Additionally, PM2.5 instillation was found to cause liver damage and inflammation. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were significantly elevated by PM2.5 instillation. PM2.5 also triggered IL-6 and TNF-α transcription but inhibited mRNA synthesis and suppressed signaling activation of the insulin-phosphoinositide 3-kinase- (PI3K-) Akt-glucose transporter 2 (GLUT2) pathway in the rat liver by reducing the ratio of phosphorylated Akt to phosphorylated insulin receptor substrate 1 (IRS-1). Thus, PM2.5-induced inflammation activation and insulin signaling inhibition in the rat liver contribute to the development of systemic insulin resistance.To elucidate the mechanism of how the liver participates in PM2.5-caused insulin resistance. A novel Wistar rat model was developed in this study by instilling a suspension of lyophilized PM2.5 sample  collected from the atmosphere. Systemic insulin resistance indicators, including serum fasting blood glucose (FBG), fasting insulin (FINS), Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), and hemoglobin A1 (HbA1), were upregulated by the PM2.5 instillation. The area under the curve (AUC A strong association between PM2.5 exposure and diabetes prevalence has been established, suggesting that ambient air pollution may contribute to the increased prevalence of diabetes in the adult U.S. and Asian populations [glu) calculated by intraperitoneal glucose tolerance testing (IPGTT) in the different PM2.5 instillation groups was also measured. In addition, the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) as well as the hepatic mRNA expression of IL-6 and TNF-α were determined. Furthermore, the impact of PM2.5 exposure on the synthesis of insulin-phosphoinositide 3-kinase (PI3K-) Akt-glucose transporter 2 (GLUT2) signaling pathway molecules in the rat liver at the mRNA level was analyzed. Finally, the PM2.5-induced phosphorylation of Akt and insulin receptor substrate 1 (IRS-1) in the rat liver was evaluated and compared by western blotting.According to the World Health Organization, the global prevalence of diabetes in adults over 18 years old increased from 4.7% in 1980 to 8.5% in 2014, while the prevalence of diabetes in China had already reached 9.7% in 2010 . Meanwhiulations –8, but tμm.The PM2.5 samples were collected from December 2016 to March 2017 at Xinxiang Medical College, which is located close to the center of Xinxiang City . The sampling site is surrounded by residential areas and is far away from major roads and factories. After sampling, the filter of the PM2.5 high-volume air sampler  was cut off and sonicated in sterile double-distilled water using a UC-6B ultrasonicator  for 3 × 15 min. The PM2.5 eluted from the filter was then lyophilized, weighed, and stored at −20°C until further use. Scanning electron microscopy was also conducted to confirm that the particulate matter collected had a diameter of no more than 2.5 ad libitum access to filtered water and food, 12 h light/12 h dark cycle). Sixty rats were then randomly allocated into five groups (12 rats/group), including control, 0.75 mg/kg, 2.5 mg/kg, 5 mg/kg, and 10 mg/kg PM2.5 instillation groups. Before exposure, the PM2.5 lyophilized powder taken from a refrigerator was weighed, and the corresponding concentration of PM2.5 was resuspended in saline. To avoid aggregation, particle suspensions were always sonicated for 3 × 15 min and vortexed. By using noninvasive inhalation tracheal instillation, the rats in each dosage group were infused with PM2.5 three times a week for 8 weeks [Sixty healthy Wistar rats, weighing 180–220 g, were purchased from Charles River, China. To avoid sex-dependent differences, only male mice were included in the study. All experiments described in the experimental protocol were approved by the Institutional Review Committee for Animal Care and Use at Xinxiang Medical College , and all experimental procedures were performed to minimize animal suffering. The health and behavior of the animals were checked daily. The rats were housed together and acclimatized in a freestanding clean room for 1 week /24 + (5 min + 10 min)/24 + (10 min + 30 min)/6 + (30 min + 60 min)/4 + (60 min + 120 min)/2. After IPGTT, the animals were anesthetized with ether and sacrificed by femoral artery terminal exsanguination. The blood was collected for serum isolation and stored at −70°C until use. At the same time, the liver was removed and spliced into two equal parts. After weighing, one part was fixed in 4% buffered paraformaldehyde, and the other part was frozen at −70°C.After PM2.5 instillation, IPGTT of rats was performed in each group, and the AUClculated . Venous μm thick slices using a rotary microtome  and subjected to HE staining with hematoxylin for 5–7 min at room temperature. After rinsing, the slices were stained with eosin for 2 min at room temperature. Micrographs under 200x magnification were randomly selected and captured using a light microscope.Briefly, rat liver samples fixed in 4% buffered paraformaldehyde were dehydrated in a series of ethanol solutions, cleared in xylene, and embedded in paraffin. The paraffin-embedded tissues were sectioned into 4 α levels were measured by enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's instructions . The HbA1c level was determined by ELISA, according to the manufacturer's instructions . The HOMA-IR was calculated by the following formula: HOMA‐IR = FBG × FINS/22.5 [The FBG, ALT, AST, and TBIL levels were determined by Chemoray . The FINS, IL-6, and TNG-INS/22.5 .β-actin gene was used as an internal control to normalize the expression of target genes, and the specific primers are listed in E = 2C−△△t algorithm were analyzed by LightCycler software (Roche Diagnostics).Total RNA was isolated from whole liver lysates by TRIzol, according to the manufacturer's instructions . RNA was reverse-transcribed using the reverse transcriptase M-MLV (RNase H) . The synthesized cDNA was subjected to qPCR using 2x All-in-One qPCR Mix  in triplicate, according to the manufacturer's instructions. The Ser473 , and β-actin . Following washing, the membranes were incubated with goat horseradish peroxidase-conjugated secondary antibodies . The reactions were detected by using an Enhanced Chemiluminescence Western Blot detection kit , and the detected bands were visualized via exposure to an X-ray beam in a dark room.Total protein of rat liver tissue in each group was extracted using radioimmunoprecipitation assay lysis buffer. The protein concentration was determined using a bicinchoninic acid protein concentration quantification kit . Denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat dry milk in tris-buffered saline containing 0.05% Tween 20 or 1–3% bovine serum albumin and probed with primary antibodies overnight at 4°C. The primary antibodies used included IRS-1, Akt , p-IRS-1 , p-Aktp < 0.05 was set to indicate a significant difference.Data were analyzed by SPSS20.0 . For the normally distributed measurement data, the results were presented as the mean ± standard error, and the Levene test was used for the homogeneity of variance. One-way analysis of variance was used for multiple group comparisons, and the least significant difference test was used for pairwise comparisons between groups. Nonparametric tests were used for measurement data that did not meet the assumptions of equal variance. p < 0.05). Compared to the 0.75 mg/kg PM2.5 group, the FBG, FINS, and HOMA-IR levels in the 2.5, 5, and 10 mg/kg PM2.5 groups were all significantly elevated (p < 0.05). The FINS and HOMA-IR levels in the 5 mg/kg PM2.5 group were significantly higher (p < 0.05) than those in the 2.5 mg/kg PM2.5 group. The FBG and HOMA-IR but not the FINS level in the 10 mg/kg PM2.5 group were significantly elevated compared to those in the 2.5 mg/kg PM2.5 group. There were no significant differences in the FBG, FINS, and HOMA-IR levels between the 5 and 10 mg/kg PM2.5 groups. Only the HbA1c level in the 10 mg/kg PM2.5 group was significantly increased when compared to each of the other four groups (p < 0.05). Next, we performed IPGTT and calculated the AUCglu for the rats in each group administered with different dosages of PM2.5 instillation . Similarly, the AUCglu in the 2.5, 5, and 10 PM2.5 groups were also significantly higher than that of the control group (p < 0.05). Thus, PM2.5 instillation in rats induced insulin resistance in a dose-dependent manner.To study the effect of PM2.5 instillation on systemic insulin resistance in rats, the levels of FBG, FINS, HbA1c, and HOMA-IR were examined in all five groups Figures . Compare Figures . Compareα , starting from 2.5 mg/kg PM2.5 instillation. The serum level of TBIL started to be significantly increased with 5 mg/kg PM2.5 instillation (p < 0.05). Compared to the 0.75 mg/kg PM2.5 group, the ALT and IL-6 serum levels started to be significantly higher with 2.5 mg/kg PM2.5 instillation (p < 0.05), while the serum levels of AST, TBIL, and TNF-α started to be significantly elevated with 5 mg/kg PM2.5 instillation (p < 0.05). Compared to the 2.5 mg/kg group, the ALT, AST, TBIL, and TNF-α levels in the 5 and 10 mg/kg PM2.5 groups were all significantly higher (p < 0.05), whereas the serum level of IL-6 was only significantly increased in the 10 mg/kg PM2.5 group (p < 0.05).At first, we found that PM2.5 instillation induced liver damage in a dose-dependent manner according to the pathological analysis with HE staining of the liver sections Figures . Compare Figures . Compareα in the rat liver of each group by qPCR ; TNF-α mRNA expression was significantly higher in the 2.5, 5, and 10 mg/kg PM2.5 groups (p < 0.05). Compared to the 2.5 mg/kg group, IL-6 and TNF-α mRNA expressions were both significantly elevated in the 5 and 10 mg/kg PM2.5 groups (p < 0.05). There was also an elevation in the IL-6 and TNF-α mRNA expression in the 10 mg/kg PM2.5 group, compared to the 5 mg/kg PM2.5 group (p < 0.05), indicating that PM2.5 induced proinflammatory cytokine transcription in the rat liver.To confirm the protein level changes of the proinflammatory cytokines in PM2.5-instilled rats, we further examined the mRNA expression of IL-6 and TNF- Figures . Comparep < 0.05), while the insulin receptor (INSR), IRS-1, PI3K, and Akt mRNA levels were significantly lower in the 5 and 10 mg/kg PM2.5 groups (p < 0.05). Compared to the 0.75 mg/kg PM2.5 group, the INSR, IRS-1, Akt, and GLUT2 mRNA levels in the 5 and 10 mg/kg PM2.5 groups were significantly decreased (p < 0.05), while the PI3K mRNA level was significantly decreased in the 10 mg/kg PM2.5 group (p < 0.05). Compared to the 2.5 mg/kg PM2.5 group, the INSR, IRS-1, and Akt mRNA expression levels in the 5 and 10 mg/kg PM2.5 groups were significantly lower, while the PI3K and GLUT2 mRNA levels were significantly lower in the 10 mg/kg PM2.5 group (p < 0.05). Compared to the 5 mg/kg PM2.5 group, there was no significant difference in the INSR, IRS-1, PI3K, Akt, or GLUT2 mRNA level in the 10 mg/kg PM2.5 group. Decreased expression of these molecules at the mRNA level revealed that PM2.5 instillation may induce insulin resistance by inhibiting the transcription of insulin-PI3K-Akt-GLUT2 signaling pathway molecules.Inhibition of the insulin-PI3K-Akt-GLUT2 signaling pathway has been reported to be associated with insulin resistance –16. To fp < 0.05), while the p-Akt/Akt ratio started to be significantly decreased in the 2.5 mg/kg PM2.5 group (p < 0.05). Compared to the 0.75 mg/kg PM2.5 group, the ratios of p-IRS-1/ISR-1 and p-Akt/Akt in the 2.5, 5, and 10 mg/kg PM2.5 groups were all significantly different (p < 0.05). Compared to the 2.5 mg/kg PM2.5 group, the ratios of p-IRS-1/ISR-1 and p-Akt/Akt in the 5 and 10 mg/kg PM2.5 groups were all significantly different (p < 0.05). There was also a significant difference in the ratios of both p-IRS-1/ISR-1 and p-Akt/Akt between the 5 and 10 mg/kg PM2.5 groups (p < 0.05). Collectively, our results indicate that the phosphorylation of insulin signaling pathway proteins induced by PM2.5 instillation contributed to insulin resistance in the rat liver.Phosphorylation of key serine residues is another indicator of insulin-PI3K-Akt-GLUT2 signaling pathway function. For example, serine phosphorylation of Akt is accompanied by insulin receptor activation. In contrast, serine phosphorylation of IRS proteins can decrease their capacity to attract downstream PI3K, resulting in signaling inhibition –16. Thusglu calculated by IPGTT with different dosages of PM2.5 instillation was also significantly greater than that of the control group.Increasing numbers of experimental animal research studies have demonstrated the impact of PM2.5 inhalation on the development of insulin resistance and diabetes . For exaThe liver is one of the most important organs involved in insulin resistance, and the inhibition or defects of the insulin-PI3K-Akt signaling pathway in the liver may be the underlying mechanism –16. Onceα stimulation leads to serine phosphorylation of IRS-1, which attenuates its ability to transduce insulin-mediated cellular events [α or the TNF receptor 1 gene do not develop insulin resistance, even under elevated fatty or obese circumstances [α also led to reduced expression of the INSR, IRS-1, and GLUT2 genes as well as decreased insulin-stimulated glucose uptake [α at both the protein and mRNA levels. These observations are consistent with some previous studies, in which IL-6 has been reported to be elevated after PM2.5 exposure [It has become increasingly evident that insulin resistance is often associated with the proinflammatory cytokine response in insulin-sensitive tissues including the liver, which may lead to a decreased insulin sensitivity –21. For r events . Furthermstances . Treatmee uptake . Here, wexposure . Future in vitro cell biology experiments to observe and study the effect of PM2.5 on the insulin signaling pathway in human liver cells to determine if the results from our rat study are consistent or to clarify its potential clinical significance. Nevertheless, to the best of our knowledge, this is the first systematic study investigating the impact of PM2.5 on insulin resistance-related hepatic inflammation and the insulin signaling pathway in a rat model. Our findings also suggest a link between air pollution and hepatic metabolic abnormalities, which is important information for public health agencies to assess risk to humans.However, our study had several limitations. For example, due to the constraints of their availability, more advanced methods, such as a versatile aerosol concentration enrichment system, were not used. Furthermore, we did not perform


Escherichia coli (E. coli) O157:H7 can cause many food safety incidents, which seriously affect human health and economic development. Therefore, the sensitive, accurate, and rapid determination of E. coli O157:H7 is of great significance for preventing the outbreak and spread of foodborne diseases. In this study, a carbon dots-Fe3O4 nanomaterial (CDs-Fe3O4)-based sensitive electrochemical biosensor for E. coli O157:H7 detection was developed. The CDs have good electrical conductivity, and the surface of carbon dots contains abundant carboxyl groups, which can be used to immobilize probe DNA. Meanwhile, the CDs can be used as a reducing agent to prepare CDs-Fe3O4 nanomaterial. The Fe3O4 nanomaterial can improve the performance of the electrochemical biosensor; it also can realize the recovery of CDs-Fe3O4 due to its magnetism. As expected, the electrochemical biosensor has excellent specificity of E. coli O157:H7 among other bacteria. The electrochemical biosensor also exhibited good performance for detecting E. coli O157:H7 with the detection range of 10–108 CFU/ml, and the detection limit of this electrochemical biosensor was 6.88 CFU/ml (3S/N). Furthermore, this electrochemical biosensor was successfully used for monitoring E. coli O157:H7 in milk and water samples, indicating that this electrochemical biosensor has good application prospect. More importantly, this research can provide a new idea for the detection of other bacteria and viruses. Escherichia coli (E. coli) O157:H7 can cause some diseases such as hemorrhagic colitis and hemolytic uremic syndrome. What is more, low-dose E. coli O157:H7 can make humans sick, and in severe cases, it can even cause kidney failure and death. Currently, more than two million acute foodborne diseases worldwide are attributed to E. coli O157:H7 each year   , lateral (ELISA) , polymer (ELISA) , and so accuracy . TherefoE. coli O157:H7 detection, such as graphene/AuNPs  have some advantages of good biocompatibility, simple synthesis, and excellent conductivity , Listeria, SARS-CoV-2, Hepatitis B virus, etc.In this study, a CDs-Fereliable . The CDs3O4 nanocomposite was based on the method of instrument preparation of our group  to prepare a CDs-Fe3O4/GCE working electrode. Second, the CDs-Fe3O4/GCE electrode was immersed in 1 ml of 0.1 mM PBS, 8.00 mM EDC, and 8.00 mM NHS mixed solution for 3 h to activate the electrode, then it was rinsed with TE buffer solution. Third, the above electrode was immersed in 1 ml of 0.1 μM probe DNA to react for 24 h, and it was rinsed with TE buffer solution to prepare the DNA/CDs-Fe3O4/GCE probe electrode. Finally, the prepared electrode was stored at 4°C.First, 5.0 μl of CDs-Fe3O4/GCE was immersed into different concentrations (C) of E. coli O157:H7 solutions and reacted for 40 min at 42°C in a water bath. After the reaction, TE buffer solution was used to wash off non-specifically adsorbed E. coli on the surface to obtain the E. coli O157:H7-DNA/CDs-Fe3O4/GCE. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) have characterized the layer-by-layer assembly of each modified electrode: the [Fe (CN)6]3-/4- has been applied as the electrolyte. For the test parameters of CV, the potential scanning range was −0.2–0.8 V, and the scan rate(5) was 0.1 V/s. DPV was used to record the electrochemical signal changes before and after the identification of the electrode and different substances, and the potential interval was −0.2–0.8 V, the amplitude was 0.05 V, the pulse width was 0.05 s.The prepared DNA/CDs-Fe3O4 nanomaterial. As shown in 3O4 nanomaterial was in the range of 15–40 nm. The HRTEM characterization of CDs-Fe3O4 (3O4 and (002) of the carbon. The above morphology characterization can indicate that CDs-Fe3O4 nanomaterial has been successfully prepared.Transmission electron microscopy (TEM) has characterized the morphologies of CDs and CDs-FeDs-Fe3O4  shows th6]3−/4− is often used as a supporting electrolyte for electrochemical biosensors (6]3−/4− and 0.1 M KCl solution (I) has good reversibility, and the oxidation peak potential (Ipa) and reduction peak potential (Ipc) are −6.616 and 5.958 A, respectively. When the CDs are modified on the surface of GCE, the Ipa and Ipc are promoted to −12.29 and 12.06 A, respectively, indicating that the prepared CDs have good conductivity. When the electrode is CDs-Fe3O4/GCE, the Ipa and Ipc are further enhanced, indicating that the introduction of Fe3O4 can improve the conductivity of the electrode. However, when the probe DNA is self-assembled on the surface of CDs-Fe3O4/GCE through carboxyammonia coupling reaction, the I decreases; the main reason is that the DNA molecules will hinder the diffusion of [Fe(CN)6]3−/4− on the surface of CDs-Fe3O4/GCE. At the same time, the results indicate that the probe DNA was successfully modified on CDs-Fe3O4/GCE’s surface.It is well known that [Fe(CN)osensors . The worsolution . On the v on the electrochemical response of DNA/CDs-Fe3O4/GCE in [Fe(CN)6]3−/4− has also been studied. v, the I also keeps increasing. As shown in Ipa and Ipc show an excellent linear relationship with ν1/2, and the linear regression equations are Ipa = 3.598 + 80.265 v1/2 and Ipa = −2.551–85.187 v1/2, respectively. The results show that the electrochemical behavior of [Fe(CN)6]3−/4− on the surface of DNA/CDs-Fe3O4/GCE is mainly affected by the diffusion-controlled process , and Listeria. 3O4/GCE has almost no effect on other bacteria. However, the DNA/CDs-Fe3O4/GCE has a very strong response to E. coli O157:H7, and the Ipa drops by 53.42%. As shown in E. coli O157:H7. The above results show that the electrochemical biosensor based on DNA/CDs-Fe3O4/GCE for detecting E. coli O157:H7 has very excellent specificity and is expected to realize the determination of E. coli O157:H7 in real samples.The specificity of the electrochemical biosensor is a necessary factor to ensure the accuracy of the biosensor. Therefore, the interferences of different bacteria with 10E. coli O157:H7 interacts with DNA/CDs-Fe3O4/GCE, Ipa decreases as the C of E. coli O157:H7 increases. The main possible reason is E. coli O157:H7 has poor electrical conductivity, which affects the electrical conductivity of the working electrode’s interface, leading to a weakening of the electrochemical signal. When the C of E. coli O157:H7 is in the range of 10–107 CFU/ml, the lgC and Ipa show a good linear relationship  can reach 6.88 CFU/ml. Compared with other electrochemical biosensors for monitoring E. coli O157:H7 (3O4/GCE has very good sensitivity.As is known, DPV has been extensively applied in the construction of electrochemical sensors because it has good detection sensitivity. This study explores the sensitivity and detection range of the electrochemical biosensors via DPV. tionship : Ipa  O157:H7 , this el3O4/GCE working electrodes were prepared, and they have been applied to detect 105 CFU/ml of E. coli O157:H7 respectively. 3O4/GCE for E. coli O157:H7 detection has good reproducibility. The stability of the electrochemical biosensor has been also studied; the DNA/CDs-Fe3O4/GCE was placed in a dryer at 4°C, and the above working electrode was applied to measure 105 CFU/ml of E. coli O157:H7 every 3 days. As shown in Ipa was only dropped by 2.7%, which shows that the electrochemical biosensor based on DNA/CDs- Fe3O4/GCE has good stability.To investigate the reproducibility of this biosensor, six DNA/CDs-FeE. coli O157:H7, it is very necessary to efficiently determine E. coli O157:H7 in milk and water. In this study, to verify the practical applicability of the biosensor, the electrochemical biosensor is used to determine the E. coli O157:H7 in milk and water. 3, 104, and 105 CFU/ml of E. coli O157:H7 were added to the samples, and the recovery rates are between 95.2% and 102.0%, At the same time, the results were consistent with ELISA results, indicating that the method is feasible for monitoring E. coli O157:H7 in milk and water samples.Since milk and water are often contaminated by E. coli O157:H7 by CDs-Fe3O4 nanomaterial. Under the best conditions, when the C of E. coli O157:H7 is in the range of 10–108 CFU/ml, the lgC has a good linear relationship with the Ipa, and the LOD reaches 6.88 CFU/ml. At the same time, the electrochemical DNA biosensor has been successfully used to determine E. coli O157:H7 in milk and water samples. Therefore, this research provides an effective biosensor manufacturing strategy, which is expected to provide a reference for the preparation of electrochemical DNA biosensors for other bacteria or viruses detection.In conclusion, a sensitive electrochemical DNA biosensor has been prepared for monitoring


Salmonella Typhimurium. Salmonella enterica subsp. enterica serovar Typhimurium ATCC 19585 (STWT) and clinically isolated S. Typhimurium CCARM 8009 (STCI) were treated with ceftriaxone (CEF), chloramphenicol (CHL), ciprofloxacin (CIP), erythromycin (ERY), norfloxacin (NOR), and tetracycline (TET) in autoinducer-containing media in the absence and presence of phenylalanine-arginine β-naphthylamide (PAβN) to compare efflux pump activity with biofilm-forming ability. The susceptibilities of STWT and STCI were increased in the presence of PAβN. ERY+PAβN showed the highest decrease in the minimum inhibitory concentration (MIC) of ERY from 256 to 2 μg/mL against STWT and STCI. The antimicrobial activity of NOR against planktonic cells was significantly increased in the presence of PAβN, showing the lowest numbers of STWT (3.2 log CFU/cm2), and the TET+PAβN effectively inhibited the growth of STCI (5.2 log CFU/cm2). The lowest biofilm-forming abilities were observed at NOR+PAβN against STWT  and CEF+PAβN against STCI (BFI = 0.32). The bacteria swimming motility and relative fitness varied depending on the antibiotic and PAβN treatments. The motility diameters of STWT were significantly decreased by NOR+PAβN (6 mm) and TET+PAβN (15 mm), while the lowest motility of STCI was observed at CIP+PAβN (8 mm). The significant decrease in the relative fitness levels of STWT and STCI was observed at CIP+PAβN and NOR+PAβN. The PAβN as an efflux pump inhibitor (EPI) can improve the antimicrobial and anti-biofilm efficacy of antibiotics against S. Typhimurium. This study provides useful information for understanding the role of efflux pump activity in quorum sensing-regulated biofilm formation and also emphasizes the necessity of the discovery of novel EPIs for controlling biofilm formation by antibiotic-resistant pathogens.This study was designed to assess the influence of efflux pump activity on the biofilm formation in  Salmonella strains [Over the last few decades, antibiotics have been indiscriminately overused and misused in the treatment of infectious diseases. However, the extensive use of antibiotics has accelerated the emergence and spread of multidrug-resistant pathogens, leading to frequent treatment failure due to the limited chemotherapeutic options . Salmone strains .Chromobacterium violaceum is commonly known as an acyl-homoserine lactone (AHL) producer, which regulates the production of proteases or pigment compounds such as violacein [C. violaceum can transfer across bacterial cells and induce the QS-controlled virulence and pathogenicity [Salmonella Typhimurium ATCC 19585 (STWT) and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 (STCI) in the presence of phenylalanine-arginine β-naphthylamide (PAβN) and also investigate the relationship between efflux pump activity and biofilm formation of STWT and STCI in C. violaceum-cultured cell-free supernatant (CFS).The antibiotic substrates of single-component efflux pumps pass through the inner membrane into the periplasmic space in Gram-negative bacteria, and the transported antibiotic substrates traverse the outer cell membrane through multiple-component efflux pumps consisting of an inner membrane transporter, periplasmic membrane fusion, and β-barrel channel proteins . In addiiolacein . The AHLgenicity . In addigenicity . Furthergenicity ,10. The genicity . Efflux genicity ,12. Hencgenicity . HoweverWT and STCI used in this study have different antibiotic resistance profiles, as shown in WT and STCI were evaluated in the absence and presence of EPI, phenylalanine-arginine β-naphthylamide (PAβN 120 μg/mL) (WT and STCI exposed to PAβN varied in the classes of antibiotics. The antibiotic activities of ceftriaxone (CEF), chloramphenicol (CHL), ciprofloxacin (CIP), erythromycin (ERY), norfloxacin (NOR), and tetracycline (TET) against STWT and STCI were increased in the presence of PAβN  and was significantly decreased in the presence of PAβN (ST0 μg/mL) . The ant of PAβN . The min of PAβN .WT and STCI were evaluated at the sub-MICs of antibiotics, CEF (0.02 and 0.03 μg/mL), CHL (0.5 μg/mL), CIP (0.004 and 0.008 μg/mL), ERY (0.5 μg/mL), NOR (0.25 and 1 μg/mL), and TET (1 and 8 μg/mL), respectively, in the absence and presence of PAβN. The sub-MICs of CEF, CHL, CIP, ERY, NOR, and TET were not effective on the inhibition of biofilm formation of STWT and STCI, which were considerably inhibited in the presence of PAβN  of Chromobacterium violaceum-cultured media with or without PAβN. The number of STWT and STCI biofilm cells in CFS without PAβN were 5.8 and 6.6 log CFU/cm2, respectively, after 24 h of incubation at 37 °C, while the numbers of STWT and STCI biofilm cells in CFS were significantly decreased when treated with PAβN . The numbers of STWT treated with CEF and CHL were increased at the late stage of incubation, showing 8.5 and 7.3 log CFU/cm2 after 24 h of incubation, respectively. However, the antibiotic activities of CEF and CHL against STWT were enhanced by PAβN, showing 7.0 log CFU/cm2 at CEF+PAβN and 5.5 log CFU/cm2 at CHL+PAβN, respectively. Compared to the control, CIP+PAβN most effectively inhibited the growth of STWT (3.4 log CFU/cm2) after 24 h of incubation. PAβN restored the antimicrobial activity of ERY against STWT, showing 4.2 log CFU/cm2 at 12 h of incubation. However, the bacterial re-growth was observed for ERY and ERY+PAβN at 24 h of incubation. STWT was susceptible to NOR in the presence of PAβN, showing the lowest number of 3.2 log CFU/cm2. The growth of STWT was significantly decreased by TET (6.9 log CFU/cm2) and TET+PAβN (5.4 log CFU/cm2) at 8 h of incubation. However, the rapid growth of STWT was observed at TET and TET+ PAβN after 12 h of incubation, showing 7.8 and 6.5 log CFU/cm2, respectively (The inhibitory effect of 1/2 MICs of CEF (0.125 and 0.25 μg/mL), CHL (2 and 8 μg/mL), CIP (0.015 and 0.031 μg/mL), ERY (64 and 128 μg/mL), NOR (1 and 4 μg/mL), and TET (4 and 256 μg/mL) on the growth of STectively .2) and CEF+PAβN (7.0 log CFU/cm2) against STCI after 24 h of incubation. The numbers of STCI had no significant difference between CHL and CHL+PAβN at the late stage of incubation, showing 7.0 log CFU/cm2 at 24 h of incubation. Compared to the control, the significant decrease in the numbers of STCI was observed at CIP and CIP+PAβN treatments, showing no significant difference between CIP (7.2 log CFU/cm2) and CIP+PAβN (6.7 log CFU/cm2) at 24 h of incubation. PAβN enhanced ERY activity and inhibited the growth of STCI up to 24 h of incubation, showing 6.5 log CFU/cm2 at ERY+PAβN. Compared to the control, the growth of STCI was rapidly decreased to 5.3 and 5.2 log CFU/cm2 by NOR and NOR+PAβN, respectively, after 24 h of incubation. The growth of STCI was significantly decreased by TET+PAβN up to 12 h (5.2 log CFU/cm2), which is the most effective treatment to inhibit the growth of STCI, whereas the steady growth was observed after 12 h of incubation, showing 5.1 log CFU/cm2 , CHL (19 mm), CIP (13 mm), and ERY (12 mm), were not significantly different from CEF+PAβN (13 mm), CHL+PAβN (22 mm), CIP+PAβN (14 mm), and ERY+PAβN (11 mm), respectively. However, the motility diameters of STWT were significantly decreased by NOR+PAβN (6 mm) and TET+PAβN (15 mm) when compared to NOR (12 mm) and TET (21 mm) (The biofilm-forming abilities and the swimming motilities of ST of PAβN . The bion of CIP . The pre (21 mm) .CI was decreased in the combination treatments of antibiotics and PAβN , CEF+PAβN (18 mm), ERY (22 mm), ERY+PAβN (19 mm), NOR (12 mm), and NOR+PAβN (17 mm). The significant decrease in swimming motility of STCI was observed at CHL (26 mm), CHL+PAβN (9 mm), CIP (12 mm), CIP+PAβN (8 mm), TET (19 mm), and TET+PAβN (9 mm) . The noticeable reduction in relative fitness of STWT and STCI was observed at CIP+PAβN and NOR+PAβN.Compared to the control, the BFI of STand PAβN . The BFIN (9 mm) . The relWT and STCI were compared in the absence and presence of PAβN (WT (r = 0.89) and STCI (r = 0.72) in the absence of PAβN. STWT planktonic and biofilm cells in the presence of PAβN were highly correlated at 0.70. However, no correlation between STCI planktonic cells and biofilm cells was observed in the presence of PAβN (r = 0.23).The correlation coefficients between the numbers of planktonic and biofilm cells of ST of PAβN . The plaWT and STCI. The bacterial efflux pumps are transmembrane transport proteins targeting multiple substrates, which are known as major determinants of multidrug resistance in bacteria [S. Typhimurium [S. Typhimurium leads to the enhanced susceptibility to quinolones, tetracycline, and chloramphenicol, and the overexpression of acrB contributes to the reduced susceptibility to those antibiotics [S. Typhimurium [This study describes the interplay between efflux pump activity and biofilm formation of STbacteria . The RNDhimurium . The losibiotics . In geneibiotics . AcrAB-Tibiotics . Therefoibiotics ,13,18. Sibiotics . The inhibiotics ,21,22. Phimurium . Therefohimurium .WT and STCI were considerably decreased in the presence of PAβN  can be a possible approach to control bacterial biofilm formation in that QS inhibitors interrupt bacterial communication. Recently, the QQ has drawn more attention to enhance antibiotic susceptibility and eventually inhibit biofilm formation. The biofilm-forming abilities of STWT and STCI were inhibited by CEF, CHL, CIP, ERY, NOR, and TET in the presence of PAβN  in a cell density-dependent manner [Salmonella can detect AHLs produced by C. violaecium [Salmonella spp. cannot synthesize their own AHLs because of the absence of a luxI homolog but can recognize external AHLs by the receptor, SdiA [WT and STCI, leading to the inhibition of biofilm formation. The RND efflux pump can extrude AHLs such as 3OC12-HSL [Gram-negative bacteria synthesize various t manner . In thisolaecium . Salmoneor, SdiA . The resOC12-HSL . For insOC12-HSL . Thus, tWT and STCI. No significant differences in the bacterial numbers between the control and antibiotic treatments , which was followed by CIP+PAβN (0.35) and TET+PAβN (0.39) (The biofilm-forming abilities of STN (0.39) . This inWT treated with NOR+PAβN showed the lowest motility when compared to other treatments  and STCI (r = 0.72) were highly correlated in the absence of PAβN. The numbers of active planktonic cells are associated with the surface-attached growth during biofilm maturation [CI planktonic and biofilm cell numbers in the presence of PAβN (r = 0.23). This result suggests that the viability of planktonic cells was not influenced by PaβN, and the reduction in the biofilm cells was related to the inhibition of efflux pump developed in STCI.STeatments . At the CEF+PAβN . This im fitness . The ant fitness . In addi fitness . In part fitness . Therefo fitness . Further fitness . Thus, tturation . The adhturation . The effSalmonella enterica subsp. enterica serovar Typhimurium ATCC 19585 (STWT) and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 (STCI) were obtained from the American Type Culture Collection  and the Culture Collection of Antibiotic Resistant Microbes , respectively. Chromobacterium violaceum KACC 11542 was obtained from the Korean Agricultural Culture Collection . Strains of STWT and STCI were cultivated in trypticase soy broth  at 37 °C for 20 h. C. violaceum was cultivated in Luria–Bertani  broth at 26 °C for 20 h. The cultured cells were harvested by centrifugation at 6000× g for 10 min and diluted with phosphate-buffered saline  to approximately 108 CFU/mL for assays.Strains of WT and STCI to antibiotics and efflux pump inhibitor (EPI) were determined according to the Clinical Laboratory Standards Institute procedure with minor modifications [5 CFU/mL, 100 μL) was inoculated in the diluted antibiotic with EPI (1:2) and incubated at 37 °C for 18 h to determine MIC defined as the lowest concentrations of antibiotics where no visible bacterial growths were observed.The susceptibilities of STications . AntibioWT and STCI (106 CFU/mL each) treated with CEF, CHL, CIP, ERY, NOR, and TET were evaluated in the absence and presence of PAβN (120 μg/mL). The concentrations were determined at the 1/2 MICs of all antibiotics in the presence of PAβN, including CEF (0.0625 and 0.125 μg/mL), CHL (1 and 1 μg/mL), CIP (0.0078 and 0.0156 μg/mL), ERY (1 and 1 μg/mL), NOR (0.5 and 2 μg/mL), and TET (2 and 32 μg/mL) against STWT and STCI, respectively. The biofilm cells were enumerated using a swabbing method with slight modification [C. violaceum-cultured cell-free supernatant (CFS) in the absence and presence of PAβN (120 μg/mL). In brief, after 24 h of incubation at 37 °C, the biofilm cells were collected from the 96-well microtiter plates. Each well of the 96-well microtiter plate was gently rinsed two times with PBS to remove planktonic cells and scraped using a water-moistened sterile cotton swab. The collected biofilm cells harvested by centrifugation at 5000× g for 10 min at 4 °C were serially (1:10) diluted with PBS, plated on the TSA using an Autoplate® Spiral Plating System  and incubated at 37 °C for 24 h. The biofilm cell numbers were enumerated using a QCount® Colony Counter .The biofilm-forming abilities of STfication . The tesWT and STCI were evaluated in TSB at 37 °C for 24 h. The initial population (107 CFU/mL) of STWT and STCI was inoculated at 37 °C in C. violaceum-cultured CFS with 1/2 MICs of antibiotics in the absence and presence of PAβN (120 μg/mL). The antibiotics concentrations were 0.125 μg/mL CEF, 4 μg/mL CHL, 0.0156 μg/mL CIP, 128 μg/mL ERY, 2 μg/mL NOR, and 4 μg/mL TET for STWT in absence of PAβN, 0.0625 μg/mL CEF, 1 μg/mL CHL, 0.0078 μg/mL CIP, 1 μg/mL ERY, 0.5 μg/mL NOR, and 2 μg/mL TET for STWT in presence of PAβN, 0.125 μg/mL CEF, 4 μg/mL CHL, 0.03125 μg/mL CIP, 128 μg/mL ERY, 4 μg/mL NOR, and 256 μg/mL TET for STCI in the absence of PAβN, and 0.125 μg/mL CEF, 1 μg/mL CHL, 0.0156 μg/mL CIP, 1 μg/mL ERY, 2 μg/mL NOR, and 32 μg/mL TET for STCI in the presence of PAβN. The inoculated samples were incubated for 0, 4, 8, 12, and 24 h at 37 °C. After incubation, the collected planktonic cells were serially (1:10) diluted with PBS. Proper dilutions were plated on the TSA and incubated at 37 °C for 24 h. Viable cell numbers were enumerated as described above.The antimicrobial activities of single antibiotic and combination treatments of antibiotics and PAβN against STWT and STCI (107 CFU/mL each) were cultured in C. violaceum-cultured CFS with 1/2 MICs of CEF, CHL, CIP, ERY, NOR, and TET with and without PAβN (120 μg/mL) in a 96-well plate. After incubation at 37 °C for 24 h, the cultures were centrifuged at 5000× g for 10 min at 4 °C to collect planktonic cells. Each well was rinsed with PBS, and biofilm cells were collected by using a cotton swab and centrifuged at 5000× g for 10 min at 4 °C. The planktonic and biofilm cells were enumerated as described above. The biofilm-forming index (BFI) was expressed as the ratio of number of planktonic cells to that of biofilm cells [STlm cells .WT and STCI cells were treated with 1/2 MICs of CEF, CHL, CIP, ERY, NOR, and TET in the presence and absence of PAβN (120 μg/mL) and incubated at 37 °C for 24 h. The planktonic cells  were collected and seeded onto the center of the surface of 0.3% agar-containing plates. After 12 h of incubation at 37 °C, the bacterial migration on each plate was measured using a digital vernier caliper .Flagellum-dependent movement was determined by the swimming motility assay . STWT anWT and STCI cells treated with 1/2 MICs of CEF, CHL, CIP, ERY, NOR, and TET in the presence and absence of PAβN (120 μg/mL) for 24 h. Then, the antibiotic-treated planktonic cells were collected by centrifugation at 5000× g for 10 min at 4 °C, rinsed two times with PBS, and cultured at 37 °C for 24 h in fresh TSB in the absence of antibiotics and PAβN. The relative fitness was expressed as the ratio of the growth (OD600) of treated cells to that of untreated control in the absence of antibiotics.The relative fitness was estimated to assess the cost of resistance of STp < 0.05, p < 0.01, and p < 0.001. The correlations between planktonic and biofilm cells were evaluated using Pearson’s correlation coefficient.All experiments were carried out in duplicate on three replicates. Data were analyzed using the Statistical Analysis System (SAS). The general linear model (GLM) and Fisher’s least significant difference (LSD) procedures were used to determine significant differences between treatments at S. Typhimurium. The most significant findings were that (1) the MICs of CEF, CHL, CIP, ERY, NOR, and TET against STWT and STCI were decreased in the presence of PAβN, (2) the sub-MICs of CEF, CHL, CIP, ERY, NOR, and TET were not effective in the inhibition of biofilm formation of STWT and STCI and, (3) PAβN significantly increased the inhibitory activity of sub-MICs of CEF, CHL, CIP, ERY, NOR, and TET on the biofilm formation by STWT and STCI. The NOR+PAβN and CEF+PAβN treatments showed the highest anti-biofilm activity against STWT and STCI, respectively. The inhibition of efflux pump by EPIs can interfere with the transport of quorum-sensing molecules, leading to the decrease in biofilm-forming abilities of pathogens. PaβN have no effect on the viability of planktonic cells but only inhibited biofilm formation. The use of antibiotics combined with EPIs has a great potential of controlling biofilm formation. Accordingly, EPIs can be used as a potential control agent to inhibit biofilm formation by pathogens.This study highlights the effect of efflux pump activity on the biofilm formation of


Large granular lymphocyte (LGL) leukemia, a lymphoproliferative disease, is characterized by an increased frequency of large-sized lymphocytes with typical expression of T-cell receptor (TCR) αβ, CD3, CD8, CD16, CD45RA, and CD57, and with the expansion of one to three subfamilies of the TCR variable β chain reflecting gene rearrangements. Molecular analysis remains the gold standard for confirmation of TCR clonality; however, flow cytometry is time and labor saving, and can be associated with simultaneous investigation of other surface markers. Moreover, Vβ usage by flow cytometry can be employed for monitoring clonal kinetics during treatment and follow-up of LGL leukemia patients.+ T cells with T-cell receptor (TCR) clonality defined by molecular and/or flow cytometric analysis. Vβ usage by flow cytometry can identify clonal TCR rearrangements at the protein level, and is fast, sensitive, and almost always available in every Hematology Center. Moreover, Vβ usage can be associated with immunophenotypic characterization of LGL clone in a multiparametric staining, and clonal kinetics can be easily monitored during treatment and follow-up. Finally, Vβ usage by flow cytometry might identify LGL clones silently underlying other hematological conditions, and routine characterization of Vβ skewing might identify recurrent TCR rearrangements that might trigger aberrant immune responses during hematological or autoimmune conditions.Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder of mature T or NK cells frequently associated with autoimmune disorders and other hematological conditions, such as myelodysplastic syndromes. Immunophenotype of LGL cells is similar to that of effector memory CD8 LGL leuhenotype . In othe TCR γδ+ ,13,14,15 TCR γδ+ . Clinica TCR γδ+ ,19,20. I TCR γδ+ . In 6–22 TCR γδ+ . Converscell NHL ,25.In this review, we focus on biological significance of LGL clonality and clinical utility of TCR repertoire investigation by flow cytometry.+ T-LGL [MYC, AURKA, and AURKB, responsible for centrosome alterations, and DNMT3B, ultimately leading to hypermethylation of tumor suppressor genes [STAT3 gene can also induce constitutive activation of JAK/STAT pathway, as well as mutations on the STAT5b gene, more frequently described in CD4+ T-LGL leukemia [+CD16+CD56− LGLs and STAT3 hyperactivation, functions as a decoy receptor preventing Fas-mediated apoptosis [The exact pathogenesis of LGL leukemia is still unclear. The most accepted hypothesis is a clonal drift of a T cell population after chronic antigen exposure, as suggested by cross-reactivity to human T-cell lymphotropic virus 1 (HTLV-1) epitopes or a frequent concomitant chronic viral infection in LGL leukemia patients, especially EBV, hepatitis C virus (HCV), or cytomegalovirus (CMV), especially in CD4+ T-LGL ,27,28,29+ T-LGL ,30,31. O+ T-LGL ,33,34. I+ T-LGL . IL-15 aor genes ,37. Howeor genes . IL-6, aor genes ,40. Uponleukemia ,42,43. Spoptosis ,44,45,46poptosis ,48. In Lpoptosis . In addipoptosis . Neutroppoptosis . Indeed,poptosis .+ or CD8+ lymphocytes, to recognize a large number of antigens  or limited ones  [18 diverse TCRs meaning that an individual might recognize up to 1018 diverse antigens [+CD28+ and CD8+CD28+ T cells are activated and expand to increase the chance of having a functional antigen-specific immune clone that can fight pathogens [+CD28−CD57+ effector memory T cells become immunodominant and constitute an oligoclonal pool of memory T cells [+ T lymphocytes is observed in several autoimmune diseases and cancers and is related to poor survival [+ effector memory cells show oligoclonality with 1–3 immunodominant clones private to the disease [STAT3 or STAT5b genes [Reactive LGLs can be found at low frequencies in healthy subjects after viral infections; however, in these cases, LGLs are polyclonal while neoplastic clones are oligo- or monoclonal . The teronality) . TCR Vβ onality) ,62,63,64antigens . In earlathogens ,67. If tsurvival ,69. More disease ,70,71,72 disease . Interes disease . TCR clo5b genes ,33. Desp5b genes ,75. CDR35b genes . TherefoTCRG) rearrangements, which are more limited than those of TCRB gene [The diagnostic definition of clonality is still challenging because current technologies alone cannot conclusively define TCR oligo/monoclonality or cannot be applied in routine diagnostic settings ,75,76. FCRB gene ,80. SangCRB gene ,82,83. TCRB gene ,85. CurrCRB gene ,87.+, CD4+, and CD8+ T cells [+ and CD8+, effector CD4+CD28+ and CD8+CD28+, and effector memory CD4+CD28−CD57+ and CD8+CD28−CD57+ cells in AA [In available kits, antibodies are conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), or tandem FITC/PE; therefore, sample acquisition requires instruments equipped just with a blue laser (488 nm excitation) and sensors for detection of FITC and PE emission wavelengths  ,68. Vβ u T cells  in LGL lls in AA ,70,71. Wls in AA .The presence of 1–3 immunodominant Vβ clones does not prove clonality and the diagnosis of LGL leukemia, because flow cytometry results should be validated by DNA sequencing of both Vα and Vβ regions and supported by clinical manifestations and immunophenotype compatible with LGL leukemia ,91. Vβ sRelevant literature using the key word “large granular lymphocyte leukemia” was searched in PubMed database from 1977 to July 2021. Limiting factors were “large granular lymphocyte leukemia”, and full text available in English language. Two investigators independently reviewed the reference list for potential eligible manuscripts, and selected articles were then reviewed independently for inclusion in the analysis. Studies were included when: (1) the year of publication was between 2011 and 2021; (2) the studies were conducted on humans and reported clinical data and Vβ usage by flow cytometry; and (3) the studies were on T-LGL leukemia .n = 20), data were collected into a standardized form including publication year, source, number of total patients and divided by sex, number of subjects with autoimmune disorders, hematological malignancies, or cancers, and Vβ usage by flow cytometry , and autoimmune polyglandular syndrome type 1. Among hematological diseases, PRCA was the most frequent disorder associated with T-LGL leukemia , followed by plasma cell disorders , especially monoclonal gammopathy of uncertain significance (MGUS), and myelodysplastic syndromes . Other reported hematological conditions associated with T-LGL leukemia were: AIHA ; T- and B-cell NHL ; acute leukemias, both myeloid and lymphoblastic; immune thrombocytopenic purpura (ITP); or AA. Solid neoplasms were also frequently associated with T-LGL leukemia . Reported frequencies are slightly higher especially for AIHA, ITP, and RA likely because Vβ usage by flow cytometry is not routinely performed, and TCR clonality is mostly confirmed by PCR without indicating expanded Vβ family but only reporting LGL phenotype and clinical manifestations [From selected articles , and 1–3 Vβ subgroups were expanded in 12 of them (4.5% of cases). Among studies, the most frequent expanded Vβ family was the Vβ13.1 , followed by Vβ3 , Vβ17 , and Vβ2 and Vβ14  (n = 18), and Vβ1 in 5.7% (n = 15). Other Vβ families were expanded in less than 5% of cases, and in less than 1% of subjects (case reports) for Vβ5.2, Vβ5.3, Vβ6.1, Vβ6.7, Vβ11, and Vβ13.3. Of total cases, the immunophenotype of LGLs was detailed only in 520 subjects, and 89 cases (17.1%) were CD4+ T-LGLs, 19 (3.7%) CD4+CD8+/dim, and the remaining cases were CD8+ T-LGLs. Of the total CD4+ T-LGL leukemia cases, in 48 of them, Vβ usage was described in detail, and there was no Vβ skewing in most cases  or Vβ13.1 expansion . Other reported expanded Vβ families were: Vβ3; Vβ8; Vβ17; and anecdotical Vβ1, Vβ2, Vβ5.2/5.3, Vβ12.2, Vβ14, and Vβ22. Of the total 33 cases with expanded Vβ13.1, 16 of them (48.5%) were CD4+ T-LGLs, three (9.1%) were CD4+CD8+, and the remaining 14 cases (42.4%) were CD8+ T-LGLs. These cumulative data are similar to those reported in single case series showing a preferential Vβ13.1 expansion in CD4+ T-LGL leukemia [+CD8dim T cells expressing the killer lectin-like receptor NKG2D, a CD4+ T cell subset present in RA, human T-lymphotropic virus 1-associated myelopathy, and Wegener granulomatosis [+CD8dimNKG2D+ T cells bearing the HLA-DRB1*0701 allele and restricted to Vβ13.1 have a highly homogeneous TCR repertoire [+CD8dimNKG2D+ T cells can produce FasL while protected from Fas-mediated growth arrest because of NKG2D protective functions [+ leukemic cells might add evidence to the antigen-driven clonal drift pathogenetic theory in T-LGL leukemia.TCR clonality is a major criterion for LGL leukemia diagnosis and can be defined using a molecular or flow cytometric analysis, as described above, and clonal kinetics have shown clinical utility in monitoring disease progression and responsiveness to treatment . TCR repl cases) . Vβ8 repleukemia ,75. Vβ13leukemia ,109,110.omatosis ,111. Intunctions . TherefoAn exact association between Vβ usage and underlying hematological disease rather than T-LGL leukemia was identified in only 32 subjects out of 189 patients . There was no recurrent Vβ expansion, despite Vβ14 represented the immunodominant clone in six cases . Vβ14 was not associated with a specific disease, as it was expanded in subjects with MDS, acute leukemias, NHL, or chronic myeloid leukemia; however, the number of evaluable patients was too small to draw any significant conclusion. Conversely, these data could be a starting point for implementing Vβ usage analysis by flow cytometry in subjects with hematological conditions with concomitant increased LGL count. Indeed, case series report concomitant Vβ skewing in MDS patients ; however+ T cells with aberrancy and TCR clonality, mostly TCR αβ and less commonly TCR γδ clonal rearrangements [+CD8+ T cells. Indeed, small T-LGL clonotypes might be diluted on total CD8+ T cells that appear polyclonal, as elegantly described in AA by both flow cytometry and NGS [LGL leukemia is classified as a lymphoproliferative disorder of mature T or NK cells; however, this clinical entity is considered among other BMF syndromes and is frequently associated with autoimmune disorders and other benign and malignant hematological conditions, such as PRCA or acute leukemias ,2,3. Thengements ,73. Demongements ,94,95,96ngements ,114. Howngements ,75. Vβ ungements ,75. Despngements ,74,88,89 and NGS . In addi and NGS . Therefo


Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNFα signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNFα mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.Tumor necrosis factor alpha (TNFα) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNFα in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U/mL TNFα induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [ The intestinal epithelial paracellular barrier is essential since it prevents the immune system from being overly induced by a wide variety of exogenous antigens and microorganisms. Tight junctions (TJs), which are formed in strands and located at the apicolateral membrane of adjacent cells, represent the functional correlate of the epithelial paracellular barrier function. These TJ strands are defined by the common presence of several integral membrane proteins, such as occludin , junctioThe proinflammatory cytokine tumor necrosis factor α (TNFα) plays a key role in the pathogenesis of IBD, and thus has been shown to be increased in patients with Crohn’s disease (CD) or ulcerative colitis (UC) ,7. One pThe permanent porcine intestinal epithelial cell line IPEC-J2, a non-transformed cell line obtained from jejunal epithelia of an unsuckled piglet, has been characterized as a suitable in vitro intestinal model . Due to 3H]-D-Mannitol flux during treatment with the cytokine. Subsequent to the incubation experiments, the TJ composition was analyzed in detail and the impact of TNFα on the density of tumor necrosis factor receptor 1 and 2  was investigated. As MLCK has been discussed in context with barrier perturbation in a series of studies, further experiments were carried out using a specific blocker against MLCK to analyze the signaling cascade of TNFα on IPEC-J2 cells. This provides new experimental data, showing that (i) the non-transformed cell line IPEC-J2 represents a useful model for the exploration of inflammation-induced changes in porcine intestinal epithelial barrier function and (ii) explains the self-enhancing effects of TNFα resulting in TJ impairment in the intestinal epithelium on a functional, molecular and regulatory level.The barrier-weakening effect of TNFα has been evaluated on various cell culture models, such as the rat ileal intestinal epithelial cell line IEC-18  or the h3H]-D-Mannitol flux were measured. After 48 h, 1000 U/mL and 5000 U/mL TNFα showed a marked decrease in TEER compared to the control group  = 9.22, p < 0.0001, n = 14–22; ctrl: 101.06 ± 2.89%; 1000 U/mL: 88.45 ± 2.56%, p = 0.0113; 5000 U/mL: 78.74 ± 3.19%, p < 0.0001, p = 0.91; 100 U/mL: 102.21 ± 5.41%, p = 0.99; 500 U/mL: 98.49 ± 2.35%, p = 0.96). Hence, the following experiments were carried out using concentrations of 1000 U/mL TNFα. After 24 h, 1000 U/mL did not have significant effects on TEER . The same effect could be observed in the second flux period from 24 to 48 h . The same effect could be observed for claudin-3 , as well as for occludin expression . The expression of the TJ proteins claudin-4, -7 and ZO-1 did not significantly change during TNFα treatment .Western blot analysis was performed to investigate whether the incubation with TNFα has any effect on the expression of TNFR-1 or -2. After normalization on total protein amount, the control groups were set to 100% and the TNFα-treated groups were compared to them, respectively. After 48 h with 500, 1000 and 5000 U/mL TNFα, a marked up-regulation of TNFR-1 expression could be observed  = 9.70, In addition to changes on the expression of TNFR-1, we aimed to analyze whether TNFα also leads to an altered localization of TNFR-1 in IPEC-J2. Therefore, cells were stained with TNFR-1 and ITGβ1, an integral membrane protein that is located at the basolateral membrane of cells . With rip < 0.0001, n = 7–29; TNFα: 92.38 ± 2.66%; TNFα + ML-7: 113.76 ± 5.69%, p = 0.0053). Comparable results could be seen between cells treated with TNFα and control groups . While the expression of claudin-1 was also notably higher with TNFα and ML-7 compared to cells treated only with TNFα, just a tendency towards significance could be shown  = 5.30, p = 0.03, n = 4; TNFα: 69.34 ± 5.08%; TNFα + ML-7: 94.31 ± 11.17%, p = 0.079). Surprisingly, treatment with the MLCK blocker did not affect the expression of occludin  = 7.65, p = 0.02, n = 4; TNFα: 50.76 ± 10.04%; TNFα + ML-7: 50.28 ± 12.91%; p = 1.00). The strong increase in TNFR-1 due to TNFα incubation was also inhibited by ML-7  = 9.09, p = 0.01, n = 4; TNFα: 160.70 ± 12.04%; TNFα + ML-7: 117.57 ± 11.32%; p = 0.026).We used ML-7, a specific MLCK blocker, to analyze the signaling of TNFα-induced changes in epithelial barrier function in more detail. After 48 h, cells treated with TNFα together with ML-7 showed a significant difference compared to cells treated solely with TNFα  = 15.37, Subsequently, confocal laser scanning immunofluorescence microscopy was carried out after 48 h to see whether ML-7 is also capable of inhibiting the changed localization of TJ proteins caused by TNFα. As shown in p = 0.0047, n = 7–13; TNFα w/o Recovery: 77.36 ± 11.41%; TNFα w/ Recovery: 115.88 ± 15.65%; p = 0.076). The control groups showed a strong significant difference compared to the cells treated with TNFα for 96 h, . To analyze the effects a recovery period from TNFα might have on the changes in TJ proteins and TNFR-1, Western blot analysis and confocal laser scanning immunofluorescence microscopy were performed subsequently. A recovery period of 48 h after incubation with the cytokine led to a marked increase in claudin-1 expression in IPEC-J2 cells  = 61.35, p = 0.0001, n = 3; TNFα w/o Recovery: 38.11 ± 2.34%; TNFα w/ Recovery: 63.20 ± 6.47%; p = 0.01; p = 0.002). Densitometric analysis of claudin-3 also revealed a rise after the removal of the cytokine, yet not to the same extent as claudin-1  = 5.84, p = 0.039, n = 3; TNFα w/o Recovery: 69.77 ± 8.10%; TNFα w/ Recovery: 95.08 ± 8.33%; p = 0.08). For occludin expression, the recovery period led to a strong increase compared to the cells treated with TNFα for 96 h  = 16.24, p = 0.004, n = 3; TNFα w/o Recovery: 58.32 ± 8.40%; TNFα w/ Recovery: 81.70 ± 3.18%; p = 0.04). The increased expression of TNFR-1 due to TNFα incubation was not affected by the recovery period  = 4.78, p = 0.09, n = 3). Because TNFα has apoptotic potential ,30, we pMoreover, confocal laser scanning immunofluorescence microscopy revealed that the paracellular TJ strands seemed to reconstitute after a recovery period of 48 h, as the claudin-1 signal appeared to be integrated again in the lateral membrane after a loss due to TNFα incubation A. Forty-In addition to the recovery experiments, an ApoTox-Glo assay was performed and the apoptotic potential of TNFα was further analyzed to ensure that the changes in barrier function are not due to the induction of apoptosis by the cytokine. Despite all this, neither the apoptotic or the cytotoxic rate nor the viability of IPEC-J2 cells was significantly changed after treatment with 1000 U/mL TNFα for 48 h. Hence, apoptosis could be eliminated as a cause for the decrease in transepithelial resistance in IPEC-J2 cells (data not shown).The intestinal mucosa provides one of the most important barriers to the outside environment, and an intact barrier is maintained by TJs linking adjacent epithelial cells and the immune system ,32. One In our current work, we analyzed the effects of TNFα on the intestinal epithelial barrier function of the non-transformed porcine intestinal epithelial cell line IPEC-J2. Although this cell line has been described as a suitable in vitro model so far , the cenAs already assumed, the use of TNFα showed a highly significant decrease in TEER, as well as an elevation in paracellular permeability to [³H]-D-Mannitol, both representing epithelial barrier function. To analyze this barrier-weakening effect more precisely, immunoblots and confocal laser scanning immunofluorescence microscopy of TJ proteins including claudin-1, -3, -4, -7, occludin and ZO-1 were subsequently carried out. In our study, strong yet differential effects of the cytokine on different TJ proteins were observed. Hence, the participation of the sealing TJ proteins claudin-1, as well as claudin-3 and occludin, on the disruption of epithelial barrier function could be shown, as the decrease in these integral membrane proteins after incubation with TNFα was in accordance with functional changes. In addition, not only a marked reduction in protein expression could be shown, but also a disruption of those TJ proteins, as the signal of claudin-1, -3 and occludin was either much reduced, disintegrated or located more intracellularly than in the cells treated without the cytokine. Because TNFα can bind two specific receptors, namely TNFR-1 and TNFR-2, we aimed to analyze if the observed changes are linked to the altered density of these receptors. Hence, we were able to detect a dose-dependent increase in TNFR-1 due to the incubation with TNFα, while TNFR-2 remained unchanged. One reason for this might be the expression of TNFR-1 in most tissues, while TNFR-2 appears to be found mostly in the lymphoid system. TNFR-1 is also the key signaling receptor for TNFα, as it is activated by both soluble and membrane-bound TNFα and mediates the signaling of apoptosis and inflammation ,36. FurtTo exclude the possibility that the observed changes are associated with an activation of apoptosis by TNFα ,30, we pBecause the disruption of TJ proteins has been explained to be in accordance with an increased expression of MLCK ,14,15,37Incubation with TNFα in the presence of the specific MLCK blocker ML-7 prevented the decrease in TEER and claudin-3 expression. However, in our experiments, occludin expression was not significantly altered. Whether the effects of MLCK are related to a role for myosin transport of the affected proteins, or if MLCK might have other, e.g., structural roles ,43, coulIn our current study, the pharmacological perturbation of MLCK was performed by the small molecule inhibitor ML-7. Although additional results such as (i) MLCK knockdown during TNFα treatment and (ii) MLCK overexpression in the absence of TNFα might even have strengthened the study, the blocking experiment has proven reliable for a convincing conclusion on the role of MLCK. Other additional mechanisms and signaling events might be still involved, though.In a publication from Xiao et al., IPEC-J2 cells were stimulated with TNFα to analyze whether TGF-β1 may have protective effects. After TNFα challenge, a reduction in occludin and ZO-1 of IPEC-J2 monolayers was observed, while the TJ protein claudin-1 remained unaffected . This isAn effect of TNFα not only on sealing but also on pore-forming tight junction proteins has been described previously . In diff2 culture flasks in Dulbecco’s MEM/Ham’s F-12  containing 3.15 g/L glucose, 2 mM stable glutamine, 10% porcine serum  and 1% penicillin/streptomycin . Cells were cultured at 37 °C in a humified 5% CO2 atmosphere. The medium was changed every 2–3 days and cells were split once a week at a ratio of 1:3. For electrophysiological measurements, cells were seeded at a density of 2 × 105 cells/mL on semipermeable cell culture inserts with a diameter of 12 mm and a pore size of 0.45 µm  and placed into multi-well plates. Routinely, 500 µL of media was added to the apical compartment, and the basolateral compartment was filled with 1 mL of media to guarantee an equal hydrostatic pressure, as specified by the manufacturer. Experiments were performed after cells reached similar Rt values (~14 days) to ensure a functional barrier. Therefore, recombinant human TNFα  was added in various concentrations  to the basolateral side of the cell culture inserts, and transepithelial resistance was monitored for 48 h. The cells were further processed and used for immunoblotting and immunohistochemistry, as described below. Resistance values were corrected with the blank values of the filters and the media used in the experiments. Only cells with passages between 7 and 12 were used for experimental purposes.Confluent monolayers of the porcine jejunal cell line IPEC-J2  were grown in 25 cmTransepithelial electrical resistance (TEER) measurements, presenting the epithelial barrier function, were assessed by using a chopstick electrode and an Epithelial Volt/Ohm Meter . Permeability studies were carried out using [³H]-D-Mannitol to examine the unidirectional paracellular tracer flux from the apical to the basolateral side of the cell filters. Cells were seeded on semipermeable cell culture inserts as described previously. A total of 0.18 µCi of [³H]-D-Mannitol  was added to the apical side of the filters, and samples of 50 µL were taken directly and 48 h after addition of the tracer. Subsequently, the specific activity of the tracer was calculated using Equation (1).For permeability measurements, samples of 300 µL were taken every 24 h during the incubation with the cytokine from the basolateral side, resulting in two flux periods. The removed media were replaced with fresh media containing the corresponding TNFα concentration. Following the sampling, Aquasafe 300plus liquid scintillation cocktail  was added and each sample was examined using TriCarb 4910TR liquid scintillation counter . By using Equation (2), the resulting paracellular flux was calculated.After incubation of the cells with TNFα, IPEC-J2 monolayers were washed in PBS with calcium and magnesium and lysed in RIPA buffer, containing 25 µM HEPES pH 7.6, 25 µM NaF, 2 µM EDTA, 1% Sodium Dodecyl Sulfate (10%), H2O and enzymatic protease inhibitors . Cells were then scraped off the permeable supports and the suspension was transferred into Eppendorf tubes. Samples were homogenized after incubation on ice for 30 min. Protein quantification was performed by using Bio-Rad DC Protein Assay  as prescribed by the manufacturer. For the detection, EnSpire Multimode Plate Reader  was used.Western blot analysis was performed to identify the TJ protein expression after TNFα treatment. Protein (20 µg) and Laemmli buffer  were mixed and loaded onto 10% TGX Stain-Free FastCast gels . Electrophoresis was carried out for 60 min at 150 V. Subsequently, samples were transferred onto a PVDF membrane for 90 min at 100 V and blocked for 60 min in 5% milk . Membranes were then incubated with specific antibodies raised against TJ proteins  claudin-1 cat. #51-9000), -3 (cat. #34-1700), -4 (cat. #32-9400), -7 (cat. #34-9100), occludin (cat. #33-1500) and ZO-1 (cat. #33-9100) following the manufacturer’s instructions at 4 °C overnight, respectively. To bind the primary antibodies, horseradish-peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies were used for 1 h at room temperature. Specificity was shown in detail previously ,51,52. F-9000, -3Confocal laser scanning immunofluorescence microscopy was performed for the detection and localization of TJ proteins after TNFα treatment. Therefore, cells were washed twice with PBS and fixated in ice-cold methanol for 10 min at −20 °C. Permeabilization was carried out using Triton X-100 for 10 min at room temperature. Cells were then blocked for 60 min in PBS containing 1% bovine serum albumin and 5% goat serum and subsequently incubated with primary antibodies raised against claudin-1, -3, occludin and ZO-1 for 60 min at 37 °C , -3 (cat. #34-1700), occludin (cat. #33-1500) and ZO-1 (cat. #33-9100)). Again, cells were washed with PBS and then incubated with secondary goat anti-rabbit Alexa Fluor-488  or goat anti-mouse Alexa Fluor-594 (cat. #A-11005) for 60 min at 37 °C. Nuclei were stained with DAPI (1:2000) for 5 min at room temperature. Following this, filters were mounted with ProTaqs Mount Fluor  and slides were analyzed by using Zeiss 710 confocal microscope .To examine whether the treatment with TNFα affects the expression level of tumor necrosis factor receptors 1 and 2  in IPEC-J2 cells, Western blot analysis was carried out as mentioned above. Membranes were incubated with rabbit polyclonal antibodies raised against TNFR-1  and TNFR-2 . After detection, specific bands were normalized on total protein amount and analyzed compared to the untreated groups, respectively. Moreover, immunohistochemistry of IPEC-J2 cells, treated with rising TNFα concentrations, was performed for examination of expression level and localization of TNFR-1. Therefore, a staining using specific antibodies raised against TNFR-1 together with a marker for the basolateral membrane, namely Integrin beta-1 , was carried out as mentioned above. To analyze the location of TNFR-1 in more detail, Z-stacks were performed additionally. For a more detailed characterization of the signaling in TNFα-induced barrier changes, ML-7 , a specific blocker against MLCK, was used. Therefore, IPEC-J2 cells were incubated with TNFα (1000 U/mL) in the presence or absence of ML-7 (10 µM). Stock solutions of ML-7, dissolved in dimethyl sulfoxide , were diluted in medium and added simultaneously with TNFα to the apical and basolateral compartment of the cell filters. Respectively, 0.1% of DMSO was added to controls as well as to cell filters incubated with TNFα without ML-7, to exclude a DMSO-dependent effect on IPEC-J2 cells. TEER measurements were carried out before addition and after 48 h. Subsequent to TEER measurements, cells were either fixated for immunohistochemistry or proteins were extracted and further processed for immunoblotting, analyzing localization and expression of TJ proteins after experiments with or without ML-7. Because TNFα has been described to have apoptotic potential, we wanted to investigate if the IPEC-J2 cells can recover from the TEER decrease after a TNFα-challenge. Therefore, cells were incubated with 1000 U/mL TNFα for 48 h as described above. Subsequently, a medium exchange was carried out and fresh media without TNFα were added to the cells that were treated with the cytokine before. Following TEER measurements, possible alterations of TJ proteins were examined using Western blot technique or immunohistochemistry as described above.5 cells/mL on PET cell culture inserts of a 24-well Transwell system with 6.5 mm membrane diameter and a 0.4 µm pore size . After a cultivation period of 14 to 16 days, cells were treated with TNFα (1000 U/mL) and further incubated for 48 h. To measure the viability, cytotoxicity and apoptosis of the IPEC-J2 cells, the ApoTox-GloTM Triplex Assay  was carried out according to the manufacturer’s specifications. After the incubation period with TNFα, the viability/cytotoxicity reagent, containing both the bis-AAF-R110 substrate and GF-AFC substrate, was added to each well and mixed for 30 s on an orbital shaker. After an incubation of 30 min at 37 °C, the fluorescence was measured at 400EX/505EM for viability and 485EX/520EM for cytotoxicity. Following this, the apoptosis reagent, containing the Caspase-Glo 3/7 substrate, was added to each well, mixed gently and incubated for another 30 min at constant room temperature. Luminescence was measured to detect cell apoptosis. All measurements were carried out using EnSpire Multimode Plate Reader .IPEC-J2 cells were seeded as triplicates at a density of 2 × 10t-test was used. Statistical analysis for densitometry of Western blots was performed with one-way ANOVA for normally distributed data and with Kruskal–Wallis test for non-normally distributed data. For the signaling experiments, Tukey–Kramer method was used as post hoc test for pairwise comparisons. Values of p < 0.05 were considered to be statistically significant, being presented as * p < 0.05, ** p < 0.01 and *** p < 0.001. Data are always expressed in means and standard error of the mean (SEM). N represents the number of cell culture inserts used unless stated otherwise. For TEER measurements, statistical analysis was performed using one-way ANOVA and Dunnett’s test for the correction of multiple testing. For comparison between two groups, unpaired Student’s Employing the non-transformed intestinal epithelial cell line IPEC-J2, our study demonstrates, for the first time, that the exponential nature of barrier impairment by TNFα in porcine intestinal inflammatory processes can be explained by the finding that its own receptor, TNFR-1, is upregulated by TNFα itself.


The benefit of adjuvant therapy in synovial sarcoma (SS) treatment is under debate. Long-term follow-up data are missing.SS patients treated in the consecutive trials CWS-81, CWS-86, CWS-91, CWS-96, CWS-2002-P, and the SoTiSaR-registry till 2013 were analyzed.Median age of 185 patients was 13.9 years (0.1–56)—with median follow-up of 7.4 years for 163 survivors. Most tumors (76%) were located in extremities. Size was < 3 cm in 58 (31%), 3–5 cm in 59 (32%), 5–10 cm in 42 (23%), and > 10 cm in 13 (7%) (13 missing). In 84 (45%) tumors, first excision was complete (R0 corresponding to IRS-I-group) and in 101 (55%) marginal (R1 corresponding to IRS-II-group). In a subsequent surgical intervention during chemotherapy, R0-status was accomplished in 23 additional IRS-II-group patients with secondary surgery. Radiotherapy was administered to 135 (73%), thereof 62 with R0-status and 67 R1-status (6 missing information). Adjuvant chemotherapy was administered to all but six patients. 5-year event-free (EFS) and overall survival (OS) was 82.9% ± 5.7 (95%CI) and 92.5% ± 3.9. Local and metastatic relapse-free survival was 91.3% ± 4.3 and 92.3% ± 4.1 at 5 years, respectively. In the multivariate analysis, tumor size and no chemotherapy were independently associated with EFS. Size and site were associated with OS. In a detailed analysis of local and metastatic events, tumor size was associated with an independent risk for developing metastases. No independent factor for suffering local recurrence could be identified.Omission of chemotherapy in a non-stratified way seems not justified. Size governs survival due to high linear association with risk of suffering metastatic recurrence in a granular classification.The online version contains supplementary material available at 10.1007/s00432-021-03614-6. Soft-tissue sarcomas represent nearly 8% of childhood malignancies. Synovial sarcoma (SS) is the most common non-rhabdomyosarcoma soft-tissue sarcoma Goldblum , charactWhereas pediatric oncologists assumed that chemotherapy might play an important role, and administered adjuvant chemotherapy regardless of risk factors  classification  in CWS-81  patients, where it was only recommended in CWS-86 and CWS-91. According to the respective CWS-protocol radiotherapy at doses of 32–54.4 Gy (when accelerated hyperfractionated) and 40–50 Gy if conventional fractionated dependent on response to chemotherapy and resection status was to be administered in analogy to recommendations for patients with rhabdomyosarcoma . Comparison of distribution was performed with the χAmong 330 patients with localized SS, 185 had undergone initial gross resection, thereof 84 with primary free margins IRS-I/(R0) and 101 with primary positive margins IRS-II/(R1). Gender distribution was balanced  received the VAIA, 1 the EVAIA, and 30 (16%) the VACA regimen.In the IRS-II/(R1) group, 23 of 101 patients underwent second surgical intervention only after the start of chemotherapy to obtain a tumor cell-free primary site. Therefore, in total, 107 (58%) achieved a R0-status as best surgical result at any time.Radiotherapy was administered to 135 (73%): 45 out of 84 (54%) IRS-I/(R0)-patients and 89 out of the 101 (88%) IRS-II/(R1)  were alive. Median follow-up for survivors was 7.4 years (0.7–31.1). Twenty patients died of disease, two of treatment toxicity .3-, 5-, and 10-year EFS was 87.9% ± 4.7, 82.9% ± 5.7 and 75.2% ± 7.4. 3-, 5- and 10-year OS was 95.0% ± 3.1, 92.5% ± 3.92 and 84.6% ± 6.7, respectively.39 patients developed recurrences, 16 local, 14 metastatic, 4 at local and distant sites combined. For 5 patients, type of relapse was not specified Table . 3-, 5-,The proportion of metastatic recurrences increased with larger tumor size. The rate of local relapses did not differ with size Table .Table 3TIn the univariate analysis, factors associated with adverse EFS were large tumor size and no application of chemotherapy Table , Fig. 1.In the Cox regression analysis, large tumor size and no application of chemotherapy were associated with adverse EFS. Large tumors and tumors localized at head–neck were associated with adverse OS Table .Table 4 In the detailed evaluation of local and metastatic events, size and application of chemotherapy correlated with LRFS. Size, invasiveness (T-status) and chemotherapy correlated with MFS. In the Cox regression analysis, no independent factors for LRFS were identified. Size was associated with independent risk for MFS.p < 0.001).When we analyzed patients treated with cyclophosphamide-based regimens (VACA) versus those treated with ifosfamide-based regimen (VAIA) versus those without chemotherapy, patients treated with the VAIA or the VACA scheme did not show different outcomes , whereas 6 patients without chemotherapy did significantly worse  and 0.7 years (size missing) after first diagnosis. One suffered local relapse at 1.8 years (primary size 5–10 cm); he is reported alive in second complete remission 10.2 years after first diagnosis. Two suffered combined relapses at 0.3 years (primary tumor size 5–10 cm) and 0.5 years (size missing); both are dead of disease 0.5 and 2.8 years after diagnosis. One patient presented with metastatic relapse 1.5 years (primary tumor size 3–5 cm) after diagnosis and was reported alive at 3.8 years.Univariate analysis showed no significant correlation between radiotherapy and survival rates for the entire cohort.The analysis of merely IRS-I/(R0)-patients revealed that 5-year EFS was 81.8 ± 11.4% for patients receiving radiotherapy and 72.2 ± 15.5% for those who did not (supplementary table 1). The application of radiotherapy was also not associated with local or metastatic relapse risk.n = 89) and 77.8 ± 13.9% for non-irradiated patients (n = 9), while 5-year OS was 96.3 ± 2.1% and 77.8 ± 13.9%, respectively. There was no significant association with local or metastatic recurrences (supplementary table 2).For IRS-II/(R1)-patients, EFS was 86.7 ± 3.7% for irradiated (In the subgroup of 23 IRS-II/R1)-patients with secondary R0-resection while on chemotherapy, 6 did not receive radiotherapy. One of those 6 suffered combined relapse, whereas the 5 remaining were reported in ongoing first remission for 4.9 years (2.2–10.4). 17 did receive additional radiotherapy to the secondary R0-resection, and one suffered metastatic and one local relapse , 3-year-EFS was 94.6% ± 5.9. 5-year-EFS was 90.5% ± 8.0, 10-year-EFS 79.1% ± 12.9, and the 5-year-OS 98.2% ± 3.5. 52/56 received chemotherapy . 2 did not receive chemotherapy, while in 2 information was missing. With median follow-up of 7.5 years (0.6–31.1), 9/56 suffered relapse, thereof 5 local, 3 metastatic, 1 unspecified. 4/9 events occurred after 5.2 years.In the subgroup of 63 patients with tumors < 5 cm and resection with positive margins IRS-II/(R1), 3-year-EFS was 93.3 ± 6.3, 5-year-EFS 91.3 ± 7.3, 10-year-EFS 86.1 ± 9.8, and the 5-year-OS was 100%. This does not differ to the results of the IRS-I/(R0)-patients. All 63 patients received chemotherapy  with 1 missing information. 8 suffered relapse with a median follow-up of 6.8 years (1.2–16.6), thereof 5 local, 2 metastatic, 1 unspecified. Median time to relapse was 3.2 years (1.3–16.0).Secondary malignancy was documented in 4 patients: . All patients achieved a remission of their secondary tumor.Information on late effects was available for 142 patients. Thereof 72 patients (51%) did not suffer any late effect. Among those 70 other patients, mainly late sequelae in the extremities was generally documented by the participating centers. Among the 70 patients in the whole, 12 were documented with renal dysfunction, 8 with neuropathy, and 2 with cardiomyopathy. 57 of those 70 patients were irradiated. In 10 irradiated patients, specifically loss of function of their extremity was documented and in 3 other irradiated patients leg length differences or growth problem of the limb.Whereas pediatric and adult studies mostly agree on factors influencing SS outcome , for which the omission of adjuvant chemotherapy might be recommended , 3-year EFS was 94.6% ± 5.9. This does not differ from the results without chemotherapy. In our series, 9/56 suffered relapse, thereof 5 local, 3 metastatic, 1 unspecified with median follow-up of 7.5 years. 4/9 events occurred after 5.2 years. Median follow-up for the Ferrari series is merely 5.2 years. Long-term results are therefore highly anticipated. International cooperation with biological accompanying investigation would be needed to finally resolve this question.In our subgroup of 63 patients with tumors < 5 cm and resection with positive margins (IRS-II/R1), 3-year-EFS was 93.3% ± 6.3. This does not differ from the IRS-I/(R0)-group. There is neither a difference between IRS-I/(R0) and IRS-II/(R1)-group, nor between best surgeries obtained in a second intervention during chemotherapy. However, patients with positive margins mostly received irradiation. Nevertheless, it should be underlined that the risk of metastatic relapse does not differ. Obviously, distant spread occurs before resection. Therefore, omission of chemotherapy might be an approach worth discussing in grossly resected SS < 5 cm regardless of resection margins.There are very few evaluations that deal with the question of which systemic anti-cancer drugs specific to SS improve outcome best , where it was only recommended in CWS-86 and CWS-91. There is no clear evidence of the role of radiotherapy in IRS-I/(R0). A favorable trend with no statistically significant difference was shown , the indication for radiotherapy is a matter of debate. Generally, it is considered as indicated. In the common analysis , ultrasound abdomen/pelvis (at least every 6 months), and a bone scan  were recommended in the first year. Chest X-ray was recommended at 6-month intervals in the second year, followed by annual checks until 5 years after completion of therapy. From the 6th year onwards, controls by sonography/cross-section imaging of the primary tumor region and lung imagings were recommended with frequency at the discretion of the responsible physician. Interestingly, despite the known heterogeneity of soft-tissue sarcoma and in particularly of the group of NRSTS, there are no detailed subtype-specific follow-up recommendations. In this series of grossly resected SS, median time to local failure was 2.5 years. Median time to distant failure without involvement of the primary region was 2.7 years. This result might suggest regular imaging follow-up examinations at closer intervals  until 3 years after the end of therapy, and then an extension of the intervals. The latest local recurrences were documented at 11.5 and 16.0 years and the latest occurrence of metastases at 6.2 and 8.6 years, respectively. Annual control examinations until ~ 10 years after the end of therapy might seem reasonable. The possibility of a very late relapse should be known by the treating physicians. Most relapses are, however, detected due to clinical signs and symptoms and the patients should be educated to contact their oncologist immediately in case of unclear symptoms. It seems reasonable to incorporate the identified risk factors in the follow-up recommendations, e.g., patients with tumors < 5 cm have a significantly lower risk of suffering metastatic recurrence Supplementary file2 (DOCX 33 kb)Below is the link to the electronic supplementary material.