factor that must be considered when establishing or reviewing measures for the control and prevention of legionellosis. Legionella, a causative agent of pulmonary infection, is ubiquitous in aquatic ecosystems. It was first described from an outbreak of pneumonia in Philadelphia (United States) during the American Legion convention in 1976  as an intracellular pathogen, although it can also be free-form in aquatic ecosystems  subspecies, and more than 78 serotypes of terature . Legioneof Spain . Althoug sources . Taking ogroup 1 . Thus, idetected .Legionella detection in urine samples demonstrated that a large number of cases were identified by PCR assay rather than by antigen immunochromatography test  genes of Legionella spp. (The gold standard technique (isolation by culture) and pioneering methods (immunofluorescence) for the diagnosis of legionellosis are rarely used nowadays . In contphy test . Therefo methods . Some oflla spp. .mip gene of L. feeleii as an alternative molecular method for environmental and clinical samples and (ii) to use it to verify the possible presence of this Legionella species as a respiratory pathogen, as well as an environmental contaminant of waters from DWTPs in the central area of Spain.Therefore, the aims of this study were, (i) to develop a PCR assay in order to detect the Legionella strains  were used in this study. All strains were cultured on Legionella selective medium GVPC at 37°C for 3–12 days. Other respiratory pathogens were cultured on their specific agars such as blood-agar media, MacConkey, cetrimide, chocolate and sabouraud agar. From Gram-negative bacteria, genomic DNA was extracted by heat shock (99°C for 20 min) and purified using the NucleoSpin® Gel and PCR Clean-up extraction kit . Gram-positive bacteria and fungal pathogens genomic DNA were extracted using the NucleoSpin® Genomic DNA from tissue kit , according to the manufacturer’s instructions. The genomic DNA was eluted with 100 μL of Milli-Q® water and stored at −80°C until PCR assay.Nine (i)Oligonucleotide primers: Lmip_F (5′-GGCAATGTC AACGACAATTGC-3′) and Lmip_R (5′-TGGCCAT CAATTAACTTGCCAGT-3′) were designed from partial amplification of the mip gene of L. feeleii serogroups 1 and 2. Primers amplified from 417 to 863-bp from Genebank sequence accession numbers U92205 and AF022341.in silico analysis through Bioedit 7.2 was performed to evaluate the binding of the primers Lmip_F and Lmip_R with the DNA sequences of the mip gene Legionella species (An  species . These s(ii)PCR conditions: Five microliter of extracted DNA was used in 20 μL reaction mixture that included 0.5 μL of each primer 20 μM, 9 μL of Milli-Q® water, and 10 μL of 2X Phusion flash high-fidelity PCR master Mix . Thermal cycling was performed with a GeneAmp® PCR System 9700 , and cycling conditions were: 1 cycle at 98°C for 15 s, 35 cycles at 98°C for 1 s, 60°C for 5 s and 72°C for 15 s, with a final extension at 72°C for 1 min.(iii)Detection of amplified products: PCR products were visualized by electrophoresis in 2% agarose gels with ethidium bromide staining (0.5 μg/mL). DNA fragments of 447-bp were purified using NucleoSpin® Gel and PCR Clean-up extraction kit , following the manufacturer’s instructions.(iv)Sequencing: Purified PCR amplicons were sequenced at both ends with PCR primers through Macrogen laboratories (South Korea) sequencing service. The sequences were analyzed with Bioedit Sequence Alignment Editor 7.2 and were confirmed by similarity using the BLAST .(v)Control material: An environmental strain of L. feeleii ; (ii) 50 L of water were collected and concentrated using IDEXX® Filta Max system, according to the manufacturer’s instructions.Samples were obtained using two methods: (i) 1 L of water was collected in a sterile plastic bottle and concentrated following ISO standard 11,731-2:2004 method .L. feeleii by culture isolation and PCR . The genomic DNA was purified using NucleoSpin® Gel and PCR Clean-up extraction kit  and eluted in 50 μL Milli-Q® water. The extracted DNA was stored at −80°C until analysis.Hundred microliter of each environmental sample were cultured in selective medium GVPC plates and incubated at 37°C for 10–12 days. These cultures were monitored periodically. Suspect colonies were incubated in buffered charcoal yeast extract agar (BCYE) with and without TM kit  from 200 μL of each concentrated water sample and homogenized biofilms, modifying the manufacturer’s protocol. Briefly, to each Fastprep tube another 1/4-inch ceramic sphere was added, and lysing cycles were performed in triplicate. The extracted DNA was stored at −80°C until PCR assay.Genomic DNA extraction was performed with FastDNAEscherichia coli. The plates were incubated at 28°C for 14–20 days. Isolation of Acanthamoeba spp. was performed according to the protocol described by Eighty microliter of environmental samples were inoculated onto 2% Neff’s saline non-nutrient agar plates seeded with heat shock inactivated ® Gel and PCR Clean-up extraction kit . DNA elution was performed with 50 μL Milli-Q® water and stored at −80°C until PCR assay.Genomic DNA was extracted from 100 μL of diluted FLA culture by heat shock (99°C for 20 min) and purified using NucleoSpinA case of atypical pneumonia was identified according to criteria established by the Spanish Society of Pneumology and Thoracic Surgery (SEPAR) .Urine and sputum samples were collected from a patient with pneumonia.(i)Legionella and Streptococcus pneumoniae by urinary antigen test: The urine sample was analyzed with Legionella urinary antigen card kit and Streptococcus pneumoniae antigen card kit  following the manufacturer’s instructions. These commercial kits, immunochromatography tests, are commonly used in hospitals for the qualitative detection of L. pneumophila serogroup 1 and pneumococcal infection.(ii)Sputum culture: An optimal specimen was graded using the Murray and Washington system  blood agar and chocolate agar/incubation at 37°C in CO2 (5–7%) for 2 days.n system . The spu(iii)DNA extraction from sputum and urine samples: Genomic DNA was extracted from both samples by NucleoSpin® Genomic DNA from tissue kit , following the manufacturer’s instructions. DNA was eluted in 50 μL Milli-Q® water and stored at −80°C until PCR assay.(iv)L. feeleii DNA and sequencing confirmation:Identification of  The PCR assay developed in this study was performed for specific detection of L. feeleii DNA. Additionally, a semi-nested PCR described by Legionella spp. partially. Positive controls were genomic DNA of L. pneumophila serogroup 1 (NCTC12821) and L. feeleii [collection of environmental bacteria from San Pablo CEU University . We were authorized to sample raw water, finished water, and decanter samples to detect This clinical research was carried out in compliance with fundamental ethical principles, including those set out in the Charter of Fundamental Rights of the European Union and the European Convention on Human Rights. The patient attested her agreement to participate in this epidemiological survey through a written informed consent, which was evaluated and approved by the Research Ethics Committee of San Pablo CEU University, following the recommendations of the Spanish Bioethics Committee, the Spanish legislation on Biomedical Research  and Personal Data Protection . The patient’s identification data was kept on a condition of anonymity.mip gene of L. feeleii. A specific 447-bp DNA fragment amplified by Lmip primers was observed in 0.05 pg of L. feeleii DNA using the present PCR protocol  to that of L. feeleii , might show a weak amplification of a non-specific target of 700 and 1,000-bp, respectively . On the other hand, L. feeleii as an Acanthamoeba endosymbiont was found in 30.7% of raw water (8 out of 26 samples), 11.5% of decanter biofilm (3 out of 26 samples), and 32% of finished water samples (8 out of 25 samples). Legionella state (free-living or endosymbiont).The detection of L. feeleii was only identified at the entrance of the water treatment line of DWTP1 (raw water). On the other hand, the presence of FLA in DWTPs seemed to increase the presence of L. feeleii in raw water (20–50%) and finished water (0–60%). Regarding the decanting system, the development of biofilms in DWTPs 2 and 3 seemed to facilitate interaction between L. feeleii and FLA  in October 2013, presenting sudden onset of fever (38°C) with shivering of 48 h evolution, productive cough, dyspnea, and pleuritic chest pain. The patient had undergone ligation of esophageal varices, without complications, except for the appearance of pulmonary symptoms at 12 h after surgery. In addition, medical history revealed a diagnosis of diabetes mellitus type II in 1992 (insulin-dependent), hepatitis C virus genotype 1b infection in 1993 (untreated), and moderate mitral insufficiency.The first case of Legionnaires’ disease caused by On physical examination, pulmonary auscultation revealed hypoventilation and crackling in the right lung. Chest radiography showed condensation at the base of the right lung , and com4 cells/μL with 84% of neutrophil granulocytes, mild thrombocytopenia (8.6 × 104 cells/μL), and a C-reactive protein level of 16.25 mg/L . Arterial blood gas analysis showed decreased pO2 level (59 mmHg) and a 91% oxygen saturation . Samples were collected for microbiological analysis, and the patient was treated empirically with ceftriaxone (2 g/day) and azithromycin (500 mg/day) through an intravenous line (IV), combined with a bronchodilator (Atrovent® 500 μg/8 h). The following day, intravenous azithromycin was replaced orally.Laboratory analysis revealed a white blood cell count of 1.27 × 10Legionella and Streptococcus pneumoniae urinary antigen tests were negative. In order to establish the presence of Legionella, extraction of genomic DNA from urine and sputum samples was performed. Semi-nested PCR for amplification of Legionella spp. DNA (16S rRNA gene) was positive in the sputum sample. Also, a 447-bp DNA band was amplified from urine and sputum samples by conventional PCR targeting the mip gene of L. feeleii. The amplified product was sequenced, and alignment tests were carried out revealing a 99% similarity with L. feeleii (GenBank accession number AF023174). The sequence of the amplified product from urine and sputum samples was registered under accession number MW292583.Respiratory saprophytic microorganisms were isolated from sputum culture. The patient evolved favorably, without complications, and 4 days post-admission, was discharged with oral antibiotic therapy at home (levofloxacin 500 mg/day) for 7 days. At follow-up, 1 month later, a chest x-ray showed resolution of the infection, with no infiltrates.by L. feeleii in Spain.The Legionella feeleii was described for the first time in 1981 during a Pontiac fever outbreak in an automobile assembly plant in Canada, in which a total of 317 workers were affected  after L. pneumophila (58.5%) (affected . This Lepatients . The mor (58.5%) .L. pneumophila non-serogroup 1 and other Legionella species   .mip gene has been demonstrated to be a sensitive and specific method for differential and simultaneous detection of some non-L. pneumophila species which can cause diseases in humans and can also be present in environmental sources, such as L. feeleii and L. micdadei . The results may suggest that FLA are an important environmental reservoir for the amplification, persistence, and spread of this Legionella species from DTWPs to drinking water supply facilities. Taking into account that a high frequency of Acanthamoeba spp. has been described in other DWTPs (77–87%) and water sources (40–90%) from Spain, elimination strategies and control measures should be improved to remove FLA from DWTPs , diabetes, heart disease, and recent surgery. Additionally, other possible causes of pneumonia and respiratory pathogens were dismissed. In summary, we suggest that this pneumonia episode was a probable case of Legionnaires’ disease associated with health care facilities, according to the criteria established by the European Legionnaires’ Disease Surveillance Network and the Spanish Society of Pneumology and Thoracic Surgery (SEPAR)  .L. feeleii and the first nosocomial Legionnaires’ disease produced by non-L. pneumophila species. Legionella pneumophila in Spain to the present. Although Legionellosis cases are kept under surveillance by the autonomous communities of Spain and are weekly reported by the Red Nacional de Vigilancia Epidemiológica (RENAVE), the Legionella species associated with them is not described. The current description highlights the importance of a deeper study of Legionellosis disease cases as they might be underdiagnosed.In Spain, this represents the first reported case of pneumonia by L. feeleii associated with a surgical procedure (L. pneumophila from Intensive Care Units (ICU), endotracheal intubation, mechanical ventilation, and nasogastric tubes   and 75% in Denmark . This moen test) . Higher L. pneumophila is a factor that must be considered when establishing measures for control and prevention of legionellosis and when investigating the sources of infection giving rise to outbreaks since its presence in natural reservoirs and drinking water distribution systems may represent a risk factor.The detection of non-The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Research Ethics Committee of San Pablo CEU University. The patients/participants provided their written informed consent to participate in this study.LV wrote the first draft of the manuscript. LV, TG, FI, AM, SL, DO, SS, AA, CG, CP, BB, SF, and CH-M contributed to collection of samples. LV, TG, FI, AM, BB, and CH-M performed the processing and analysis of the samples. ML contributed to the interpretation of clinical case and approved its publication. FI, CA, SF, and CH-M contributed to the conception and design of the work. CA and CH-M contributed to the funding acquisition. All authors contributed to manuscript revision, read, 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.


Silybum marianum in starter and grower rations (over the whole rearing period) made it possible to obtain the highest body weight at the lowest feed conversion per body weight gain unit, without influencing muscularity and fattening grade, at the same time improving the meat’s value for health.This study shows how introducing milk thistle seeds into broiler chicken feed rations affects rearing results , carcass composition and meat quality , the chemical composition  and organoleptic properties  of meat. Based on the results the use of milk thistle seeds in broiler chicken starter/grower diets can be recommended in the amount of 0/2% or 2/3%, respectively. However, the introduction of Silybum marianum added to feed rations over the whole rearing period (group MT23) increased above 3% the birds’ body weight on rearing day 42. (p < 0.05) and decreased about 7% the feed conversion ratio (p < 0.05) in comparison to group C. No effect of feeding on the carcass composition was observed, including on muscularity and fattening grade, although diets containing milk thistle reduced  the content of crude fat in chicken leg muscles (p < 0.05). The highest (p < 0.05) content of polyunsaturated fatty acids (PUFA) was determined in the breast (38.06%) and leg (37.63%) muscles of chicken receiving feed rations containing Silybum marianum throughout the rearing period. No effect of nutrition on the evaluated physical properties of muscles was observed, except on the decrease in lightness color (L*) and increase in values a* and C as well as a decrease of water holding capacity of the breast muscles. It was found that Silybum marianum in chicken diets had a positive effect on the evaluated meat flavor characteristics of the muscles. To sum up, based on the study results, including ground seeds of milk thistle in broiler chickens nutrition can be recommended in the amount of 2/3% in starter/grower diets, respectively.The studies aimed to evaluate the impact of milk thistle seeds in broiler chicken feed rations on rearing results, carcass composition and meat quality. The experiment involved 120 broiler chickens randomly allocated to three equinumerous groups . Each group was divided into five subgroups of eight chickens each. Over the first 21 days of life the birds were fed starter rations, and over the following 21 days received grower rations. Chicken starter/grower diets in groups MT02 and MT23 were supplemented with ground seeds of milk thistle in the amount of 0/2% (MT02) and 2/3% (MT23). It was demonstrated that  The European Union’s prohibition of using antibiotics in feeds as growth stimulants made breeders and feed producers look for new nutritional solutions. Apart from probiotics, prebiotics, organic acids, antioxidants, and feed enzymes, various preparations of plant origin—so-called phytobiotics—have raised more and more interest ,2,3,4,5.Herbs and herbal preparations contain active biological ingredients, i.e.,: essential oils, tannins, glycosides, flavonoids, terpenes, mucilages, and organic acids ,8,9,10. Silybum marianum [L.] Gaertn) is increasingly popular in animal nutrition  Gaertn.) contained 219 g/kg of total protein and 238 g/kg of crude fat with an advantageous fatty acid composition . The content of MUFA (monounsaturated fatty acids) exceeded 24.60% and PUFA (polyunsaturated fatty acids) were predominant—54.99%. Among all monounsaturated acids, milk thistle contained the largest share of oleic acid—23.59%, and the polyunsaturated acids profile was dominated by linoleic acid—54.64%. As a result, nearly 85% of total fatty acids (FA) in milk thistle seeds were neutral and hypocholesterolemic acids (DFA).The inclusion of milk thistle seeds in starter/grower diets in the amount 2/3% (MT23 group) significantly (by more than 3%) increased the body weight of broilers on the 42nd day of rearing in comparison to group C .p ≤ 0.05) conversion of feed. Differences in FCR between groups MT02 and MT23 and group C were 3% and 7%, respectively, throughout the rearing period. The type of feed rations used did not affect the post-slaughter performance, except the share of drumstick muscles (Milk thistle in starter diets (group MT23) significantly decreased feed conversion in comparison to other groups. In the second rearing period and throughout the entire rearing period—chickens fed diets with milk thistle showed more efficient ( muscles .Silybum marianum in starter and grower diets (MT02 group) significantly increased the share of drumstick muscles in cold carcass compared to muscles of birds from MT23 group (9.03% vs. 8.13%).The presence of ground seeds of The nutrition used significantly affected the content of crude ash in breast muscles and the level of crude fat in leg muscles .p < 0.05) the content of crude fat in leg muscles.The breast muscles of birds from group MT02 contained significantly less crude ash than the muscles of birds from group C. Milk thistle seeds in chicken diets (groups MT02 and MT23) contributed to decreasing  in leg muscles only. Milk thistle introduced into chicken diets increased the content of linoleic acid (C18:2) in both evaluated muscle types, but a higher content of this acid was found in the muscles of chickens from group MT23 in comparison to the control group. Breast muscles of birds receiving feed rations with milk thistle in both rearing periods (MT23 group) contained by about 42% more linoleic acid than in group C (p < 0.05). A high (p < 0.05) content of PUFA was determined in the breast (38.06%) and leg (37.63%) muscles of chicken receiving feed rations containing ground seeds of Silybum marianum throughout the birds’ rearing period in comparison to group C. In addition, the breast muscles of birds from group MT23 featured a lower (p < 0.05) ratio of n–6:n–3 fatty acids in comparison to chickens fed rations without the phytobiotic.Feeding chickens with feed rations containing 2% (starter) and 3% (grower) of Silybum marianum into chicken diets did not affect the evaluated physical properties  of thigh muscles, but it changed the color and WHC of breast muscles (The introduction of  muscles .p < 0.05). In addition, the use of milk thistle in the diets of birds intensified meat color toward red (a*) and increased chroma (C) of breast muscles compared to the C group. The water holding capacity (WHC) was lower (p < 0.05) in the breast muscles of chickens receiving diets containing Silybum marianum.Breast muscles of chickens from group MT02—fed with rations containing the evaluated phytobiotic at the grower stage only—were darker (p < 0.05) impact on the evaluated taste characteristics  of breast and thigh muscles, except on juiciness in breast muscles (p > 0.05) (Adding ground seeds of milk thistle both in starter diets (20 g/kg) and grower diets (30 g/kg) and only in grower diets (20 g/kg) had a significant ( > 0.05) .Breast and thigh muscles of chickens fed with rations containing milk thistle in both rearing periods (group MT23) scored the highest.Silybum marianum [L.] Gaertn.) ranged from 161 to 250 g/kg as reported by many researchers [The content of total protein in the evaluated seeds of milk thistle  and oleic acid (23.59% FA). Similar content of fatty acids was measured by Garaev et al. [Silybum marianum seeds were found by Khan et al. [An analysis of the fatty acids profile of v et al. , Růžičkov et al.  and Harrv et al. , who in n et al. , whereasn et al.  and Majin et al. , respectSilybum marianum depending on the year of harvest were revealed by Sadowska et al. [Grela at al.  demonstra et al. .p < 0.5) higher for group MT–15 (rations 15 g/kg of milk thistle) compared with other experimental groups. In turn, Gharahveysi [Silybum marianum was 3.440 and 3.407 kg, respectively, and body weight was 1.810 and 1.793 kg, respectively. Other authors who demonstrated a reduction in the productivity of broiler chickens after including Silybum marianum in their diets were Suchý et al. [Silybum marianum introduced into feed rations for broiler chickens decreased (by about 10%) daily weight gain and increased (by 4.5%) the feed conversion rate (FCR) in comparison to birds fed diets without milk thistle. The weight gain of chickens decreased after using 5% or 15% of milk thistle (Silybum marianum) oil cake in feed rations, as noted by Šťastník et al. [A positive impact on weight gain and feed conversion after introducing milk thistle into the feed rations for broiler chickens was observed by Tedesco et al.  and Muharahveysi  found thý et al. , Kalantaý et al.  and Šťasý et al. . Suchý eý et al.  found thý et al.  showed tk et al. .Silybum marianum to broiler diets was observed by Schiavone et al. [p < 0.05) in the dressing percentage of chickens after adding Silybum marianum in the amount of 15 g/kg of feed rations was observed by Ahmad et al. [A decreased carcass yield after adding e et al.  and Šťase et al. . Šťastníe et al.  using 5%e et al. . On the d et al. .Silybum marianum in broiler chicken diets on the share of breast muscles in the carcass is consistent with the findings of Šťastník et al. [Silybum marianum in diets had no impact on the share of abdominal fat. A significant decrease in the share of abdominal fat in the carcasses of birds fed with rations containing milk thistle extract (250 mg/kg) was found by Tavakolinasab et al. [In the production of broilers breast muscle weight in relation to carcass weight is of economic significance. Breast muscles account for about 30% of edible meat in the whole carcass , which ik et al.  and Rashk et al. . Rashidik et al.  also demb et al. .Silybum marianum. Similarly, Tavakolinasab et al. [Šťastník et al.  reports b et al.  report tPoultry meat available on the market should be of proper quality, perceived by consumers as a collection of many features, including nutritional value as one of the most important characteristics ,74,75. PSilybum marianum extract (3.81% live weight) compared to a control group (4.79% live weight) and birds fed 80 ppm dried SM fruit extract (4.22% live weight). Dry matter, protein and ash content of breast and thigh meat did not significantly differ between the control and dried Silybum marianum extract fed groups. In turn, Grela et al. [longissimus lumborum muscle of fatteners fed with rations containing 3% or 6% of milk thistle seeds noted a significant reduction in the content of this ingredient only when the content of milk thistle in the diet was reduced.The absence of an impact of milk thistle in broiler chicken feed on the total protein content in breast and leg muscles corroborates the results obtained by Šťastník et al. . Howevera et al.  analyzinlongissimus lumborum muscle. The increased share of PUFA in the muscles of birds fed with diets containing Silybum marianum is contrary to the findings of Kralik et al. [Own studies showed an increased share of stearic and linoleic acids in the muscles of chickens receiving feed rations containing milk thistle seeds. In turn, Kralik et al. , using fk et al.  but consk et al.  in studik et al. .Silybum marianum) to broiler chicken feed rations.The studies and experiments carried out by Kralik et al.  and ŠťasColor is an important attribute taken into account by consumers when buying meat, and an important element of evaluating meat dishes during their consumption . Meat ofSilybum marianum was observed by Šťastník et al. [The obtained L color values in the evaluated muscles were characteristic of normal muscles, since Van Laack et al.  classifik et al. . In turnk et al. , having Silybum marianum point to an impairment of the water holding capacity, but Kralik et al. [The WHC results obtained for muscles of chickens fed with rations containing k et al.  noted a Silybum marianum in diets as compared to other groups.In these studies, a positive impact of diets containing milk thistle seeds on the organoleptic (sensory) characteristics of muscles was noted. However, Šťastník et al. , having Higher notes for respective sensory characteristics of thigh muscles should be associated with a higher content of fat in comparison to breast muscles. Komprda et al.  underlinSilybum marianum in both types of feed rations made it possible to obtain the highest body weight at the lowest feed conversion per body weight gain unit, without any influence on muscularity and fattening grade. At the same time, muscles of chickens fed diets containing the phytobiotic featured the healthiest fatty acid profile and good taste characteristics.Based on the results of studies, the use of milk thistle seeds in broiler chicken starter/grower diets can be recommended in the amount of 2/3%. The introduction of


To evaluate the safety and efficacy of a filter embolic protection device (FEPD) in endovascular interventions of the femoropopliteal arteries.TM FEPD was applied were included in this retrospective study. Clinical and angiographic characteristics, filter macroembolization (FME), device-related complications, distal embolization, as well as the early clinical and hemodynamic outcome, were assessed. Potential risk factors for FME were evaluated by multivariate analysis.Patients who underwent endovascular interventions of the femoropopliteal arteries between 2008 and 2016 and in whom the SpiderFXp = 0.02).A total of 244 cases were identified . Balloon angioplasty ± stenting (BAP), directional atherectomy ± balloon angioplasty ± stenting (DA) and rotational thrombectomy ± balloon angioplasty ± stenting (RT) were performed in 141, 61 and 42 cases, respectively. FEPD placement and retrieval were successful in all but one case each. Permanent filter-related vessel damage was not observed. The rate of FME was 37.3% . Risk factors for FME in the BAP- and DA-group were total occlusion, lesion length > 19 cm, visible thrombus and diabetes mellitus. The distal embolization rate despite filter protection was 4.1 %  and was higher in cases with FME compared with those without FME (8.7% vs. 1.5%, TM device is safe and effective in capturing embolic debris during femoropopliteal interventions. A residual risk of peripheral embolization remains.The Spider FXIII, Cohort study Peripheral embolization is a well-recognized, potentially limb threatening complication of lower extremity endovascular procedures, resulting in clinically significant perfusion impairment at the cruropedal level in 1.6 to 4% of patients, with higher numbers reported in the treatment of acute thrombotic lesions up to 24%) [4% [2]. LWe sought to evaluate the procedural safety and efficacy of a FEPD in a large contemporary cohort of patients with acute, subacute and chronic obstructions of the femoropopliteal arteries, who either underwent balloon angioplasty ± stenting (BAP), directional atherectomy ± balloon angioplasty ± stenting (DA) or rotational thrombectomy ± balloon angioplasty ± stenting (RT). We further aimed to determine potential predictors of peri-procedural filter macroembolization (FME).The study protocol followed the principles of the Declaration of Helsinki. Patients who underwent endovascular interventions of the femoropopliteal arteries  between 2008 and 2016 were retrospectively identified. Patients aged ≥ 18 years suffering from symptomatic peripheral arterial disease Rutherford stage 2–6 were eligible for study inclusion, provided that the target lesion was successfully crossed by a guidewire.Clinical data  were obtained from the medical records. Laboratory values as well as pre- and post-interventional hemodynamic parameters  were recorded.All procedures were performed by a single, experienced interventional radiologist (M.T.). The endovascular procedures were carried out using 6–8F sheaths  and after intraarterial administration of 5.000IU. of heparin.The choice of a single revascularization technique or a combination of more than one technique was at the discretion of the operator, based on symptom duration and angiographic appearance. In general, DA was performed in patients with chronic symptoms resulting from arteriosclerotic lesions (mainly stenoses), whereas RT was applied in (sub-)acute occlusions of native arteries and prosthetic bypass grafts with high clinical suspicion of thrombotic material. BAP was used for treatment of both chronic arteriosclerotic lesions and (sub-)acute occlusions. DA and RT procedures all were performed under mandatory filter protection. In BAP procedures, utilization of the FEPD was indicated when, based on clinical information  and angiographic appearance , an increased periinterventional embolization risk was anticipated.TM-family . RT was carried out with RotarexTM-catheter systems . DA and RT were followed by adjunctive BAP, if necessary. If required , adjunctive stenting with self-expanding nitinol stents (open-cell design) of various manufacturers was performed. Stent implantation was preceded by lesion preparation in all cases. In some acute and subacute thromboembolic occlusions, aspiration thrombectomy and/or local thrombolysis was applied in addition to the above-mentioned recanalization procedures.Standard balloon catheters were used for BAP. DA was carried out with devices of the HawkTM  was inserted. The filter basket  was deployed at least 2 cm below the target lesion either in a popliteal or, in case of single vessel crural run-off, in a below-the-knee vessel segment. After release of the FEPD, digital subtraction angiography images were obtained in two projections in order to confirm adequate contact of the FEPD to the vessel wall. After completed revascularization, the FEPD was retrieved using the manufacturer-provided capturing catheter. Final completion angiography of the treated lower extremity was performed in order to document technical success and to assess distal run-off with regard to peripheral embolization.Following lesion crossing with a 0.018 V-18TM ControlWire  and prior to revascularization, the SpiderFXPost-interventional medical treatment consisted of dual antiplatelet therapy with aspirin and clopidogrel for three to six months, with the loading dose of clopidogrel (300 mg) given immediately after the procedure. In patients with an indication for oral anticoagulation , aspirin was combined with oral anticoagulants at therapeutic doses instead of clopidogrel.An experienced interventional radiologist (M.T.) reviewed the digital subtraction angiography sequences. Lesion characteristics  were analysed according to a predefined protocol. Peri-procedural FEPD-related complications were recorded, including problems with filter management, filter-induced local vasospasm and permanent filter-related permanent vessel damage.Debris within the filter was classified using a modified semiquantitative scoring system based on the appearance of the filter basket during final magnified angiography, as follows: (0) no visible material; 1) sludge with partial occlusion of filter meshes or single debris particles (2) macro-debris filling less than one-third of the filter; (3) macro-debris filling more than one-third of the filter; (4) completely filled filter basket .- Hemodynamic improvement: increase in ankle brachial index > 0.15 and/or significant improvement of forefoot pulse volume recordings by semiquantitative assessment .- Peripheral embolization: occurrence of a new angiographic filling defect in a tibial, fibular or pedal artery observed during the procedure or at final completion angiography.- FME of potential clinical significance: debris within the filter ≥ grade 2 was considered to be FME of potential clinical relevance, given its potential to occlude a tibial or fibular artery.p values < 0.05 were considered statistically significant.Statistical analysis was performed with the R software for statistical computing . Results for categorical variables are presented as absolute numbers/percentages, and continuous variables are displayed as mean ± standard deviation. For univariate analysis, Fisher’s exact test and Wilcoxon’s rank sum test were applied. Correction for multiple testing was done using the Bonferroni method. To identify independent predictors of FME, multiple logistic regression models were calculated. Finally, decision tree analysis was performed. Two-sided The main clinical and angiographic characteristics of the 244 included cases (203 patients) are summarized in Table DA and RT were performed in 61 (25%) and 42 (17.2%) patients, respectively. The remaining 141 patients underwent BAP without any debulking procedure. Adjunctive aspiration thrombectomy and/or local thrombolysis was performed in 28.3 and 17.6% of cases, respectively. Additional endovascular treatment of iliac artery inflow or crural artery outflow was performed in 2.0 and 31.1% of cases.Of note, the rate of patients with acute symptoms was higher in the groups treated by RT (71.4%) and BAP (55.6%) compared with DA (29.8%). Correspondingly, the rate of patients with total occlusions was substantially higher in these groups (RT 40.5% vs. BAP 55.3% vs. DA 3.3%). Aspiration thrombectomy and/or thrombolysis was performed more frequently in the RT-group (40.5%) and BAP-group (55.3%) compared with the DA-group (3.3%).Deployment of the FEPD was successful in all but one case. This patient was consequently treated with unprotected RT which was complicated by peripheral embolization. In another single case, retrieval of the FEPD was not possible due to an overfilled filter, necessitating FEPD removal in conjunction with the introducing sheath without further complications. Thus, the rate of successful filter management was 99.2%.Permanent filter-related vessel damage was not observed. However, vasospasm at the filter site requiring intraarterial spasmolysis with nitroglycerine occurred in 11 cases (4.5%), including seven cases with filters at the crural level and four cases with filters at the popliteal level.Technically successful revascularization was achieved in 96.7% of cases. The corresponding rate of post-procedural hemodynamic improvement was 93.0%. Revascularization failed in eight cases (3.3%), including two patients with primary technical failure and one patient in whom perforation of the popliteal artery following RT required urgent surgical revascularization. Five patients experienced early re-occlusions of the treated vessel segment within 24 hours of the procedure.n = 7; RT n = 2; DA n = 1), including nine total occlusions. The mean symptom duration in these patients was 80 days , and mean lesion length was 17.2 ± 16.4 cm (range 8–42 cm). In five of these cases, aspiration thrombectomy was attempted prior to filter placement. In eight of the ten cases with peripheral embolization distal to the FEDP, macroemboli were also found in the filter basket. Treatment consisting of aspiration thrombectomy (n = 3) and/or thrombolysis (n = 8) was successful in nine of the ten cases. After the procedure, hemodynamic improvement was documented in all ten cases. Within a mean follow-up time of 2.8 + 2.9 years, freedom from target lesion revascularization was documented in only two of these ten cases. Eight re-occlusions occurred after a mean of 0.9 ± 1.4 years, prompting revascularization procedures in six patients (two of whom required bypass surgery). One patient subsequently underwent lower limb amputation.Despite filter protection, peripheral embolization distal to the FEDP occurred in ten cases .Overall, peri-procedural FME considered to be of potential clinical significance (≥ grade 2) occurred in 37.3% of cases. FME was more common in patients who underwent RT compared with DA and BAP (47.7% vs. 36.2% vs. 32.8%). Peripheral embolization distal to the FEPD was documented in 4.1% of all patients and was significantly more common in cases with FME compared with those without FME . Notably, there was a remarkable overlap between lesion lengths in patients with and without FME .Given the excessive risk of FME associated with RT, further analysis evaluating risk factors for peri-procedural embolism was restricted to the groups with DA and BAP only. Results of the univariate analysis are listed in Table FME Fig. . A markeLogistic regression analysis favoured a model including the following variables: total occlusion, lesion length > 19 cm, visible thrombus, DA procedure and chronic antiplatelet therapy. However, comparison between patients with and without chronic antiplatelet therapy showed significantly worse patient- and lesion-based risk profiles in the 73.9% of patients who were under chronic (> 14 days of treatment) antiplatelet therapy prior to the procedure . Therefore, the variable antiplatelet therapy was excluded from the final model, which included the following parameters: total occlusion, lesion length > 19 cm, visible thrombus, DA procedure and diabetes mellitus. There was a significant interaction between visible thrombus and diabetes mellitus, leading to a marked risk increase for FME (83%) for this small subset of patients  with both factors.The model showed a substantial discriminatory value for patients with high, intermediate and low embolization risk. In the group of patients treated with BAP, the model allowed exact identification of a low-risk group (embolization risk < 12%) and a high-risk group (embolization risk > 75%) Fig. . These fFEPD appeared to be safe and effective in capturing embolic debris during complex endovascular interventions of the femoropopliteal arteries . Distal embolization despite filter protection occurred in 4.1% of cases. The comparatively high rates of both FME and distal embolization are mainly attributable to the cohort characteristics, with large proportion of cases presenting with acute symptoms and/or complete occlusions particularly in the groups treated by BAP or RT (more than 50% of the cases).RT of (sub-)acute femoropopliteal occlusions was associated with a rate of FME approaching 50% and a distal embolization rate of 4.8%. Data on the risk of FME during mechanical thrombectomy procedures are scarce. Not surprisingly, Karnabatidis et al. identified mechanical thrombectomy procedures as independent risk factors of FME . In olde++ study, distal embolization occurred at a lower rate in three out of 111 patients with chronic limb ischaemia (2.3% of the study cohort) who underwent filter-protected DA [n = 508) with patients who underwent femoropopliteal DA with utilization of a FEPD, Krishnan et al. were the first to propose an algorithm for the rational use of FEPD in DA procedures [In previous DA series, the reported rates of FME varied widely, ranging from 9 to 91% , 6–8. InThe role of FEPD in the setting of BAP of femoropopliteal lesions is less well defined. In large series without filter protection, the rates of clinical significant distal embolization were between 1.6 and 2.4% . In the In our analysis, thrombotic and long lesions, as well as chronic occlusions, appeared to be associated with a particularly increased risk of FME also in the subgroup of patients with BAP, whereas other parameters such as symptom duration, lesion calcification and number of run-off vessels were not. Chronic total occlusions, lesion length, as well as thrombus burden, were positively correlated with the amount of captured particles in the series by Karnabitides et al. . AccordiTM in our study, it is worth discussing the utilization of FEPD at least in subjects considered to be at high risk for distal embolization or with a predicted poor outcome in case of distal embolization.In patients with compromised crural run-off, FEPD may be used even more liberally to avoid clinically relevant outflow deterioration . The potHowever, the level of evidence regarding the rational use of FEPD in peripheral interventions remains low and the additional costs must be taken into account. A huge discrepancy exists between high rates of FME and the remarkable lower rate of clinically significant distal embolism in unprotected procedures. Noteworthy, in our study, the rate of distal embolization was significantly higher in cases with FME than in those without 8.7 vs. 1.5%). A consensus definition of how to define “clinically significant” FME does not exist. While some studies provided no clear definitions of FME , 6, 8 an vs. 1.5%Our retrospective study has some limitations, mainly the inhomogeneous patient cohort, performance of endovascular procedures by a single operator who was involved in analysis of procedural parameters and the lack of a core laboratory analysis. The comparatively high rates of both FME and distal embolization are mainly attributable to an obvious selection bias, as stated above.As most studies with large patient numbers relied on retrospective data with the inherent risk of bias , 8, and TM to effectively capture macro-debris during different types of femoropopliteal interventions. A residual risk of distal embolization remains, and the discrepancy between high rates of FME and much lower rates of distal embolization in unprotected cases represents an unsolved problem. Our results may inform future research aiming at determining the clinical benefit of FEPD in certain clinical scenarios of femoropopliteal interventions.In summary, our study showed an excellent safety profile and the ability of the SpiderFX


P < 0.001), the square root of the mean squared differences of successive NN intervals (P < 0.001), and the percentage of NN intervals that are more than 50 ms different from the previous interval (P < 0.001) were associated with the occurrence of AF in univariate analysis. Premature atrial contractions burden, lower baseline heart rate, age, hemodialysis, coronary artery disease, and chronic heart failure were also associated with AF. In Cox regression analysis, higher HRV (representing excessive autonomic fluctuation) was an independent risk factor for AF. Excessive autonomic fluctuation represented by higher HRV in patients with hypertension was associated with an increased risk of AF.The autonomic nervous system (ANS) plays an important role in the initiation and maintenance of atrial fibrillation (AF). However, the meaning of higher heart rate variability (HRV) in predicting AF remains unclear. Among 2100 patients in the Holter registry, a total of 782 hypertensive patients were included in this study. Baseline HRV was measured by time domain and frequency domain methods using 24-h Holter monitoring. The primary outcome was the development of AF. During an average follow-up of 1.1 years, 44 patients developed AF. Higher HRV parameters including high-frequency ( Various pathophysiological mechanisms for the development of AF have been studied2. Recently, there has been an increasing evidence that the dysfunction of the autonomic nervous system (ANS) including the sympathetic and parasympathetic nervous systems, and the interaction between sympathetic and parasympathetic nervous systems are involved in the pathogenesis of AF3.Atrial fibrillation (AF) is the most common cardiac arrhythmia requiring medical therapy6. However, another study reported that higher HRV value was associated with the incident AF7. The electrocardiography (ECG) recording time used to analyze HRV and analysis methods differed among the studies, leading to inconsistent results.Despite the increasing evidence of an association between dysfunction of ANS and AF, whether abnormalities in the ANS can predict the development of AF remains unclear. Several population-based studies showed that lower heart rate variability (HRV) was associated with an increased risk of new-onset AF8. Previous studies reported that HRV was not only associated with cerebrovascular diseases such as stroke and myocardial infarction but also relatively mild disease such as hypertension and anxiety disorders11. The objective of this study was to investigate whether HRV using 24-h Holter monitoring could predict the development of AF in patients with hypertension.Hypertension is the most common cardiovascular risk factor in patients with AFThe study population was selected from the Kangwon National University Holter registry. A total of 2100 patients underwent 24-h Holter monitoring between May 2018 and April 2019. Patients were eligible for this study if they were in sinus rhythm at baseline 24-h Holter monitoring and had HRV data. The exclusion criteria were: (1) < 18 years of age, (2) had persistent AF or paroxysmal AF lasting more than 30 s at baseline 24-h Holter monitoring, (3) had temporary or permanent pacemaker, (4) had complete atrioventricular block, or (5) < 22 h of recording. After excluding 480 patients who met the exclusion criteria, 1620 patients were identified. Among the 1620 patients, 782 patients with hypertension were finally analyzed.This retrospective observational cohort study was conducted in accordance with the principles of the Declaration of Helsinki. The Institutional Review Board of Kangwon National University Hospital approved the study protocol (KNUH-2020-04-021-001). The need for informed consent was waived for this retrospective study by the Institutional Review Board of Kangwon National University Hospital. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines.Baseline demographic data, cardiovascular risk factors, medications, and clinical outcomes were retrospectively analyzed by reviewing the medical records. The study population was divided into two groups according to the presence of hypertension. Holter data including the HRV parameters and cardiovascular risk factors were compared. Whether HRV parameters could predict the occurrence of AF during the follow-up period in patients with hypertension was also determined.The study outcome was the development of AF during follow-up. The development of AF was defined as a standard 12-lead ECG recording or Holter recording with ≥ 30 s of AF. The development of AF was evaluated by reviewing the medical records from our hospital. All ECG and 24-h Holter monitoring data were interpreted by one cardiologist.HRV data were acquired by 24-h Holter monitoring  and measured by frequency domain and time domain methods. Fast Fourier Transform (FFT)  was used to convert single-lead ECG signals to power spectral densities. The cubic spline-interpolated NN interval function was sampled at 1024 samples/300 s or 3.413 samples/s. NN interval ratios of < 0.80 or > 1.20 and NN intervals of < 150 ms or > 5000 ms were excluded prior to HRV analysis. The power spectral density included very low-frequency (VLF) component (0.0033–0.0400 Hz), low-frequency (LF) component (0.0400–0.1500 Hz), high-frequency (HF) component (0.1500–0.4000 Hz), and the ratio of two components (LF/HF ratio). The time domain methods included the standard deviation of the NN interval (SDNN), the standard deviation of all 5-min average NN interval (SDANN), the average standard deviation of all 5-min NN interval (ASDNN), the square root of the mean squared differences of successive NN intervals (rMSSD), the percentage of NN intervals that are more than 50 ms different from the previous interval (pNN50), and the count of intervals that were more than 50 ms different from the previous interval (BB50).P-value less than 0.05 was considered statistically significant.Continuous variables are expressed as mean ± standard deviation or median and interquartile range. Categorical variables are expressed as frequency and percentage. To evaluate the difference in HRV values and clinical characteristics according to the occurrence of AF, Student’s unpaired t test was used for normally distributed data and Mann–Whitney test was used for skewed data. Categorical variables were analyzed with chi-square test or Fisher’s exact test. Receiver operating characteristic (ROC) curve analysis was used to select the cut-off value between the HRV parameter and the occurrence of AF. Cox regression analysis was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for the risk factors associated with the occurrence of AF. Calculations were performed using the Statistical Package for the Social Sciences version 20.0 for Windows . A Among the 2100 patients who underwent 24-h Holter monitoring between May 2018 and April 2019, 480 patients were excluded due to the following: 120 patients were younger than 18 years old, 191 patients had persistent AF, 73 patients had paroxysmal AF, 5 patients had no HRV data, 3 patients had permanent pacemaker, 14 patients had complete atrioventricular block, 2 patients had insufficient recording, and 72 patients performed Holter monitoring repeatedly . However, the burden of premature atrial contractions (PAC) was significantly higher in AF occurrence group (P < 0.001).LF and HF component in the frequency domain method and ASDNN, rMSSD, pNN50, and BB50 in the time domain method were higher in the AF occurrence group Table . Mean heDuring an average follow-up of 1.1 years, 44 patients developed AF. Among the 44 patients who developed AF during the follow-up period, 27 (61.4%) patients were found to have paroxysmal AF and 17 (38.6%) patients were found to have persistent AF at the initial AF identification. Of the 27 patients found to have paroxysmal AF, 10 patients had a history of AF, and 10 of the 17 patients found to have persistent AF had a history of AF.P = 0.002), LF (P < 0.001), HF (P < 0.001), rMSSD (P < 0.001), and pNN50 (P < 0.001) were associated with AF occurrence. History of AF, the mean heart rate, and PAC count was also associated with AF occurrence. ROC analysis for HF, rMSSD, pNN50, and PAC count as predictors of AF occurrence revealed areas under the curve of 0.7033, 0.7045, 0.6911, and 0.7060, respectively . Cox regression analysis showed that older age, higher PAC burden, hemodialysis, coronary artery disease, history of AF, and any risk of HRV parameters were independent predictors for the occurrence of AF  ECG recording was used. Therefore, the HRV parameters measured by time domain methods were more appropriate in our study. Third, a closer look at the previous Atherosclerosis Risk in Communities (ARIC) cohort study reported that lower HRV was associated with an increased risk of AF, a figure in that report showed that higher HRV values were also associated with the occurrence of AF. However, this association between higher HRV values and the occurrence of AF was not addressed in previous ARIC cohort study4.Two previous long-term follow-up studies showed that lower HRV was associated with an increased risk of AF19. Generally, HF components are predominantly modulated by the parasympathetic nervous system, whereas LF components are considered a marker of sympathetic modulation21. HRV represents the degree of autonomic fluctuations rather than the mean level of autonomic tones. Therefore, relatively higher HRV could be considered excessive fluctuations in the ANS rather than a physiologic condition. In other words, even though hypertensive patients generally had low HRV than those without hypertension, hypertensive patients with comparatively higher HRV might be considered to have disproportionate fluctuations in the ANS. A previous study7 showed that higher rMSSD was associated with incident AF could be interpreted from this point of view.Parasympathetic dominance is associated with an increased propensity for AF, and vagal-mediated paroxysmal AF is preferentially seen in young individuals who have structurally normal heartWe enrolled patients with sinus rhythm at baseline 24-h Holter monitoring. Therefore, patients with paroxysmal AF were enrolled in this study. Sixty-six of 782 (8.4%) hypertensive patients had a history of AF. Perhaps more patients with a history of AF might be included due to its asymptomatic feature. Among 66 patients with a history of AF, AF recurred in 20 patients. A history of AF was an independent risk factor for AF occurrence as well as higher HRV in this study. When we examine a patient in an outpatient clinic, it is difficult to know whether the patient has a history of AF. Therefore, it is important to predict the occurrence of AF regardless of a history of AF, and the HRV value would be one of the factors that could predict the occurrence of AF.This study had several limitations. First, this was a retrospective cohort study. We could not fully evaluate many other risk factors for AF, including obstructive sleep apnea, body weight, alcohol consumption, or physical activity. Second, the duration of follow-up was relatively short and the degree of effort to identify AF varied from patient to patient. Since most patients with AF are asymptomatic, it is difficult to detect AF with an intermittent ECG test. Regular 24-h Holter monitoring could not be performed due to the limitations of the retrospective study. Therefore, AF incidence in our study was low. Third, our study enrolled patients undergoing hemodialysis. Because blood pressure and autonomic tone change before and after dialysis, short-term ECG recordings are generally preferred to avoid environmental influences. However, since the number of dialysis patients in our study was very small, we analyzed HRV data using 24-h Holter recordings. Finally, since HRV parameters measured by frequency domain methods were made by 24-h Holter recording, there might be a problem of stationarity.In conclusion, higher HF, rMSSD, and pNN50 in patients with hypertension as surrogate markers for excessive autonomic fluctuation could predict the occurrence of AF. Frequent AF screening should be considered for hypertensive patients with higher HRV values and other risk factors for AF. Further large scale, prospective studies are needed to verify our findings.Supplementary Tables.


Leptomeningeal metastasis (LM) is a severe complication of advanced non-small cell lung cancer (NSCLC). This retrospective study aimed to investigate the potential use of osimertinib for preventing LM in patients with advanced epidermal growth factor receptor (EGFR)-mutated NSCLC.Patients with advanced NSCLC harboring EGFR mutations who underwent tyrosine kinase inhibitors (TKIs) therapy for at least 8 weeks between September 2016 and September 2019 were eligible for this study. All included patients were divided into two groups based on whether they received osimertinib, the osimertinib group (patients treated with osimertinib) and the control group (patients not treated with osimertinib). Propensity score matching  was used to account for differences in baseline characteristics. The cumulative incidence of LM and the overall survival (OS) were evaluated.p = 0.009). Multivariate analysis indicated that osimertinib was an independent, statistically significant predictor for determining the risk for LM, with an HR of 0.33 (p = 0.042). At present, the OS rate data are too immature for statistical analysis.A total of 304 patients were included in the study population. Among them, 116 patients received osimertinib, and 188 did not. A total of 112 patients remained in each group after PSM, and the baseline characteristics were not significantly different between the two cohorts. LM developed in 11 patients (9.82%) in the osimertinib group and 24 patients (21.42%) in the control group (hazard ratio [HR] 0.38, 95% confidence interval [CI] 0.19–0.79, Real-world data demonstrate that osimertinib can significantly reduce the incidence of LM in patients with advanced NSCLC harboring common EGFR mutations. Given this result, osimertinib should be encouraged in clinical practice for specific patient populations. Leptomeningeal metastasis (LM) is the seeding of malignant cells within the subarachnoid space and leptomeninges (pia and arachnoid mater) . LM is ap < 0.001) [Due to the existence of blood-brain barrier (BBB) and the low penetration of therapies into the cerebrospinal fluid (CSF), traditional therapies are often considered futile for preventing and treating brain metastasis (BM) and LM , 8, 9. H< 0.001) . Another< 0.001) . Osimert< 0.001) , 14. In < 0.001) . The NatHowever, considering all relevant progress made in this setting, no study on the relationship between osimertinib treatment and LM development has been reported. Despite significant advances in systemic and local approaches, establishing preventive strategies for LM is more rewarding than administering treatment. Therefore, we conducted a propensity-matched retrospective study to investigate the role of osimertinib therapy for preventing LM in patients with advanced EGFRm NSCLC.This single-center, retrospective study was approved by the Institutional Ethics Committee of Second Affiliated Hospital of Nanchang University. Written informed consent was not required due to the retrospective nature of the study. From the clinical records database of our center, we reviewed a total of 1505 patients diagnosed with NSCLC between September 2016 and September 2019 with adequate follow-up data. The eligibility criteria included the following: (1) histological diagnosis of postoperative recurrence or stage IV NSCLC; (2) the presence of activating EGFR mutations; (3) age older than 18 years; and (4) treatment with at least 8 weeks of TKIs as a first-line treatment . Major exclusion criteria included patients with NSCLC who presented with LM at the time of diagnosis or recurrence. All of the patients with recurrent NSCLC were retrospectively restaged. After the screening procedure, 304 individuals were selected for the analysis. Patients who met the inclusion criteria were divided into two groups: the osimertinib group and the control group. Oligometastatic disease was defined as the presence of ≤5 lesions in 1 to multiple organs at the initiation of TKIs, while polymetastatic disease was defined as > 5 metastatic lesions.Between September 2016 and September 2019, a total of 1505 patients with NSCLC were hospitalized at our center. Three hundred four patients were included in the study population. Among them, 116 patients received osimertinib, and 188 patients did not. In the 1:1 match, patients who received osimertinib therapy were individually matched with one control patient. Variables used for the propensity score matching (PSM) included chemotherapy and antiangiogenic therapy. Baseline laboratory characteristics  were evaluated every 4 weeks. Magnetic resonance imaging of the brain, chest computed tomography (CT) and upper abdominal CT were performed every 2 months until death or last follow-up. The LM diagnosis at our center was based on the cytological presence of malignant cells in the CSF or typical clinical signs along with positive radiologic findings.P values ≤0.10 in the univariate analysis were included in the multivariate analysis. P-values < 0.05 were considered to indicate significant differences. The statistical software packages R  and Empower Stats  were used to analyze all data.The primary endpoint of the present study was the cumulative incidence of LM, with time to development computed from the date of NSCLC diagnosis or recurrence to the date of LM diagnosis. Patients who did not develop LM were censored on the date of last follow-up or death. The secondary endpoint was OS. Data represent the median  and n (%). Baseline characteristics were compared using Fisher’s exact test for categorical variables and the two-sample t-test or Mann-Whitney U-test for continuous variables, as appropriate. Actuarial rates of freedom for LM development and OS were evaluated by Kaplan-Meier plots and the Cox log-rank test. The cumulative risk for LM was estimated using the cumulative incidence curve. The Cox proportional hazards model was used to run univariate and multivariate analyses to evaluate factors that influenced the occurrence of LM. Variables with Between September 2016 and September 2019, a total of 1505 patients with NSCLC were hospitalized at our center. The flow chart of the screened patients is summarized in Fig. , with LM developing in 24 patients (21.42%). The median time to LM development was 23.6 months (95% CI 17.9–28.2 months), with 1-year and 2-year cumulative incidence rates of LM of 5.36 and 11.60%, respectively. There was a significant difference in the cumulative risk for LM between the two groups   , which was comparable to the median follow-up time of the control group (23.5 months 95% CI 19.3–24.0 months). In the osimertinib group, LM developed in 11 patients (9.82%), and the median time to LM development was 20.3 months (95% CI 7.4–21.4 months), with 1-year and 2-year cumulative incidence rates of LM of 3.57 and 8.03%, respectively. Findings from the control group were differentAt present, the available OS rate data are too immature (42.41% maturity) for statistical analysis. At the endpoint of the study, 33 deaths (29.46%) had occurred in the osimertinib group and 62 (55.36%) in the control group , EGFR mutations (19del vs Others)  and metastatic status (Polymetastasis vs Oligometastasis)  were independent prognostic factors for LM in the multivariate analysis . The cumulative risks for LM in the osimertinib group at 1 year and 2 years were 3.57 and 8.03%, respectively, whereas the rates were 5.36 and 11.6%, respectively, in the control group. In the multivariate analysis, a Cox regression model also showed that osimertinib was an independent, statistically significant predictor for determining the risk for LM . This suggests that osimertinib can prevent the occurrence of LM in patients with advanced EGFRm NSCLC.Osimertinib, a third-generation TKI designed to target both EGFR sensitizing mutations and T790M, was approved for the first-line treatment of patients with EGFRm advanced NSCLC and for the treatment of EGFR T790M mutation-positive NSCLC patients with progressive disease after prior EGFR TKI therapy , 20. OurOsimertinib significantly prolonged the progression-free survival in treatment-naïve patients with EGFRm advanced NSCLC and in patients with EGFR T790M NSCLC after disease progression with prior EGFR-TKIs treatment –23. OsimThe number of T790M confirmed cases in control group is only 12 after PSM. Patients with acquired T790M mutation had a prolonged survival compared to patients with negative T790M . Thus, ip = 0.021). This finding was consistent with that of a prior study that revealed that 19del potentially predicts a lower risk for LM than L858R [p = 0.002). One probable reason is that patients with oligometastases may survive longer than those with polymetastases [Previous studies showed that LM occurs in 9.0 to 9.4% of patients with EGFR mutations , 4; howean L858R . Our dattastases . As patip < 0.001) [p = 0.296). The therapeutic and prophylactic effects of radiotherapy  for LM need to be studied further.The goal of radiotherapy for LM is to palliate symptoms and to improve CSF flow. There were conflicting findings on whether whole brain radiation therapy (WBRT) improved survival for patients with NSCLC and LM , 16, 30.< 0.001) . Cranios< 0.001) , 18. In < 0.001) . In our Although this study provides meaningful data, we acknowledge several limitations. First, this study was based on a retrospective analysis performed at a single center with potential hidden biases. The adoption of PSM balances baseline patient characteristics between groups, and multivariate regression was used to minimize some weaknesses of a retrospective study. The second limitation is the low number of patients treated with osimertinib as the first-line treatment; osimertinib was not covered by health insurance before March 2021 as per the local government policy.In conclusion, we observed that osimertinib can effectively decrease the incidence of LM in patients with advanced common EGFR mutations NSCLC in clinical practice. Interestingly, patients harboring deletion mutations of EGFR exon 19 were less likely to experience LM than those with other EGFR mutations. Therefore, osimertinib might be a suitable option for specific patient populations with advanced common EGFR mutations NSCLC. However, additional studies are needed to confirm these findings.