Purpose: Sex differences in conduct disorder (CD) pathophysiology have yet to be resolved. In this study, we applied the amplitude of low-frequency fluctuations (ALFF) and fractional ALFF (fALFF) to compare spontaneous brain activity in male versus female adolescents diagnosed with CD in light of the gender paradox hypothesis.Materials and Methods: Resting-state functional magnetic resonance imaging (rs-fMRI) examinations were conducted with 51 CD patients  and 53 age-matched healthy controls . Pearson analysis was conducted to detect relationship between ALFF/fALFF values in gender-differentiated regions and clinical characteristics.Results: We observed that male CD patients showed significant increased ALFF in the bilateral superior temporal gyrus (STG)/insula, and significant decreased ALFF in the left anterior cingulate cortex (ACC), left middle frontal gyrus (BA8 andBA11), left middle temporal gyrus and left inferior/middle temporal gyrus relative to female CD patients. The fALFF in male CD patients was significantly increased in the right STG/insula, decreased in the right superior frontal gyrus, left middle frontal gyrus, right inferior frontal gyrus, and right postcentral gyrus relative to female CD patients. Considering the sex-by-diagnosis interactions in CD patients, the male CD patients had significantly higher fALFF in the left putamen, lower fALFF in the right postcentral gyrus relative to the female CD patients.Conclusion: The brain regions whose activity index values differed in relation to sex should be further explored in CD pathophysiology studies, particularly with respect to sex differences in clinical symptoms, emotional features, cognitive features, and prevalence rates in CD. The present findings are consistent with the gender paradox hypothesis. Conduct disorder (CD) is a common mental disorder of childhood and adolescence characterized by behaviors that are dismissive of others’ basic rights and societal norms . CD may Clarifying sex differences in brain activities is important to gaining a full understanding of antisocial and aggressive behaviors and deciphering the neural mechanisms related to the atypical emotional processing in CD in particular. The majority of fMRI studies have found that the CD adolescents  exhibited abnormal activities in insula , anterioResting-state functional magnetic resonance imaging (rs-fMRI) is a reliable neuroimaging technique in which subjects keep their eyes either closed or fixated on a crosshair during scanning in a task-free paradigm. In rs-fMRI, low frequency (0.01–0.08 Hz) fluctuations in blood oxygen level-dependent (BOLD) signals during rest are highly associated with spontaneous neuronal activity . The ampTo our knowledge, no previously published rs-fMRI studies of CD have included female subjects , let aloSD = 0.76) were recruited from out-patient clinics affiliated with the Second Xiangya Hospital of Central South University . A group of 53 gender- and age-matched HCs  were recruited from middle schools in Changsha. All participants were aged between 12 and 17 years old.Adolescents with CD  . DetaileFor all participants, the exclusion criteria were a history of attention-deficit/hyperactivity disorder, any psychiatric or emotional disorder , any pervasive developmental disorder , any chronic neurological disorder , persistent headaches, head trauma, alcohol/substance abuse in the past year, contraindications to magnetic resonance imaging, or an intelligence quotient (IQ) ≤ 80 on the Wechsler Intelligence Scale for Children-Chinese revision (C-WISC) examination . All CD The study was approved by each school’s administration and the Ethics Committee of the Second Xiangya Hospital of Central South University. All subjects and their parents were informed of the study’s goal and the participant’s parents provided written informed consent.All participants completed the Barratt Impulsivity Scale-version 11 (BIS-11), including three subscales attention impulsiveness (attention and cognitive instability dimensions), non-planning impulsiveness (self-control and cognitive complexity dimensions), motor impulsiveness (motor and perseverance dimensions) , and theAll participants underwent magnetic resonance imaging in a Philips, Achieva, 3.0-T scanner. The functional images were acquired with an echo-planar imaging (EPI) sequence . Noise and head movement were reduced by fitting ear plugs and padding around the head.1) (Table 1). Meanwhile, we excluded the subjects whose head motion exceeded “mean FD + 2SD” (Data preprocessing was conducted with Data Processing Assistant for Resting-State fMRI (DPARSF1) . The fir1) . Spatial1) , there wD + 2SD” . FinallyAmplitude of low-frequency fluctuations and fALFF analysis were conducted in DPARSF. After the above preprocessing, the fMRI data were temporally band-pass filtered (0.01–0.08 Hz) to remove physiological high-frequency noise and low-frequency drift. The time series of each voxel was converted into the frequency domain, and its power spectrum was calculated. The square root was calculated at each frequency of the power spectrum, and the average square root was then obtained at each voxel across the frequency range of 0.01–0.08 Hz . The ave2 was used to investigate main effects of diagnosis, sex, and sex-by-diagnosis interactions based on ALFF and fALFF values with sex and diagnosis as factors and IQ and age as covariates. We defined the regions that showed significant differences in sex effects and sex-by-diagnosis interactions as potential regions of interest, and the post hoc t-tests were used to detect the significantly different regions between female and male CD patients. All significance thresholds were set to p < 0.05 with false discovery rate correction and a 10-voxel extension threshold.Demographic and clinical characteristics were compared between groups with two-way analyses of variance (ANOVAs), with sex and diagnosis as between-group factors, in Statistical Package for Social Sciences for Windows 16.0  . Full-faPearson correlation was conducted in SPSS 16.0 to explore associations between ALFF and fALFF values in gender-differentiated regions and clinical characteristics in CD patients subgroups separately. For multiple comparison, a Bonferroni correction was conducted.Table 1. The four groups were well-matched in terms of age and FD. There were no significant differences in demographic or clinical characteristics between females and males. Relative to HCs, subjects with CD had a significantly lower mean IQ scores and had higher mean impulsivity  and CU trait  scores. Female CD patients had higher impulsivity and CU trait scores than the other three groups.The demographic and clinical characteristics of the four groups are summarized in Figure 1A). Additionally, we observed a main effect of diagnosis (CD vs. HC) on ALFF in the left insula, right precuneus, left lingual gyrus, and left middle frontal gyrus. There was a sex-by-diagnosis interaction of ALFF in the right superior parietal lobule/precuneus . Regarding the main effects of sex, relative to the female CD group, the male CD group had higher ALFF in the bilateral STG/insula and lower ALFF in the left ACC, left middle frontal gyrus (BA8 and BA11), left middle temporal gyrus, and left inferior/middle temporal gyrus . Regarding the sex-by-diagnosis interaction, there was no significantly different regions in male CD and female CD patients.We observed a main effect of sex  on ALFF in the left middle frontal gyrus, left superior frontal gyrus, right inferior frontal gyrus, right STG, left middle temporal gyrus, left middle occipital gyrus, left parahippocampal gyrus, and left cerebellum posterior lobe . Additionally, we observed a main effect of diagnosis (CD vs. HC) on fALFF in the left insula, left precentral gyrus, right cerebellum posterior lobe, left inferior parietal lobule, and left precuneus. There was a sex-by-diagnosis interaction of fALFF in the left thalamus, left putamen, and right postcentral gyrus . Regarding the main effects of sex, relative to the female CD group, the male CD group had higher fALFF in the right STG/insula and lower fALFF in the right superior frontal gyrus, left middle frontal gyrus, right inferior frontal gyrus, right postcentral gyrus . Regarding the sex-by-diagnosis interaction, relative to the female CD group, the male CD group had higher fALFF in the left putamen, lower fALFF in the right postcentral gyrus .We observed a main effect of sex  on fALFF in the bilateral middle frontal gyrus, bilateral inferior frontal gyrus, right middle temporal gyrus, right temporal lobe, right precentral gyrus , left middle temporal gyrus, left inferior/middle temporal gyrus, and right postcentral gyrus. Regarding the sex-by-diagnosis interactions, the fALFF in male CD patients was significant increased in the left putamen and decreased in the right postcentral gyrus relative to female CD patients.Our finding of lower ALFF and fALFF in the bilateral STG of female, relative to male, CD patients should be considered in the context of prior STG findings in CD patients. Structural deficits in the STG have been reported in both males  and femaRelative to the male CD patients, the female CD patients in our study had increased ALFF in the left ACC. Functionally, the ACC has been associated with the processing social information  and rewaOur findings of higher impulsivity and CU trait scores in females with CD (the lower incidence gender) than in males with CD are consistent with the gender paradox hypothesis. Current findings implied that gender differences in ACC among CD patients may be associated with higher impulsivity and CU trait levels in female CD patients than in male CD patients. Our findings underscore the importance of exploring this region’s structure and function in relation to gender in CD patient populations.Female CD patients in our study also showed increased ALFF and fALFF in the right superior frontal gyrus, right inferior frontal gyrus, left middle frontal gyrus, and left inferior/middle temporal gyrus, relative to those observed for male CD patients. Previous rs-fMRI studies have reported decreased activity in the MFG and middle temporal gyrus in CD patients relative to activity seen in HCs . The patConsidering the sex-by-diagnosis interactions in CD patients, the fALFF in female CD patients was significant increased in the right postcentral gyrus and decreased in the left putamen relative to male CD patients, but the opposite pattern of sexual differentiation in the two regions existed in typical population. The postcentral gyrus is the primary somatosensory cortex, located in lateral parietal lobe . The putThe current study had several limitations. First, the small sample size of females with CD may limit the generalizability of the current results. Larger CD samples are needed to confirm the presently reported sex differences. Second, because both of our CD groups, male and female, had lower IQ scores than the two HC groups, IQ scores were not matched between CD patients and HCs in the main analysis. To reduce the effect of IQ on our results, we included IQ as a covariate in the analyses. Third, the study did not discuss early-onset CD samples, so whether the observed sex differences in spontaneous brain activity were limited in adolescent-onset CD needs to be explored. Finally, in the future, multi-parametric neuroimaging studies with larger, more diverse samples should be used to explore the pathophysiology of sex differences in CD.The present rs-fMRI findings provide insights into gender differences in the pathophysiology of CD. Using ALFF and fALFF as indexes of spontaneous neuronal activity, we observed resting-state activity differences between male and female CD patients in frontal lobe, temporal lobe, left ACC, right postcentral gyrus, and left putamen. These regions have been associated with clinical symptoms, emotion, and cognition. Elucidating these regions’ gender-associated differences in CD may be useful for resolving the reasons underlying gender differences in CD diagnosis prevalence rates. The present findings are consistent with the gender paradox hypothesis.SY and BH conceived and designed the study. WC, XS and DD collected and analyzed the data. XS and DD helped to draft the manuscript. WC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To compare perioperative and pathological results in different approaches of robotic or laparoscopic radical prostatectomy. We retrospectively reviewed 206 patients diagnosed with prostate cancer (PC) from June 2016 to October 2017 in the First Affiliated Hospital of Nanjing Medical University. A total of 132 cases underwent robot-assisted laparoscopic radical prostatectomy (RLRP) including 54 patients on transperitoneal robot-assisted laparoscopic radical prostatectomy (Tp-RLRP) and 78 on extraperitoneal robot-assisted laparoscopic radical prostatectomy (Ep-RLRP). Meanwhile, 74 patients performed with extraperitoneal laparoscopic radical prostatectomy (Ep-LPR) were also included. Perioperative and pathological data were compared among these groups.In Tp-RLRP vs. Ep-RLRP: Significant differences were found in the comparison in total operation time , estimated blood loss (EBL) , postoperative pelvic drainage time  and postoperative length of stay  while no significant differences were detected in other variables. In Ep-RLRP vs. Ep-LPR: Longer total operation time was observed in Ep-RLRP when compared to Ep-LPR . No significant differences were observed in other variables. All operations were completed without conversion. There was no significant difference in basic and pathological characteristics of patients between each two groups.  In RLRP, Ep-RLRP was proved a safe and effective approach based on the perioperative results compared to Tp-RLRP. Ep-RLRP and Ep-LPR provides equivalent perioperative and pathological outcomes. Prostate cancer (PC) is the second most common malignant tumor in men and an important cause of cancer-related morbidity and mortality worldwide. In 2017, the estimated new PC cases and deaths were 161.360 and 26.730 in the United States, respectively  had been a good alternative for the treatment of PC. However, high incidence of iatrogenic diseases caused by open surgery have led people to look for minimally invasive ways to improve perioperative and postoperative conditions.Laparoscopic radical prostatectomy (LRP) technique was first systematically reported in 1997 by Schuessler  and releAlthough there are some previous comparisons between LRP and RLRP -10, the From June 2016 to October 2017, patients diagnosed with organ-confined PC who underwent LPR or RLRP in our institution were included in our study. Patients were excluded from this research if they had had any other malignant tumors and serious diseases. All patients were newly diagnosed and had not received other treatments for PC before, such as brachytherapy, external radiotherapy, chemotherapy, etc. A total of 206 patients were selected into 3 groups . Detailed basic characteristics of patients in each group are summarized in Ep-LRP: Patients were placed in the supine position; five ports were used in the operation. The position for each trocar and general surgical procedures were described previously .Tp-RLRP: Patients were placed in the half lithotomy position with their legs outreached at 30° higher than head level. The position for each trocar and general surgical procedures were described previously .Ep-RLRP group: Unlike the transperitoneal approach, five ports were used lower in the pelvis. The position for each trocar and general surgical procedures were described previously .Laparoscopic technique has been carried out for nearly ten years and robot-assisted operation was developed successfully in our institution based on the mature laparoscopic technique. There was no specific indication for one technique to another, and main influence factors for choosing surgery techniques were patient’s will and figure. Whether transperitoneal or extraperitoneal surgical approach (with or without robotic assistance) were all common surgery styles without learning curve effect.Patients diagnosed with PC by biopsy underwent surgery at 6-8 weeks after biopsy in order to reduce the difficulty of surgery and postoperative complications. Additionally, patients who underwent transurethral resection of prostate (TURP) should wait 12 weeks for further surgery. All operations were performed by the same surgeon who has worked for 30 years and has been involved in PC surgery. Since 2009, he has completed more than 1.000 cases of LRP (more than 100 cases of RLRP). Additionally, postoperative management for each patient was the same, regardless in LRP or RLRP. In all surgeries, pelvic lymph node dissection (PLND) was performed in patients with a serum prostate specific antigen (PSA) greater than 10 ng / mL, or biopsy Gleason score (GS) more than 7. In general, the range of PLND includes the nodes covered on external iliac arteries and veins, the nodes within the obturator and the nodes overlying internal and external side of internal iliac arteries . MoreoveAll data analyzed in this study were based on the documentations of our PC database including age, body mass index (BMI), preoperative PSA, biopsy GS, prior history of abdominal surgery, estimated blood loss (EBL), total operation time, postoperative pelvic drainage time, the indwelling catheter time, postoperative length of stay, extra-prostatic extension (EPE), lymph node invasion (LNI) and cases of seminal vesicle and vas deferens involved. The pathological results including postoperative GS and positive surgical margin (PSM) were also documented.Stata software  was used for the statistical analysis. Pearson’s chi-square test was used for the comparison of nominal data while the numeric parameters were compared utilizing Student’s t-test. For all analyses, two-sided P value < 0.05 was considered as statistical significant.All operations were completed successfully without conversion. No significant differences in preoperative data were detected between every two groups except the comparison in age between Tp-RLRP and Ep-RLRP which may due to the relatively small sample size .Detailed information of comparison between each two groups is shown in Longer total operation time  was found in Ep-RLRP when compared to Ep-LRP. Additionally, no statistical difference was found in other variables.Significant differences were detected in the comparison of EBL , total operation time , postoperative pelvic drainage time  and postoperative length of stay  between Tp-RLRP and Ep-RLRP while difference in the comparisons of other variables showed no statistical significance.PC is a male malignant tumor with high incidence . DefinitThere are some typical features favored in RLRP such as 3D viewing, improved ergonomics, elimination of hand tremor and refined dexterity , 18, whiTraditionally, radical prostatectomy can be performed via transperitoneal and extraperitoneal approach. Either a transperitoneal or an extraperitoneal approach have been proved to be safe and eﬀective, and each approach has its advantages and short comes. The main advantages for transperitoneal approach are summarized as following:  easier fIn our study, operation time, postoperative pelvic drainage time, postoperative length of stay and less EBL was favored in Ep-RLRP when compared to Tp-RLRP. Comparison between Ep-LRP and Ep-RLRP showed no statistical difference except the longer total operation time in Ep-RLRP.The operation time, defined as a period of time from the incision of the skin to the end of the skin suture, was different in various surgical approaches. Significant difference (P < 0.0001) between Ep-RLRP and Ep-LRP could have been caused by the extra time for disposition of robot arms. Longer operation time in Tp-RLRP when compared to Ep-RLRP may have occurred due to faster placement of trocars (P < 0.0001).In terms of EBL, patients with Tp-RLRP had more blood loss than those of Ep-RLRP . However, no significant difference was observed in the comparison between Ep-RLRP and Ep-LRP. Therefore, a preliminary conclusion can be drawn that more EBL is tightly associated with the transperitoneal route, similar results can be found in some previous studies which compare EBL between Ep-LRP and Tp-LRP . One posIn the comparison between Tp-RLRP and Ep-RLRP, we can conclude that the postoperative length of stay and pelvic drainage duration time was significant longer in Tp-RLRP. This might be explained by the disadvantages of Tp-RLRP mentioned above. However, the difference between Ep-LRP and Ep-RLRP showed no statistical significance which indicated that the robot-assisted technique did not have especially obvious effect on postoperative recovery.Generally, postoperative pathological results were tightly to PSM and postoperative GS. PSM is an independent predictor of tumor progression which can probably be prevented by appropriate patient selection and meticulous surgical technique . In our In This was a single-instruction, retrospective study, and no strict selection criteria were applied when choosing the surgery technique . The surgeon has already been an experienced operator, and we thought the bias of experience accumulation can be minimized. The limitation for this study could be overcome by expanding the number of cases in each group with a longer follow-up period in future studies.In RLRP, Ep-RLRP was proved to be a safe and effective approach because of the shorter operation time, postoperative pelvic drainage time, postoperative length of stay and less EBL when compared to Tp-RLRP. Ep-RLRP and Ep-LPR provides equivalent perioperative and pathological outcomes.


Supplemental Digital Content is available in the text Recent findings from an animal experiment suggest a modest association between silymarin and decreased risk of contrast-induced nephropathy. However, the relationship between silymarin and contrast-induced nephropathy in patients with liver cirrhosis remains unclear.From 1997 to 2007, we identified 3019 patients with liver cirrhosis who were administered silymarin and matched them with 3019 patients with liver cirrhosis who were not administered silymarin. Each patient was followed up for a minimum of 4 years. After adjusting for age, gender, hepatitis B, hepatitis C, alcoholic hepatitis, and Charlson comorbidity index, we considered death occurrence and used the Fine and Gray regression models to calculate subdistribution hazard ratios (sHRs) for contrast-induced nephropathy. Sensitivity analyses were also performed using the same model on the subgroups classified by comorbidity.P = .791). On the basis of sensitivity analyses results classified by comorbidity, a nonsignificant decrease in risk of contrast-induced nephropathy was found.Using the Fine and Gray regression models and with death as the competing risk, we observed that sHR for contrast-induced nephropathy was 0.94-fold higher in the silymarin cohort than in the nonsilymarin cohort (95% confidence interval = 0.61–1.47, Silymarin shows no nephron-protective positive effects on contrast-induced nephropathy. Silymarin did not play a nephron-protective role according to Longitudinal Health Insurance Database of Taiwan. Clinical trials are necessary to further assess the nephron-protective effects of silymarin of contrast-induced nephropathy. CM-adverse reactions (ARs) and those originating from mild symptoms can potentially be life-threatening. Although low-osmolarity nonionic CMs have been introduced since the mid-1970s to reduce CM-ARs, CM-ARs have still been reported.–5 CM-induced nephrotoxicity (CMIN) is one of the major causes of acute kidney injury (AKI) among hospitalized patients. CM-ARs cannot always be predicted, but various studies indicated that CMIN pathophysiology is closely related to renal hemodynamic changes and medullary ischemic injury, reactive oxygen species (ROS)-induced oxidative stress damage, indirect damage to the tubules, and tubular obstruction.,7 Among the possible pathogenesis mechanisms of CMIN, ROS-induced oxidative stress damage is important.,8 It is currently an important target for drug intervention to prevent CMIN. To decrease and prevent CM-ARs, several guidelines have been developed to prevent AEs, but these guidelines are only partially successful.,10 Limited evidence prove the effectiveness of premedication before CM administration.Contrast medium (CM) is one of the most common pharmacological agents injected in hospitalized patients.–14 Two major mechanisms have been proposed to account for the organ-protective effects of this compound. The first mechanism is its dose-dependent antioxidant effect. The second mechanism involves its anti-inflammatory and antiapoptotic properties. Silymarin may act as a nephron-protective agent against CMIN. To date, the protective effects of silymarin on CMIN have been primarily investigated in animals, and nephroprotection was observed. However, large-scale clinical observations are needed to prove the nephroprotection effects of silymarin.Silymarin is a hepatoprotective drug. N-acetylcysteine has been recognized as a CMIN prevention drug because of its strong antioxidant effects that can prevent CMIN.,11 However, N-acetyl cysteine may slow down the blood clotting, and patients receiving CT examination require a large-sized needle for CM injection. Silymarin possesses both antioxidative and anti-inflammatory effects and is commonly used to manage hepatitis. However, few evidence prove the nephroprotective effect on CMIN. The current study aimed to determine and evaluate the nephroprotective effect of silymarin on CMIN cohorts from the longitudinal National Health Insurance Research Database.Among possible pathogenesis mechanisms of CMIN, ROS-induced oxidative stress damage is one of the most important.22.1Silymarin and nonsilymarin cohorts were obtained from the Longitudinal Health Insurance Databases (LHIDs), including LHID2000, LHID2005, and LHID2010. LHID2000, LHID2005, and LHID2010 included all the original claim data randomized from the beneficiary registry in 2000, 2005, and 2010, respectively, and the registration file of 1 million individuals (N = 23.72 million) for the Taiwan National Health Insurance (NHI) program. According to the National Institutes of Health in Taiwan, no significant difference was found in the gender distribution of enrolled students and the list of enrolled students under the National Health Plan for enrollment opportunities for undergraduates throughout the country. The LHID enables researchers to access all medical services provided to individuals registered in the database from the beginning of the 1995 NHI. Such data can be used to explore the link between silymarin and contrast-induced nephropathy. The study was expelled from the Tainan Municipal Hospital Authority Review Board because it used LHID2000, LHID2005, and LHID2010, which included secondary data released to the public for research purposes. This study was approved by the Tainan Municipal Hospital.Patients with liver cirrhosis  who were identified between 1997 and 2007 were selected from the database. For inclusion, at least one of the following criteria should be met: diagnosis of cirrhosis of one or more hospitalized patients; and diagnosis of liver cirrhosis at 3 or more outpatient visits within 6 months. Index day for the patients with liver cirrhosis was assigned as 1 year after the newly liver cirrhosis diagnosis. Prescribed use of silymarin medications in the follow-up period was also considered. Prescription records contained dates of order, dosage, route of every prescription, and number of days. Two cohorts were categorized from the patients with liver cirrhosis. The first cohort included patients who regularly use silymarin medication (silymarin cohort). The other cohort included patients who did not use any silymarin medication (nonsilymarin cohort) during the follow-up period. The nonsilymarin cohort was matched (1:1) with the silymarin cohort according to age, gender, Charlson comorbidity, and index day. Patients with diagnosis of contrast-induced nephropathy prior to the index day were excluded from the study. Comorbidities were classified as those existing prior to the index day and included Charlson comorbidity, hepatitis B, and hepatitis C. The study also categorized liver cirrhosis into alcoholic and nonalcoholic types. The end of the follow-up period for the analyses was marked on the day of contrast-induced nephropathy diagnosis and terminated on 2012 or upon death. Follow-up data were available for a minimum of 4 years for all selected subjects.2.2In this study, the definition of contrast-induced nephropathy is combine receiving CT examination (computerized tomography code) and exposure to contrast (contrast code) and within 1 week duration between the date of new nephropathy diagnosis (nephropathy code) and contrast exposure. The source code is listed at Appendix.2.3t test for continuous variables and chi-squared test for categorical variables to analyze the differences between silymarin and nonsilymarin cohorts. The baseline characteristics from the database included age, gender, Charlson comorbidity, hepatitis B, hepatitis C, and alcoholic liver cirrhosis. The number of contrast-induced nephropathy cases in the 2 cohorts during follow up was counted. The subdistribution hazard ratio (sHR) was calculated using the Fine and Gray competing risk regression models, whereas a regression hazard model was used to compare the silymarin and nonsilymarin cohorts to assess the risk of contrast-induced nephropathy. Kaplan–Meier method was used to determine the cumulative incidence of CMIN in both cohorts, and differences between cohorts were tested using the Gray test. To examine whether the main findings had different assumptions, sensitivity analyses were performed. Sensitivity analyses were also performed using the Fine and Gray regression hazard models on subgroups classified by comorbidity. All data management and sHR calculations were conducted using Statistical Analysis System (SAS) software for Windows .The study used the 3P = .791) after adjusting for age, gender, hepatitis B, hepatitis C, alcoholic liver cirrhosis, and CCI in the stratified Fine and Gray models , and index days after excluding unqualified subjects (Fig. 4 However, CMIN remains a major problem for health care. Silymarin shows no nephroprotective role according to this population-based, nested case–control study.This work is the first nationwide, population-based follow-up study that determined whether silymarin exerts significant nephron-protective effects on patients with CMIN. Hospital-acquired AKIs, including CMIN, are important causes of mortality and morbidity. Several CMIN treatment options have been proposed.,16,17 CM-AR rates range between 0.7% and 0.82%.,3,18,19 However, studies rarely reported nephrotoxic CM-ARs, and the possible reasons for result include aggressive premedication and hydration before CT examinations among high-risk patients. The total incidence of CMIN is low. In the current study, such low value may indicate the negative nephroprotection effect of silymarin on CMIN.The incidence of CMIN ranges between 2% and 7%.,20,21Silymarin is a useful hepatoprotective medication because of its antioxidant and anti-inflammatory properties. Dashti-Khavidaki et al reported the nephroprotective effects of silymarin against some nephrotoxins. Khan et al reported that silymarin treatment can increase kidney weight from renal damage status. Kaur et al reported the potent nephroprotective effect of silymarin in an animal mode.Theoretically, silymarin may positively affect patients with CMIN. Silymarin decreased renal damage and restored ROS activities in an animal model. The study of Homse et al revealed that silymarin can result in persistent oxidative stress and inflammatory processes, tubular necrosis, and apoptosis.However, silymarin can exacerbate renal damage in an animal model according to the study of Homse et al.In our study, silymarin did not play a nephroprotective role. This finding might have been affected by the following: inadequate patient numbers, inadequate dosage and duration, and inaccurate prescription timing. Further studies are needed in the future to evaluate the nephroprotective effects of silymarin against CMIN.4.1The current study used the Taiwan NHI database, which includes data from a longitudinal cohort and is a large and population-based database. The nationwide LHID 2000 is an excellent resource for evaluating patients with CMIN. Our study is relevant because it evaluated the nephroprotective effects of silymarin against CMIN.Some limitations were considered. First, several CMIN patients were not reported in LHID, and we assumed that the dataset from the NHI program are relatively accurate. Second, laboratory information about some potential bias, including coding bias, was lacking. Third, no laboratory data are available in the NHI Research Database. Therefore, we cannot determine the severity of CMIN in our current patients.In conclusion, silymarin did not exert nephroprotective positive effects on CMIN. Although CMIN remains a burden among hospitalized patients, silymarin cannot be recommended as a nephron-protective drug. After reviewing the major studies focusing on the role of silymarin in nephroprotection, silymarin administration to animals can reduce or prevent CMIN. However, silymarin did not exhibit any nephroprotective role according to the LHID of Taiwan. Further clinical trials are necessary to assess the nephron-protective effects of silymarin on CMIN.Conceptualization: Yu-Jui Kuo, Hui-Ping Chang, Yu-Jun Chang, Hsing-Hsien Wu, Chang-Hua Chen.Data curation: Hui-Ping Chang, Chang-Hua Chen.Formal analysis: Yu-Jun Chang.Funding acquisition: Chang-Hua Chen.Methodology: Yu-Jun Chang.Project administration: Chang-Hua Chen.Supervision: Hsing-Hsien Wu, Chang-Hua Chen.Validation: Yu-Jun Chang, Hsing-Hsien Wu, Chang-Hua Chen.Writing – original draft: Yu-Jui Kuo, Chang-Hua Chen.Writing – review & editing: Yu-Jui Kuo, Hui-Ping Chang, Yu-Jun Chang, Hsing-Hsien Wu, Chang-Hua Chen.


Choices regarding mode of travel have an evident effect on environment pollutants and public health. This paper makes a significant contribution by examining the differences between low-carbon and non-low-carbon travel mode choices during shopping trips, and how socio-economic characteristics impact individual travel behavior based on data gathered from a questionnaire conducted in Shenyang, China. The study found that, firstly, low-carbon travel modes were more common than non-low-carbon travel modes for shopping, and the average travel distance by non-low-carbon modes was a little longer than that of low-carbon modes. Secondly, suburban and wholesale specialized commercial centers attracted more residents travelling longer distances by non-low carbon modes, especially private car, compared to regional commercial centers in inner city areas. Thirdly, strong relationships between car ownership, gender, monthly income, and travel mode choices were identified in a binary logistic regression model. This study thus highlights the importance of sustainable transportation policies to advocate low-carbon travel modes and reduce carbon emissions. Mitigat by 2030 ,7. In de by 2030 . Accordi by 2030 .The travel behavior of urban residents is of increasing interest to researchers and planners. Choices of travel mode for daily mobility have significant effects on the future development of urban regions due to urban sprawl and structural changes to growing metropolitan areas related to transportation development ,12,13,14Previous studies that have analyzed low-carbon development in the transport sector mainly focused on technological improvement strategies and policies to reduce carbon emissions ,8,26,27.Shenyang city, located in the middle of Liaoning Province, is the provincial capital and the largest city in northeast China . It contThe data used in this study were collected through questionnaire surveys and interviews conducted with residents on shopping trips to eight typical commercial centers on weekends and weekdays. The survey consisted of three parts: one about travel behavior such as travel mode, travel time, frequency of trips and travel routes; another on personal socio-economic characteristics such as car ownership, gender, age, education, occupation, and monthly income; and a third on attitudes towards the development of public transport and their opinions on shopping by private car. A random sample of 1672 shoppers was invited to respond to the questionnaire, which was filled out by 1525 people, a 91.21% response rate.p for the respondents’ travel decisions of non-low-carbon transport mode and a value of 0 with probability 1-p for choosing low-carbon transport mode), using one or more continuous or categorical variables of socio-economic characteristics as predictors [β0 is the model constant, and rx is a continuous or categorical predictor variable. Parameter βr is the regression coefficient to be estimated.Logistic regressions have been widely used to identify the determinants of travel behavior in many contexts ,60,61,62edictors ,64. The edictors ,66.(1)lIt is necessary to sum up the socio-economic characteristics of respondents surveyed in the eight typical commercial centers . The resThe mode choice behavior of respondents for shopping trips to the eight typical commercial centers was shown in 2 = 270.220; df = 15; p = 0.000), although their explanatory power was low (Nagelkerke R2 = 0.256). Using a significance test, we found the socio-economic variables of car ownership and gender to be significant at a 99.9% confidence level, and monthly income significant at a 99% confidence level. This may explain the increase in the use of non-low-carbon transport modes for shopping.To test content validity, we developed a binary logistic regression model to explain how socio-economic factors affect transport mode choices during shopping trips. Socio-economic factors included gender, age, education level, occupation, and monthly income. The estimation results of the binary logistic regression model analysis for the choice between non-low-carbon and low-carbon transport mode are presented in As with findings from previous studies, this study confirms car availability has the largest impact on the choice of non-low carbon transport modes for residents during shopping trips. The vehicle ownership results indicate that residents having their own vehicles were 5.629 times more likely to select non-low carbon modes for shopping trips than those who do not own a car. Plaut , Choi anThis study was performed by obtaining data from questionnaire surveys on weekends and weekdays in Shenyang, China, to explore low-carbon and non-low-carbon travel mode choice behavior during shopping trips to eight typical commercial centers, examining how socio-economic characteristics impact travel patterns and carbon emissions. Our study shows that low-carbon travel modes are more frequent than non-low-carbon modes for shopping. Furthermore, travel mode choice is associated with the location and retail type of the commercial centers. Suburban and specialized wholesale market-oriented commercial centers attract many residents travelling longer distances by non-low-carbon modes of transportation, especially private cars, for shopping. The municipal commercial centers of Middle Street and Taiyuan Street in the city center witness relatively long travel distances by non-low-carbon modes. The regional commercial centers of Xita-Beishi, Beihang, and Tiexi, in the inner city, attract shoppers who almost always choose low-carbon modes, and for whom the travel distances are shorter. The results of binary logistic regression indicate that car ownership, gender, and monthly income are significantly associated with travel mode choices.The findings of the study have some implications for sustainable transport and low-carbon urban planning. First, they suggest that there should be a shift of emphasis from expanding transport networks to policies that encourage the use of low-carbon alternatives. According to previous research, the improvement of low-carbon transport infrastructure and the adoption of different strategies should depend on city size . Notably


Whole-body MRI (WB-MRI) could be an alternative to multimodality staging of colorectal cancer, but its diagnostic accuracy, effect on staging times, number of tests needed, cost, and effect on treatment decisions are unknown. We aimed to prospectively compare the diagnostic accuracy and efficiency of WB-MRI-based staging pathways with standard pathways in colorectal cancer.The Streamline C trial was a prospective, multicentre trial done in 16 hospitals in England. Eligible patients were 18 years or older, with newly diagnosed colorectal cancer. Exclusion criteria were severe systemic disease, pregnancy, contraindications to MRI, or polyp cancer. Patients underwent WB-MRI, the result of which was withheld until standard staging investigations were complete and the first treatment decision made. The multidisciplinary team recorded its treatment decision based on standard investigations, then on the WB-MRI staging pathway , and finally on all tests. The primary outcome was difference in per-patient sensitivity for metastases between standard and WB-MRI staging pathways against a consensus reference standard at 12 months, in the per-protocol population. Secondary outcomes were difference in per-patient specificity for metastatic disease detection between standard and WB-MRI staging pathways, differences in treatment decisions, staging efficiency , and per-organ sensitivity and specificity for metastases and per-patient agreement for local T and N stage. This trial is registered with the International Standard Randomised Controlled Trial registry, number ISRCTN43958015, and is complete.Between March 26, 2013, and Aug 19, 2016, 1020 patients were screened for eligibility. 370 patients were recruited, 299 of whom completed the trial; 68 (23%) had metastasis at baseline. Pathway sensitivity was 67% (95% CI 56 to 78) for WB-MRI and 63% (51 to 74) for standard pathways, a difference in sensitivity of 4% . No adverse events related to imaging were reported. Specificity did not differ between WB-MRI (95% [95% CI 92–97]) and standard pathways . Agreement with the multidisciplinary team's final treatment decision was 96% for WB-MRI and 95% for the standard pathway. Time to complete staging was shorter for WB-MRI  than for the standard pathway (13 days [11–15]); a 5-day (3–7) difference. WB-MRI required fewer tests  than did standard pathways (two [2 to 2]), a difference of one (1 to 1). Mean per-patient staging costs were £216 (95% CI 211–221) for WB-MRI and £285 (260–310) for standard pathways.WB-MRI staging pathways have similar accuracy to standard pathways and reduce the number of tests needed, staging time, and cost.UK National Institute for Health Research. Colorectal cancer is the second leading cause of cancer-related deaths in the UK, with about 16 000 deaths annually.Staging pathways are complex, relying on high technology imaging platforms such as CT, PET-CT, and MRI. In England, for example, the National Institute for Health and Care Excellence (NICE) publishes guidelines that require multiple, sequential imaging tests to complete staging and allow the first treatment decisions to be made.Evidence before this studyThe detection of metastatic disease during colon cancer staging underpins treatment strategy and is fundamental to the optimisation of patient outcomes. Staging pathways rely on high technology imaging platforms such as CT, PET-CT, and MRI, which differ in their diagnostic accuracies across individual organs. Such multimodality staging pathways are complex, resource and time intensive, involve irradiation, and increase patient anxiety. Modern MRI platforms can image the whole body within 1 h, and whole-body MRI (WB-MRI) is advocated as a more accurate, efficient, and safer alternative to multimodality staging pathways. We searched PubMed and Embase (without language restriction) for articles published between Jan 1, 1990, and Sept 30, 2018, using MeSH and full-text search-strings for “cancer”, “neoplasm” “staging”, “diagnostic accuracy”, “magnetic resonance imaging”, “whole body imaging”, “diffusion magnetic resonance imaging”, “metastasis”, “colorectal”, and “colon”. We found several meta-analyses reporting WB-MRI accuracy for cancer staging. Many combined different primary cancers in single analyses or were limited to detecting metastasis in single organs (or both). Most meta-analyses compared WB-MRI with PET-CT, and scintigraphy (in the case of bone metastasis), rather than CT alone, which is the test used most commonly in colorectal cancer staging. No meta-analysis considered colorectal cancer in isolation. Most primary studies were small, single site, and explanatory, with WB-MRI interpreted by a few specialised radiologists. They focused on single modality comparisons rather than evaluating real-world, multimodality staging pathways. We found no data regarding how WB-MRI influences the first major treatment decision or staging efficiency.Added value of this studyTo our knowledge, this is the largest prospective multicentre trial to date comparing the diagnostic accuracy of WB-MRI staging pathways to standard staging in patients newly diagnosed with colorectal cancer. We used a pragmatic trial design to better test pathway performance in routine clinical practice and investigated pathway efficiency in terms of test number, time to completion, and costs. We also contemporaneously tested the effect of alternative staging pathways on the nature and timing of the first major treatment decisions. Patient outcomes were followed up after 12 months to better evaluate pathway accuracy at the time of initial staging. We found both pathways had similar accuracies for identifying patients with metastatic disease and the nature of first major treatment decision was similar. Notably, WB-MRI was more efficient and reduced the number of tests needed, time to complete staging, and costs.Implications of all the available evidenceWB-MRI staging pathways have similar accuracy to current standard staging pathways, resulting in the same treatment decisions. However, they are more efficient and reduce test numbers, time to complete staging, and costs. WB-MRI is, therefore, more suitable for staging in routine clinical practice. Future research should investigate the use of WB-MRI treatment response assessment and cancer surveillance after curative treatments.vs PET-CT) instead of the multiple staging tests encountered in daily practice.Modern MRI scanners can image the entire body within 1 h, and whole-body MRI (WB-MRI)—which typically scans from the head to mid-thigh—is a potentially more accurate and safer alternative to standard multimodality staging pathways. WB-MRI could also accelerate staging, thereby increasing efficiency by reducing additional tests, staging time, and costs. Meta-analyses suggest accuracy for metastatic disease is equivalent to, or might exceed, standard technologies,We did two parallel prospective multicentre trials to elucidate and directly compare the diagnostic accuracy and efficiency of WB-MRI-based staging pathways with standard pathways in non-small-cell lung cancer (Streamline L)Streamline C is a multicentre, prospective trial comparing diagnostic accuracy for metastatic disease of staging pathways based on initial WB-MRI, with standard pathways in colorectal cancer. Ethics committee approval was granted on Oct 3, 2012, and the trial was coordinated by Cancer Research UK and University College London Cancer Trials Centre, with oversight from an independent data monitoring committee and a trial steering committee. All patients gave written informed consent.Patients were recruited from 16 general and teaching UK National Health Service (NHS) hospitals in England. Because eight of the 16 sites did not have the infrastructure to do WB-MRI, these sites sent patients to a nearby hospital for scanning . EligiblParticipants were identified from outpatient clinics, multidisciplinary team meetings, and inpatient wards by the local research team, who took informed consent from consecutive, unselected, eligible patients. A screening log detailed all patients approached and reasons for non-participation, where applicable. Age, performance status, sex, and request date for the first staging investigation were collected from recruited patients. Staging completion date was also recorded, defined as the date of the final test in the standard staging pathway.protocol has been publishedThe Participants had contemporaneous WB-MRI plus all standard staging investigations done as part of usual clinical care. Standard investigations were generally undertaken at the recruitment site, or a secondary hospital by referral in the case of specialised tests (such as PET-CT), and were interpreted by local consultant radiologists as per usual clinical practice. Interpretation of standard investigations was masked to WB-MRI images and findings. Although UK NICE guidelines recommend staging chest abdomen and pelvic CT, and pelvis MRI in the case of rectal cancerThe platform used for WB-MRI was in line with usual practice. A minimum dataset of sequences was acquired, including diffusion, T2-weighted, and T1-weighted (pre-intravenous and post-intravenous gadolinium containing contrast medium) imaging . WB-MRI Across all recruitment sites and imaging hubs, 19 radiologists interpreted WB-MRI and were unaware of all other standard staging investigations and clinical information . All radiologists were fellows of the Royal College of Radiologists and had interpreted at least 20 validated staging WB-MRIs. Radiologists with experience of fewer than 100 WB-MRI datasets initially had their reports validated by more experienced colleagues  and reported alone only once deemed competent by their colleague. This procedure was designed specifically to mirror how WB-MRI would be reported in NHS practice if more widely disseminated. Radiologists completed case report forms documenting the T and N stage of the local tumour  to assess the effect of lesion size on diagnostic accuracy, per-organ sensitivity, and for WB-MRI as a stand-alone investigation based on the original radiologist report.Secondary outcomes were difference in per-patient specificity for metastatic disease detection between standard and WB-MRI staging pathways, agreement between treatment decisions based on alternate pathways and the multidisciplinary team and consensus panel treatment decisions, staging efficiency , per-organ sensitivity and specificity for metastases, and per-patient agreement for local T and N stage. Additional secondary outcomes related to the effect of differing combinations of MRI sequences on accuracy, interobserver variability in WB-MRI interpretation, and the effect of adding WB-MRI to standard pathways, and will be reported elsewhere. The comparative patient experience of staging pathways and the findings of a discrete choice experiment have already been reported.28Using methods for comparative studies,We report our prespecified primary and secondary outcomes, and additional sensitivity analyses. Binary comparisons  were calculated using paired proportions  in STATA 14.2 . For the primary outcome, equivocal disease was considered positive for colon cancer and negative for rectal cancer, as specified by the independent data monitoring committee. Sensitivity analysis treated equivocal results as either negative or positive .There were no missing data for the primary outcome. Statistical significance was determined on the basis of 95% CIs from Newcombe paired proportion method;We compared the costs of WB-MRI versus standard pathways . The cosStreamline C is registered with the International Standard Randomised Controlled Trial registry, number ISRCTN43958015.The funder of the study stipulated that the study design should be a diagnostic accuracy trial using a cohort design, but was not involved in data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Between March 26, 2013, and Aug 19, 2016, 1020 patients were screened for eligibility . 370 patSensitivity of staging for patients with metastatic disease was 67% (95% CI 56–78) for WB-MRI and 63% (51–74) for standard pathways, a difference of 4%  and standard pathway . Sensitivity analysis found no significant differences between pathways when lesions reported as equivocal were treated as either all positive or all negative , or acroThe WB-MRI pathway had 54% agreement for T stage compared with 60% for the standard pathway, a non-significant difference of 6% ; a difference of 5 days  for metastatic disease was lower than published meta-analyses suggest. For example, Xu and colleaguesWe found that the WB-MRI pathway had 86% sensitivity for patients with metastatic disease of at least 1 cm. 48 (71%) of 68 patients with metastasis had liver metastasis, and the association between diameter and detection is well established. In their 2010 meta-analysis, Niekel and colleaguesWe found that WB-MRI pathways had similar accuracy for T and N staging compared with standard pathways. Although MRI appears to be promising for local staging of colorectal cancer, its superiority over CT is unproven.Generally, efficiency receives less attention than diagnostic accuracy.A strength of our trial is its pragmatic design. We recruited from a representative range of general and teaching hospitals, with all imaging done and interpreted according to usual local protocols, to increase generalisability of our results. The 19 radiologists interpreting WB-MRI were representative of those who would do so in daily NHS practice. We avoided using a smaller number of highly experienced radiologists; although we acknowledge that such individuals might achieve sensitivities greater than we report, they do not represent the national workforce. We used multidisciplinary team meetings to mirror patient care in the NHS. In doing so, we captured the entirety of standard pathways, including contemporaneous treatment decisions. We used a novel cloud-based image repository to maintain blinding and control multidisciplinary team access to WB-MRI until the appropriate time in the decision-making process. We were able to model the content and timing of WB-MRI staging pathways, and the potential effect on decision making. Conversely, previous research usually reports head-to-head comparisons between single imaging platforms, failing to capture pathway complexity. To our knowledge, our trial design is unique.Streamline C does have limitations. We masked radiologists to patient history and, for WB-MRI, to contemporaneous imaging, which was necessary to isolate diagnostic test accuracy within a pragmatic setting. Participants were representative of those undergoing staging in daily practice, although we did exclude pregnant women, patients not wanting to undergo WB-MRI, and patients with contraindications to MRI. The prevalence of metastatic disease was lower than assumed by our power calculation. However, the independent data monitoring committee recommended continuing the trial so as to achieve our original target number of evaluable patients. On independent data monitoring committee advice, equivocal findings were treated as negative for rectal cancer . Sensitivity analysis found that alternate classification of equivocal abnormalities had no meaningful effect. We modelled timing of WB-MRI staging pathways on the basis of real waiting times collated from recruitment sites during the trial, although sites had capacity to do WB-MRI. Waiting times might not be representative of those at other hospitals, and in other countries. Some of the benefits of reduced staging time by WB-MRI pathways could be negated if time to commencing treatment  are not reduced in parallel. Treatment decisions based on WB-MRI pathways were made after the multidisciplinary team was unmasked to all standard imaging tests, which could introduce bias. However, this situation was unavoidable if the full complexity of standard staging pathways was to be captured without interference from WB-MRI findings and if treatment decisions were to be recorded contemporaneously. Furthermore, alternate pathway agreement with a retrospective optimal treatment at 12 months remained very similar. Our cost analyses reflect an English NHS perspective and could differ in other settings, which might negate some of the cost advantages of WB-MRI pathways. Although WB-MRI is advocated as being safer than current standard staging investigations, new technologies are reducing radiation dose,In summary, WB-MRI staging pathways have similar diagnostic accuracy to standard pathways for identifying patients with metastatic disease in newly diagnosed colorectal cancer, and precipitate similar treatment decisions. However, they reduce staging time, test number, and costs. In a real-world NHS setting, WB-MRI-based pathways are a viable replacement for standard pathways.emailingctc.enquiries@ucl.ac.ukIndividual participant data that underlie the results reported in this Article, after de-identification , will be available for individual participant data meta-analysis beginning 9 months and ending 36 months after article publication. Data will be available to investigators whose proposed use of the data has been approved by an independent review committee (learned intermediary) identified for this purpose. Data access requires proof of relevant ethical committee approval for the specified analysis only. Data will be limited to those required for a specific analysis to protect deanonymisation. Where proposals that would compete with ongoing or planned research from the investigators within the trials team, data access will only be granted once investigator team publications are submitted. Proposals should be directed to the corresponding author; to gain access, data requestors will need to sign a data access agreement. After 36 months, there is no funded technical support. Information regarding submitting proposals and accessing data can be obtained by


Multipotent mesenchymal stem/stromal cells (MSCs) possess robust self-renewal characteristics and the ability to differentiate into tissue-specific cells. Their therapeutic potential appears promising as evident from their efficacy in several animal models of pulmonary disorders as well as early-phase clinical trials of acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). Such therapeutic efficacy might be attributed to MSC-derived products (the “secretome”), namely conditioned media (CM) and extracellular vesicles (EVs), which have been shown to play pivotal roles in the regenerative function of MSCs. Importantly, the EVs secreted by MSCs can transfer a variety of bioactive factors to modulate the function of recipient cells via various mechanisms, including ligand-receptor interactions, direct membrane fusion, endocytosis, or phagocytosis.Herein, we review the current state-of-the-science of MSC-derived CM and EVs as potential therapeutic agents in lung diseases. We suggest that the MSC-derived secretome might be an appropriate therapeutic agent for treating aggressive pulmonary disorders because of biological and logistical advantages over live cell therapy. Nonetheless, further studies are warranted to elucidate the safety and efficacy of these components in combating pulmonary diseases. The increasingly high rates of morbidity and mortality due to acute and chronic lung diseases have emerged as one of the major public health issues globally. Pulmonary diseases are mainly caused by trauma, air pollution, smoking, and various pathogens and exert a devastating effect on quality of life . Acute rAmong many destructive chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension, chronic obstructive pulmonary disease (COPD) is thought to be one of the leading causes of death worldwide. Approximately 20% of COPD patients present with emphysema, which is characterized by destruction of terminal bronchioles and alveolar walls causing overdistension of air spaces due to air trapping. Despite advances in the symptomatic treatment of emphysema, a cure remains obscure .Stem-cell-based therapy is an attractive approach for treating both acute and chronic lung diseases, such as ARDS and COPD, mainly because the cells can simultaneously target multiple pathological processes to protect lung function. Among the various types of stem cells, the therapeutic potential of mesenchymal stem/stromal cells (MSCs) has been extensively demonstrated in several animal models of pulmonary disorders –15, and In this review, we describe the characteristics of MSC-CM and MSC-EVs as well as the methods of their isolation. Next, we summarize the current state-of-the-science relating to their therapeutic potential for various lung diseases. Finally, we will discuss the advantages and limitations of these products in clinical settings.In 1968, Friedenstein and colleagues first discovered MSCs as a component of bone-marrow stromal tissue . SubsequThe initial motive for exploring their therapeutic benefits stemmed from the capability of MSCs to repair injured tissues by engrafting and replacing damaged cells through differentiation . An arraThe MSC-derived secretome is released into CM in vitro , 35–41. Recent efforts have focused on the paracrine effects of MSCs both in vitro and in vivo –52. MSC Structurally, EVs are nano- to micro-sized particles surrounded by a phospholipid bilayer. Most eukaryotes and prokaryotes have been shown to secrete a heterogeneous population of EVs. The presence of these vesicles can be detected in physiological fluids, such as plasma, urine, cerebrospinal fluid, milk as well as in the supernatant of cell cultures in vitro –55. Of nExosomes are formed by the inward budding of multi-vesicular bodies (MVBs), size ~ 40–100 nm, and rich in CD63, CD9, CD81, and tumor susceptibility gene 101 (Tsg 101). These vesicles are also enriched in annexins, ALG-2-interacting protein X (Alix), clathrin, heat shock proteins, and low amounts of phosphatidylserine (PS). Cytoskeleton activation is required for their release.Microvesicles (shedding vesicles) are formed by budding from the plasma membrane, size ~ 80–1000 nm, and rich in integrins, selectins, and CD40 ligands. Their release is dependent on cytoskeleton activation as well as calcium influx. The lipid bilayer of microvesicles is enriched in cholesterol, sphingomyelin, ceramide, and PS.Apoptotic bodies are derived from fragmentation of cells undergoing apoptosis, size ~ 1000–5000 nm, and rich in PS, nuclear fractions, and cellular organelles. While the role of cytoskeleton-associated molecules in the formation and the release of apoptotic bodies has been implicated, the mechanism responsible for relocation of fragmented DNA still remains unclear [ unclear , 62, 63.It is now established that MSCs secrete all types of EVs . The hetMSC-CM is considered the least processed cell-free product from MSCs. Based on the source of MSCs, culture condition, culture duration, and collection method, different quantities of secreted factors may be detected in the CM . GeneralThere are several approaches to determine the yield of the MSC-CM. One of the most common approaches is based on the number of MSCs from which the CM was derived , 73–76. So far, the ability of MSC-CM to significantly reduce the severity of lung injury, as effectively as MSCs, has been demonstrated in several in vitro and in vivo preclinical animal models Table . To thisEscherichia coli (E. coli) endotoxin intra-bronchially to perfused lungs, followed by the administration of MSCs or MSC-CM one hour following induction of injury. Both treatments improved lung endothelial barrier integrity and the rate of alveolar fluid transport. This study also revealed the essential role of keratinocyte growth factor (KGF), a MSC component, in alveolar fluid transport. MSC pretreatment with KGF small-interference RNA (siRNA) decreased the effect of MSC-CM on alveolar fluid clearance. Conversely, this effect was restored by the addition of recombinant KGF protein to the CM [Lee and colleagues investigated alveolar fluid transport using whole human lungs in an ex vivo lung perfusion model. The idea of such experimental studies stemmed from the notion that impaired alveolar fluid clearance is associated with higher mortality in ARDS patients. In this study, the therapeutic benefits of MSCs and MSC-CM were evaluated by the extent of their ability to restore alveolar fluid clearance in ALI. The experimental design consisted of delivery of o the CM .+ alveolar macrophages in BALF, resembling the phenotype associated with alternatively activated macrophages (M2). Insulin-like growth factor 1 (IGF-1) was detected in MSC-CM, suggesting its role in polarization of alveolar macrophages to the M2 phenotype and protection of lungs from endotoxin-induced injury [E. coli-induced pneumonia. MSCs also improved survival and bacterial clearance and preserved lung function. In contrast, MSC-CM improved animal survival without any significant mitigation of the severity of lung injury or inflammation [Further studies to elucidate the anti-inflammatory potential of MSC-CM were extended to several small animal models of ALI. In this pursuit, Ionescu et al. showed that intratracheal (IT) administration of MSCs or MSC-CM reduced the total number of infiltrated neutrophils in bronchoalveolar lavage fluid (BALF) in mice with endotoxin-induced ALI. Of note, the administration of fibroblasts or fibroblast-CM did not reproduce the same effect thus demonstrating MSC specificity. Concurrently, the flow-cytometric analysis revealed that MSC and MSC-CM administration increased the percentage of inducible nitric oxide synthase (iNOS)-Ym1d injury . In anotammation . Experimammation .Likewise, in a mouse model of asthma, Ionescu et al. demonstrated that local delivery of CM derived from plastic-adherent bone marrow cells (BMC) is effective in reducing the number of pro-inflammatory cells in the lungs and airway hyper-responsiveness while increasing the percentage of IL-10 producing T-regulatory lymphocytes and macrophages. CM derived from lung fibroblasts did not produce the same effect as BMC-CM. Interestingly, adiponectin, an anti-inflammatory cytokine found in BMC-CM but not in fibroblast-CM, was shown to play a major role in conferring the protective effects of BMC-CM .2 improved therapeutic benefits of the CM compared with control MSC-CM or lung fibroblast-CM. Correlatively, the preconditioned MSC-CM was also shown to contain a higher amount of STC-1 [Additionally, the beneficial role of MSC-CM in protecting lung tissue from hyperoxia was studied in several small animal models of bronchopulmonary dysplasia (BPD). BPD and consequent pulmonary hypertension are serious complications in premature infants caused by mechanical ventilation and oxygen toxicity. As reported by Aslam and colleagues hyperoxia-induced BPD in neonatal mice can be prevented by IV administration of concentrated mouse MSC-CM similar to MSC treatment. This study also revealed that early administration of MSC-CM significantly decreased right ventricular hypertrophy and muscularization of intrapulmonary arterioles as well as preserved the number of alveoli with normal morphology in neonatal mice exposed to hyperoxia. In parallel, MSC-CM treatment was shown to dramatically reduce the number of inflammatory cells and cytokine levels in BALF of injured mice. Another important finding was reflected in the identification of two immunomodulatory molecules, osteopontin (Spp1 or Opn) and macrophage-colony-stimulating factor 1 (M-CSF or Csf1), exclusively present in higher extent in the MSC-CM. This suggests their plausible role in the therapeutic benefits of MSC-CM . In a seof STC-1 , which iof STC-1 , 88–90.Shen and colleagues examined the anti-apoptotic and anti-fibrotic benefits of MSC-CM in a chronic model of bleomycin-induced lung injury. Histological analysis of MSC-CM-treated rats revealed a significant reduction in lung fibrosis and apoptosis 28 days post-injury. Interestingly, the MSC-CM was able to promote proliferation and prevent apoptosis of human non-small cell lung cancer epithelial cells in vitro .The therapeutic benefits of MSC-CM were also evaluated in a small animal model of cigarette-smoke-induced COPD. In addition to lung emphysema, cigarette smoke can cause apoptosis in lung fibroblasts. One of the roles of lung fibroblasts is to maintain the integrity of the alveolar structure; thus, their normal function is essential for the regeneration of the injured lung. Huh et al. showed that MSC and MSC-CM treatment significantly improved the histology of the emphysematous lungs and increased the number of small pulmonary vessels . In a seOverall, these reports strongly suggest that administration of CM derived from MSCs can significantly attenuate cell death and inflammatory responses while improving tissue healing and endogenous regeneration in various lung injury models. However, the key mediators and the optimal conditions to produce them are still largely unknown.Among the potential key mediators of MSCs, MSC-EVs are considered to bear therapeutic potential in lung injury. In order to establish a safe and effective alternative to live-cell transplantation, the therapeutic potential of different EV fractions was examined in a variety of preclinical animal models of lung injury as summarized in Table Gennai and colleagues used an ex vivo lung perfusion model to demonstrate the effect of MSC-EVs on alveolar fluid clearance rate in human lungs rejected for transplantation. MSC-EVs improved alveolar fluid clearance rate in a dose-dependent manner. Lung weight gain was also reduced by MSC-EV therapy compared to perfusion alone. Conversely, EVs derived from normal human lung fibroblasts had no beneficial effects. Further, MSC-EVs restored tracheal pressure, improved lung compliance, and reduced pulmonary artery pressure over six hours. The amount of lactate was also significantly lower in MSC-EV treated lungs compared to the control lungs. Since the anti-CD44 antibody attenuated the uptake of EVs by alveolar epithelial cells, the study suggested a key role of the CD44 receptor in internalizing EVs into recipient cells .E. coli endotoxin-induced ALI in mice. Additionally, MSC-EVs attenuated the influx of inflammatory cells and the presence of macrophage inflammatory protein 2 (MIP-2) in the alveoli. Both IT and IV administration of MSC-EVs were shown to dramatically increase the amount of KGF in BALF compared to injured untreated mice, maybe via transfer of KGF mRNA [The studies on exploring therapeutic benefits of MSC-EVs have been examined in several ALI models. In this regard, an investigation by Zhu and colleagues recently showed that IT administration of MSC-EVs was associated with a reduction in pulmonary edema in KGF mRNA . In anotKGF mRNA .E. coli-induced pneumonia. EVs harvested from MSCs preconditioned with a toll-like receptor (TLR)-3 agonist were shown to further increase KGF secretion and bacterial clearance. When examined in vitro, the MSC-EVs increased the phagocytic function of human macrophages and the ATP levels in alveolar epithelial type 2 cells. Preconditioned MSC-EVs were more effective in enhancing the anti-inflammatory and phagocytic activity of cultured macrophages, possibly by transferring cyclooxygenase-2 mRNA to activated monocytes resulting in an increase in production of PGE2 [Another report by Monsel and colleagues demonstrated that systemic administration of MSC-EVs improved survival and reduced lung inflammation and injury while promoting bacterial clearance in a murine model of  of PGE2 .The direct effects of MSC-EVs on activated macrophages has also been studied by preconditioning alveolar macrophages (AMs) with MSC-EVs and administering them to mice with LPS-induced lung injury. Administration of pretreated AMs, but not control AMs, significantly reduced total proteins, TNF-α, and the total number of neutrophils in BALF. Mitochondrial transfer via MSC-EVs was shown to play an important role in carrying the anti-inflammatory and pro-phagocytic effects of MSCs on macrophages . PhinneyThe therapeutic benefits of MSC-CM and MSC-EVs were compared in a mouse model of acute asthma. Systemic administration of both MSC-CM and EVs, but not lung fibroblast products, significantly abrogated the increase in airway hyper-responsiveness and lung inflammation. The amounts of T helper lymphocyte (Th)2- and Th17-associated cytokines were reduced in BALF while IL-10 levels were concomitantly increased. Blocking the release of secreted mediators in MSCs in vitro suppressed many protective effects of the cells .In a murine model of hemorrhagic shock, MSCs and their EVs were shown to attenuate vascular permeability in injured lungs. Both treatments were able to suppress hemorrhagic-shock-induced RhoA GTPase activity in lungs. MSC-CM and recombinant angiopoietin 1 (Ang1), but not EVs, significantly protected endothelial VE-cadherin junctions and reduced permeability in vitro. In vivo analysis of protein phosphorylation patterns also revealed some differences in signaling pathways activated by MSC-EVs compared to MSCs. Overall, these observations suggest that while MSC-EVs preserved pulmonary vasculature integrity similar to MSCs, their mechanism of action was not necessarily equivalent .The effect of MSC-EVs on pulmonary vasculature was also demonstrated by Lee and colleagues. Studies by this group indicated that the administration of MSC-EVs, but not fibroblast-EVs, can protect against the elevation of right ventricular systolic pressure and pulmonary vascular remodeling in a murine model of hypoxia-induced pulmonary artery hypertension. The MSC-EVs mediated an anti-inflammatory effect by reducing the influx of lung macrophages and early inflammatory mediators. MSC-EV treatment also abrogated the hypoxia-induced phosphorylation of signal transducer and activator of transcription 3 (STAT-3), which plays a critical role in the response of pulmonary vasculature to hypoxia . In a raSimilar to MSC-CM, MSC-EVs were shown to significantly reduce lung fibrosis, restore lung architecture, and improve alveolarization and lung function of neonatal mice exposed to 75% oxygen for 7 days. MSC-EV administration also reduced pulmonary arterial remodeling and hypertension associated with BPD. Additionally, MSC-EVs downregulated expression of genes associated with pro-inflammatory macrophages . In a stRecently, MSC-EVs were examined in a swine model of influenza virus-induced ALI. Local administration of MSC-EVs was able to significantly reduce the infiltration of inflammatory cells to the lungs, the thickening of alveolar walls, and the number of collapsed alveoli in infected swine. MSC-EVs were also able to reduce replication and shedding of influenza virus both in vivo and in vitro. It was suggested that the transfer of RNA to the infected cells might be an important factor in the observed anti-influenza activity by MSC-EVs .Overall, cumulative studies described above suggest that in order to increase cell survival, preserve physiological function, and reduce immune/inflammatory responses, MSC-EVs can transfer bioactive mediators to injured cells and regulate their pathophysiologic responses. In most experiments, EVs appeared to be as potent as MSCs, possibly with lower health-associated risks.In 2014, allogeneic MSC-EVs were administered to a patient with a steroid refractory graft-versus-host disease (GvHD). Not only the diarrhea volume of the patient was reduced after MSC-EV therapy, but also cutaneous and mucosal symptoms were improved within two weeks and the patient was in stable condition four months after EV treatments. Consequently, the dose of steroids given to the patient was reduced. Interestingly, the number of patient’s peripheral blood mononuclear cells producing TNF-α, IL-1β, or IFN-γ was reduced more than 50% after the last EV administration .In a separate study, forty patients with chronic kidney disease (CKD) were randomly divided into two groups to receive MSC-EVs or placebo treatment. Two doses of MSC-EVs were administered a week apart. The first dose was administered IV and the second intra-arterially. Of note, MSC-EV treatment, but not the placebo, was effective in improving estimated glomerular filtration rate and urinary albumin-to-creatinine ratio as well as reducing serum creatinine and blood urea. At twelve weeks after EV treatment, plasma levels of TNF-α were significantly reduced, whereas levels of IL-10 and TGF-β were increased . In bothclinicaltrials.gov evaluating the benefits of UC-MSC-EVs in type I diabetic patients [A phase II/III clinical trial is currently registered on patients . In addipatients .Overall, current data suggest that MSC-derived products can effectively mimic the therapeutic effects of MSCs in preclinical models of lung injury. The use of CM or EVs may circumvent many of the safety concerns associated with the use of live cells, such as thrombogenicity. Also, unlike cells, CM and EVs are not able to proliferate or reprogram after administration, which decreases the risk of tumorigenicity –108. FroAnother therapeutic advantage of EVs arises from their ability to protect their cargo from unfavorable environmental conditions, such as dramatic changes in pH or release of digestive (lytic) enzymes into the bloodstream and damaged tissue. Based on this concept, MSC-EVs are considered efficient vehicles for transporting delicate bioactive mediators to the distal airways. It is also thought that MSC-EVs can be used to deliver specific drugs to target organs. Such drugs can be delivered to EVs via MSCs that overexpress those factors, or can be loaded directly to the EVs after they have been released by the cells. Examples of different types of therapeutic cargo include siRNAs for silencing target genes and chemotherapeutic drugs, such as doxorubicin , 110.In addition to their biological advantages, feasibility/logistical advantages set cell-free products apart. For instance, large-scale manufacturing of cell-free products is possible by repeated harvesting of CM from cultured MSCs. MSC-CM and EVs can be prepared and stored in small volumes, which could simplify dosing studies. Another great advantage is that CM and EVs can also be stored for several months at − 20 °C to − 80 °C without the need for cryopreservatives, unlike their parent cells . NotablyThere appear to be therapeutic advantages in using MSC-CM and EVs over live whole cells; yet, to develop safe and effective cell-free therapies, a significant knowledge gap needs to be addressed, including optimization of bioactive components, dosing regimens, and production methods. A growing number of studies have identified some of the key mediators in MSC-CM and EVs; however, there is a variability in the content of the secretome due to several factors. These include donor’s condition , the type of MSCs , the type of culture (flask or bioreactor), the type of media and supplements , and the microenvironment  , 111. Al6–107 cells/kg). In order to produce sufficient pools of MSCs, the cells need to be expanded in vitro for several population doublings. Although MSCs have been shown to be safe for up to 40 population doublings [Based on currently available techniques, large numbers of MSCs are still required to produce an adequate amount of CM or EVs for clinical use, similar to the number of MSCs used for clinical trials -graded MSC-CM and EVs needs to be developed so that cell-free therapy can be safely translated from the bench to the bedside.


Recently, the interest in extracellular vesicles (EVs) released by pathogens like bacteria, fungi, and parasites has rapidly increased. Many of these pathogens actively modulate the immune responses of their host and there is accumulating evidence that pathogen-derived EV contribute to this process. The effects of pathogen-derived EV on the host immune system have been attributed to proteins, lipids, nucleic acids, and glycans contained in, or present on these EV. For example, toxins in bacterial EV can modulate pathogen clearance and antigen presentation, while EV-associated polysaccharides are potential vaccine targets because they induce protective immune responses. Furthermore, parasite EV-associated microRNA may increase parasite survival via host gene repression, and the lipid A moiety of LPS in bacteria-derived EV induces strong pro-inflammatory responses. Research on pathogen EV-associated molecules may pave new avenues to combat infectious diseases by immune intervention. This review provides an overview of the current knowledge of EV-associated molecules released by extracellular pathogens and their effects on the host immune system. The current focus and future hotspots of this rapidly expanding field will be highlighted and discussed. An increasing number of studies documenting the molecular characteristics and the function of pathogen-derived extracellular vesicles (EVs) suggest that pathogen EV play important roles in the activation and modulation of the host immune system. However, only few reports assign EV-mediated effects to specific EV-associated molecules or molecular principles. Here, we focus on defined pathogen-derived EV-associated molecules, their immunomodulatory effects, and the implications for host–pathogen interactions. We first briefly discuss pathogen–host communication via EV and EV biogenesis. Next, various classes of molecules in pathogen-derived EV—proteins, lipids, glycans (carbohydrate chains), and nucleic acids—will be addressed with respect to molecular identity and function. With this approach we aim to outline the current understanding of molecular principles through which pathogen EV modulate immune responses of their hosts.Many pathogens, including bacteria, fungi and parasites, have evolved to survive and reproduce in their host environment. For their success, modulation of the host immune system is often crucial. Simultaneously, the mammalian innate immune system has evolved to recognize and respond to the invading pathogens in order to eliminate them. Various pathogen-associated molecular patterns (PAMPs)—such as proteins, lipids, glycans, and nucleic acids—can bind to different families of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), and NOD-like receptors (NLRs), expressed by both immune and non-immune cells in the host. While the activation of PRRs by PAMPs is essential for immunity and host defense, pathogens have developed several modulatory mechanisms to interfere in PRR binding and signaling . In addiExtracellular vesicles are a collective term for exosomes, microvesicles, and other cell-derived membrane-enclosed vesicles. These vesicles transport various molecules, including proteins, lipids, and nucleic acids between cells within one organism or between organisms, such as in host–pathogen cross-talk. Mammalian EV contain a mix of endosomal and plasma membrane proteins, including tetraspanins and major histocompatibility complex (MHC) molecules, and cytosolic proteins such as cytoskeletal proteins and heat shock proteins . Lipids Vibrio cholerae . Although the biogenesis of vesicles within the MVE is better understood than vesicle biogenesis at the plasma membrane, both routes share molecular machineries for EV generation, such as ‘Endosomal Sorting Complex Required for Transport’ (ESCRT) proteins and tetraspanins. Both ESCRT-dependent and independent mechanisms have been implicated in clustering of cargo for biogenesis of EV  analysis of multicellular parasites, such as hepatica , and unima cruzi , indicatr pocket . Howeverr pocket . In addir pocket . These fon of EV . Furtheron of EV . Differeon of EV . For thion of EV . Since tFigure .There is limited knowledge on the mechanisms through which EV can interact with cell surfaces and transfer their  cargo to the recipient cell. In general, membrane-bound (glyco)proteins and (glyco)lipids drive the initial interaction with the recipient cell. First, the EV needs to dock at the recipient plasma membrane where it can subsequently remain bound to the surface, be internalized, or fuse with the cell membrane. Receptors on the target cell plasma membrane, such as integrins, can recognize the exterior cargo of the EV, which can lead to activation of signaling cascades within the cell . ReceptoTrypanosome EV to erythrocytes for prevention of anemia . In this way, we aim to assess whether there are similarities in how different pathogens use EV-associated molecules to affect host cells. This overview is restricted to studies in which clear evidence was provided for the association of a pathogen-specific molecule in/on EV and the direct involvement of this molecule in the EV-induced effects. A total overview of the selected papers, the reported EV-associated molecules, the observed effects on the host immune system, and the methodological approaches applied in these studies can be found in Figure 2A). These EV proteins have been shown to affect immune responses or the survival rates of the host or pathogen . Below, we distinguish and describe pathogen EV-associated proteins that cause cytotoxicity, increase pathogen survival, or induce pro-inflammatory responses.Proteins form the largest group of known pathogen EV-associated molecules  in human airway epithelial cells. CFTR facilitates chloride secretion by epithelial cells needed for mucociliary clearance of the pathogen in the lungs A1. UspA1 can bind to carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM-1) on epithelial cells and subsequently reduce TLR2-activated IL-8 production  in EV from an uropathogenic trophils . Anotherruginosa , 2014. Ehe lungs . In a fohe lungs . These soduction . As IL-8Francisella tularensis, the gram-negative bacterium that can cause fetal pneumonic tularemia in humans, contain a protein with lipolytic activity named FtlA. Since this bacterium primarily replicates inside alveolar macrophages and epithelial cells, the success of invasion into host cells determines its virulence. FtlA mutant bacteria showed significantly reduced lipolytic activity and virulence and reduced inflammatory cell infiltrations in a lung infection model  within macrophages such that it inhibits inflammatory responses leading to increased parasite survival. Although this is an intracellular parasite, the authors used isolated EV from in vitro extracellular parasite cultures. In vivo administration of EV from GP63-/-L. major parasites resulted in stronger pro-inflammatory effects, such as increased neutrophil and eosinophil recruitment, compared to wild type L. major. This suggests that EV-associated GP63 could dampen immune responses and may support the establishment of Leishmania infection in the host. However, these effects could also be due to other EV-associated proteins, since it was shown that GP63 additionally affects the proteomic content of EV 63 released by the protozoan parasite L. major . GP63 isnt of EV . Furtheris miRNA . These rhe liver .Opisthorchis viverrini, for example, were shown to induce IL-6 production by human cholangiocytes, an effect that could be blocked by antibodies against the EV-associated tetraspanin TSP-1  protein on the EV of this gram-negative bacterium was essential for EV uptake by human tonsillar B lymphocytes via the B cell receptor and induced IL-6 production by these cells  in TSP-1 . This sual cells . An addise cells . Interesnfection . These rStreptococcus pneumoniae. PspA was introduced into a gram-negative Salmonella enterica strain  were used, or when cells were incubated with EV from gram-positive bacteria lacking LPS. The pro-inflammatory effects were also reduced when using bacteria from a E. coli strain which released less EV due to mutations in EV biogenesis. These data suggest a role for EV in delivering LPS into the cytosol and subsequent activation of innate immune responses during an infection with gram-negative bacteria lipid associated with EV. LPS is known to bind to TLR4, but can also be recognized in the cytosol by the murine receptor protease caspase-11, and in humans by caspase-4 and caspase-5. EV from gram-negative bacteria were shown to deliver LPS to the cytosol, thereby inducing an inflammatory form of programmed cell death (pyroptosis) and increased IL-1β expression in a caspase-11-dependent manner . These ebacteria . A more bacteria . When stbacteria . Lipid AN. meningitidis are already used in a vaccine against meningococcal disease in children . Considering the increasing interest in EV and continuous improvement of EV isolation and characterization technologies, it is suspected that additional biological effects of pathogen-derived EV-associated lipids may be elucidated in the near future. For example, mass spectrometry indicated that EV from the parasitic worm Heligmosomoides polygyrus differed in lipid composition from murine cell-derived EV and were enriched in ether glycerophospholipids called plasmalogens. The authors created artificial vesicles and observed that increasing the plasmalogen content of the vesicles increased the efficiency with which they fused with cells lipids in pathogen-derived EV  showed higher protective immune responses after bacterial challenge compared to their highly acetylated counterparts  signaling and favors a reduction in IL-6 and an increase in macrophage arginase expression. As IL-6 enhances host susceptibility to H. polygyrus and arginase is a mediator of killing this parasite in mice, repression of DUSP1 may be a parasite-driven mechanism to enhance its survival in the host. This is a very interesting example of cross-organism communication in which a parasite EV-associated miRNA functions due to complementarity to the host target gene.There is an increasing number of studies that report on the composition of the small RNA repertoires in vesicles from bacteria , fungi , and paro effect . This efT. cruzi releases EV containing transfer RNA-derived small RNAs (tsRNA), that could be visualized inside HeLa cells after uptake of these EV —and can modify host cell function or induce host cell apoptosis. Although the field of research on pathogen-derived EV is still in its infancy, many advanced studies have already been performed on EV released by bacteria. The generation of mutant bacterial strains has provided great opportunities to study the function of specific EV-derived molecules. From these studies, we learned that many, if not all, gram-negative bacteria release LPS-containing EV that are well-capable of inducing pro-inflammatory reactions. Despite gaps in our understanding of the broad group of pathogen-derived EV, the currently available data indicate that pathogen EV-associated molecules can promote survival and spreading of pathogens, but can also facilitate the induction of host-immune responses . The exact role of pathogen-derived EV in host–pathogen interaction likely depends on pathogen life stage, environmental, and/or tissue specific conditions.The observed effects of EV released by pathogens underline that intercellular communication via EV is a conserved mechanism that most likely benefits pathogen survival in co-evolution with its host. Immunomodulatory components of pathogen-derived EV are represented in all molecular subclasses—proteins, lipids, glycans, and nucleic acids (The current overview does not show us yet whether EV from different pathogens contain conserved classes of molecules or act via similar principles. This is probably due to the limited number of publications on EV-associated immunomodulatory molecules from pathogens that is available at this early stage. In addition, inter-study comparability of data on both mammalian and pathogen EV is currently hampered by the use of a wide range of different techniques to isolate and characterize EV. Different EV isolation methods, for example, yield EV of different purity and can bias toward isolating certain EV subtypes . This urin silico. Computer-based models can search all available pathogen specific EV-associated molecules for family members with immunomodulatory potential that are conserved between different pathogens, and directly point toward interesting molecules for further functional studies. Increasing the knowledge on pathogen-derived EV-associated molecules and their effects on the host immune system will certainly shed further light on the importance of EV molecules in infection biology.A promising method to allow pathogen-overarching comparisons of pathogen-derived EV-associated molecules is the use of bioinformatics . The incMK wrote the manuscript. EN-‘tH, CH, and HS initiated and edited the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.