The results of Monte Carlo simulation studies show that the Bayesian method provided coverage percentages close to the nominal confidence level and shorter intervals than the other methods. Importantly, it maintained a good balance between LEP and UEP even for large variation and percentage of zero-valued observations. To illustrate the efficacy of our proposed methods, we applied them to hourly wind speed data from northern Thailand.Wind speed has an important impact on the formation and dispersion of fine particulate matter (PM), which can cause several health problems. During the transition from the winter to the summer season in northern Thailand, the wind speed has been low for longer than usual, which has resulted in fine PM accumulating in the air. Motivated by this, we have identified a need to investigate wind speed due to its effect on PM formation and dispersion and to raise awareness among the general public. The hourly windspeed can be approximated by using confidence intervals for the ratio of the medians of three-parameter lognormal distributions containing zero values. Thus, we constructed them by using fiducial, normal approximation, and Bayesian methods. By way of comparison, the performance measures for all ofthe proposed methods , and expected length) were assessed  By way of comparison, estimating the ratio of the median wind speeds in two areas is used as a starting point in this study. The median, a measure of central tendency, is the central value of a dataset (the midpoint of a distribution). Moreover, it is more efficacious to use the median than the mean in analyses when the distribution of data is skewed. In addition, the ratio of the medians of two datasets can be used to measure the difference between them.Oxygen in the air is necessary for the survival of humans and other animals. How fast the air moves past a certain point is known as wind speed (measured in km/h), which is an important phenomenon in meteorology for monitoring and predicting weather patterns in a given area. It is reported daily along with the temperature, precipitation, and humidity for all provinces on the Thai Meteorological Department website. Importantly, low wind speeds during the transition from the winter to the summer season lead to increased fine particulate matter (PM) levels. In 2020, high PMn region . Spikes y–March) . These ri.e., a TPLN distribution containing zero values).A lognormal distribution is used to represent right-skewed data when the threshold parameter (the lower bound of the data) is equal to zero. The TPLN distribution first introduced by via Monte Carlo simulation.One of statistical inference methods is the parameter estimations, including the point and interval estimations with the best-known example of the latter being a confidence interval (CI). According to In particular, some researchers have formulated methods for estimating the CIs for the parameters of the TPLN. However, the CIs for the parameters of a TPLN distribution, especially when zero values are included along with the nonzero values, have not yet been formulated. Therefore, we herein propose fiducial, NA, and Bayesian methods for constructing CIs for the ratio of the medians of TPLN distributions containing zero values. We conducted a Monte Carlo study to assess their performances in terms of their coverage percentages (CPs), lower and upper error probabilities , and expected lengths. These methods were applied to estimate hourly wind speeds during the transition from the winter to the summer season in northern Thailand. This information could be used to help design green corridors and implement other policies to reduce PM levels in northern Thailand in the future.Xi = , i = 1, 2 be random samples drawn from a TPLN distribution containing zero values, denoted as γi is the threshold parameter, μXi is the scale parameter (the mean of Xi), Xi) and ρi is the proportion of nonzero values. For Xi > 0, random variable Yi = ln(Xi − γi) is normally distributed as Xi is a lognormal random variable  are initialized as vectors of 0’s, and the Adam algorithm operates until their gradients approach zero. The bias-corrected first- and second-moment estimates are used to update parameter γi as follows: where γi is updated by Adam update rule, which is defined as Finally, the parameter δ and ϵ denote the step size and the learning rate, respectively. ϵ is fixed at 10−8 for a sufficient learning rate using the Adam optimization algorithm, as can be seen in Algorithm 1.where After obtaining ηi = ρi[γi + exp(μXi)] provides the medians of Xi, the ratio of the medians of Xi (the parameter of interest in the this study) is given by Since ηi. The concepts are elaborated in the methods for constructing CIs for ω in the next section.By substituting MLEs Here, we present constructing the CIs for the ratio of the medians in the TPLN models containing zero observations based on the fiducial methods ) and the method of variance estimates recovery-based fiducial generalized pivotal quantity , NA  and the Y =  be a random variable with probability density function fY, where λ and θ are vectors of the parameter of interest and the nuisance parameter, respectively. Moreover, let y =  be the observed values of Y, and assume that fiducial GPQ T is only a function of λ. This method is especially associated with the fiducial inference proposed by T for a parameter λ is called a fiducial generalized pivotal quantity (FGPQ) if it satisfies the following conditions: (FGPQ1) Given Y = y, the T distribution is free of all parameters. (FGPQ2) For every y ∈ R+, the observed pivotal T = λ.A GPQ T satisfies the conditions (FGPQ1) and (FGPQ2), then it is possible to construct the 100(1 − φ)% fiducial GPQ-based CI for λ is ; Tλ(φ) is the φth percentile of T.If the γi < Xi(1), we proposed the fiducial GPQ of γi based on a continuously uniform distribution and the fiducial GPQ of ρi based on a beta distribution, respectively, as follows:The fiducial GPQ CI can be constructed based on the fiducial GPQ conditions in Definition 1. Recall that (Xi(1) denotes the minimum value of Xi and ni1 is the sample size of nonzero values ni = ni1 + ni0. Furthermore, the fiducial GPQ of μXi can be obtained by using the concepts of where Wi and Vi are independently draw from a standard normal distribution and a chi-square distribution with ni1 − 1 degrees of freedom, respectively. Since the three fiducial pivots  satisfy the fiducial GPQ properties and Tηi = Tρi1−[Tγi + exp(TμXi)] is the fiducial GPQ of ηi, then we can obtain the the 100(1 − φ)% fiducial GPQ CI for ω as follows: where Tω = Tη1/Tη2, and Tω(φ) denotes the φth percentile of Tω. The steps for calculating the CP of fiducial GPQ CI (CPF) for ω can be carried out by using Algorithm 2.where ω as follows:MOVER is a well-known method for estimating the CI of the parameter of interest . Moreoveγi, μXi, ρi) can respectively be written asThe fiducial GPQ CIs for (φ)% MOVER-fiducial CI for lnηi becomes These intervals can be formulated the MOVER CI by using the concept of whereφ)% CI-based the MOVER-fiducial GPQ CI for ω as thereby providing the 100(1 − M) for ω.Algorithm 3 presents the computational steps for calculating the CP of the MOVER-fiducial GPQ CI  denotes the rth order statistic in a random sample drawn from a TPLN model with zero observations of size ni1, and MS is a consistent estimator of the variance of the median. Similarly, the variance of where Wi can be defined as where n → ∞. Subsequently, the 100(1 − φ)% NA-based CI for ω can be written as which approaches a standard normal distribution as Wφ denotes the φth percentile of a standard normal distribution. Algorithm 4 was used to compute the CP of the NA (CPN).where θ, a prior distribution p(θ) represents the subjective belief as a subjective distribution formulated before the data are seen. The posterior distribution is obtained from a prior that is updated with the likelihood function (or sample information) by using Bayes’ rule  are obtained by obtaining The posterior densities of ; the logarithm of the posterior density is approximated by using a quadratic function of κi. The second derivatives of the log-posterior density are needed for constructing the approximation. From Next, we apply NA to the posterior distribution of μXi, logσXi) respectively becomeAfter that the first and second derivatives of  are derived after setting their first derivatives to zero. Meanwhile, the variances of their estimates are respectively obtained by using an inverse Fisher information matrix as follows:The point estimates of (After using the Jacobian to back-transform from ω becomes where φ)% Bayesian-based CI for ω is Finally, the 100(1 − fω(φ) denotes the φth percentile of fω. The CP of Bayesian CI (CPB) can be computed by using Algorithm 5.where via a Monte Carlo simulation study. The settings for the simulation parameters for the two simulation studies were as follows. For each specified parameter combination, the 95% CIs for the ratio of the medians (ω) of TPLN distributions containing zero values were constructed based on 5000 randomly generated samples. In addition, 2500 Monte Carlo sampling passes were used for each generated sample for the fiducial GPQ-based method. To assess the performances of the methods, their CPs were calculated by using the proportion of 5000 simulated CIs covering ω. LEP and UEP are defined as the proportion of times that ω falls below and above the stimulated CIs, respectively. At the 95% nominal confidence level, the expected lengths (ELs) of the CIs is also needed for deciding which method performs the best. A good performance will produce CP = 95% and LEP = UEP = 2.5%. Likewise, the comparison between LEP and UEP can be expressed in terms of the relative bias, which is defined as The comparative performances of the proposed Bayesian-, the fiducial GPQ- and MOVER-fiducial GPQ-, and NA-based CI were evaluated Thus, a good balance between LEP and UEP will produce a relative bias close to zero. Last, the best-performing method will provide the shortest EL.d1, d2) = ,   and In the first simulation study, we chose a small proportion of zeros and variance ,  and In the next simulation, we were interested in scenarios with a large proportion of zeros and variance (ctively) , 5 and 62.5 and PM10 when its speed is 7.2 km/hr or higher  have strong points (We applied fiducial, NA, and Bayesian-based method to formulate CIs for the ratio of the medians of TPLN distributions containing zero values. From the results of the simulation study, the fiducial methods based on fiducial GPQ and MOVER-fiducial GPQ always provided CPs greater than the nominal 95% confidence level because the fiducial GPQ of (g points , as reveg points .Meanwhile, the Bayesian method provided suitable CPs with the shortest interval length, except for a small variance and a large sample size for which the MOVER-fiducial GPQ method performed the best. This could be because of using the uniform prior to gain information about the parameters from the data to obtain their posterior density. As such, the constructed CIs for the ratio of the medians of TPLN distributions containing zero values based on the Bayesian method with a uniform prior performed well.CIs for the ratio of the medians of TPLN distributions containing zero values were formulated by using fiducial-, NA-, and Bayesian-based methods. Since a theoretical comparison was not possible, a Monte Carlo simulation and empirical application with two real datasets of wind speed observations were used to evaluate their performances in terms of their CPs and ELs. The results of the simulation study led us to recommend the Bayesian method for constructing the CIs for the ratio of the medians of TPLN distributions containing zero values because it attained CPs close to the nominal 95% confidence level and the shortest EL in most cases, except for a small variance and a large sample size for which the MOVER-fiducial GPQ method should be used.10.7717/peerj.14194/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14194/supp-2Supplemental Information 2Click here for additional data file.


Fluid creep, including fluids administered as drug diluents and for the maintenance of catheter patency, is the major source of fluid intake in critically ill patients. Although hypoxemia may lead to fluid restriction, the epidemiology of fluid creep in patients with hypoxemia is unclear. This study aimed to address the burden due to fluid creep among patients with respiratory support according to oxygenation status.We conducted a post-hoc analysis of a prospective multicenter cohort study conducted in 23 intensive care units (ICUs) in Japan from January to March 2018. Consecutive adult patients who underwent invasive or noninvasive ventilation upon ICU admission and stayed in the ICU for more than 24 h were included. We excluded the following patients when no fluids were administered within 24 h of ICU admission and no records of the ratio of arterial oxygen partial pressure to fractional inspired oxygen. We investigated fluid therapy until 7 days after ICU admission according to oxygenation status. Fluid creep was defined as the fluids administered as drug diluents and for the maintenance of catheter patency when administered at ≤ 20 mL/h.p < 0.001 in both patients), but no significant trend was observed in those with severe hypoxemia (p = 0.159). Similar trends have been observed in the proportions of sodium and chloride caused by fluid creep.Among the 588 included patients, the median fluid creep within 24 h of ICU admission was 661 mL , and the proportion of fluid creep gradually increased throughout the ICU stay. Fluid creep tended to decrease throughout ICU days in patients without hypoxemia and in those with mild hypoxemia .The online version contains supplementary material available at 10.1186/s13613-022-01073-x. Intravenous fluid is commonly administered to critically ill patients. The aim of intravenous-fluid administration can be categorized into resuscitation and non-resuscitation  . Non-resThe prevalence of fluids administered as drug diluents and for the maintenance of catheter patency, so-called fluid creep, has recently been investigated , 20, 21.Resuscitation is challenging in septic shock patients with hypoxemic respiratory failure, since hypoxemia is one of the conditions that discourage fluid resuscitation . FurtherIn this study, we conducted a post-hoc analysis of the incidence and risk factors for phlebitis and complications due to peripheral venous catheters in critically ill patients (AMOR-VENUS study) , which wThis study was a post-hoc analysis of the AMOR-VENUS study conducted in 23 ICUs in Japan from January 1 to March 31, 2018. This study was approved by the institutional review board or medical ethics committee of each institution . The requirement for informed consent was waived, and an opt-out recruitment method was employed. This study is reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology statement .Consecutive adult patients (aged ≥ 18 years) admitted to the ICU during the study period were included in the AMOR-VENOUS study. The exclusion criteria of the original data set were as follows: refusal to participate in the study, having no intravascular catheters during ICU stay, and physician’s discretion, that is, patients who were selected for exclusion by physicians . Of these patients, this post-hoc analysis included those who underwent invasive or noninvasive ventilation upon ICU admission. In addition, patients were excluded if they died or were discharged within 24 h of ICU admission, no fluids were administered within 24 h of ICU admission, or the ratio of arterial oxygen partial pressure to fractional inspired oxygen (P/F ratio) was not recorded.Baseline data included age, admission category , the severity of illness , hypotonic crystalloids , nutrition fluids (which contained ≥ 10% dextrose or amino acids), colloids, blood products, or vehicles for drugs. The dosage of the liquid drug was defined as an unclassified fluid. Fluid creep was defined as fluids administered as drug diluents  and for the maintenance of catheter patency [The following solutions were considered the fluids for maintenance of catheter patency, which were administered at ≤ 20 mL/h: isotonic crystalloids and hypotonic crystalloids . Isotonit test, Mann–Whitney U test, one-way analysis of variance, or Kruskal–Wallis test, according to the data distribution. Dichotomous variables were analyzed using the chi-squared test or Fisher’s exact test. According to the patient’s oxygenation status, we evaluated the trend toward increasing or decreasing intravenous fluid, sodium, and chloride intake with longer ICU stays using the Jonckheere–Terpstra trend test. Multiple linear regression models were used to evaluate the relationship of septic shock and hypoxemic respiratory failure to the amount of intravenous fluid and fluid creep within 24 h of ICU admission, as well as sodium and chloride burden. Covariables known to be associated with fluid management in critically ill adults  were identified a priori and subsequently forced into the models [p < 0.05. Statistical analyses were performed using Stata 15.1 .Data are expressed as medians with interquartile ranges (IQRs) or means with standard deviations for continuous variables and numbers with corresponding percentages for dichotomous variables. In all analyses, the number of cases with missing data was reported, and these cases were excluded from each analysis. Baseline characteristics were compared between the study groups. Continuous variables were compared using the unpaired e models , 34. In A total of 3482 patients were admitted to the ICU during the study period. Among these, 842 patients underwent mechanical or noninvasive ventilation upon ICU admission, and 588 were included in this analysis after applying the inclusion and exclusion criteria Fig. . The patPatient demographics and clinical characteristics are presented in Tables A mean total intravenous-fluid volume of 2662 range, 1646–4059) mL was administered during the first 24 h after ICU admission, and the total amount of intravenous fluid gradually decreased but remained > 1000 mL per 24 h during the entire observation period , but no significant trend was observed in those with severe hypoxemia (P for trend = 0.159) . Similar trends were observed regarding the proportions of sodium and chloride due to fluid creep.The median fluid creep during the first 24 h after ICU admission was 661 mL (range: 402–984 mL), constituting 25.2% of the total intravenous-fluid volume. After 24 h, fluid creep tended to decrease throughout ICU days in patients without hypoxemia and in those with mild hypoxemia (59) Fig. . MeanwhiThe results of the multiple linear regression model for the amount of intravenous fluid, sodium, and chloride burdens within 24 h of ICU admission after adjusting for the prespecified covariates are shown in Table Among 588 patients with 2387 patient-days, the most used drug vehicle was normal saline Fig. , with thThis post-hoc analysis of a prospective multicenter observational study investigated the epidemiology of intravenous fluid therapy among patients with respiratory support. Among the 588 included patients, fluid creep was a major source of fluid volume, sodium, and chloride burdens. Fluid creep tended to decrease throughout ICU days in patients without hypoxemia and in those with mild hypoxemia, but no significant trend was observed in those with severe hypoxemia. After adjusting for the prespecified confounding factors, septic shock was associated with most of the burdens; meanwhile, hypoxemic respiratory failure was associated with a reduction in the total amount of intravenous fluid, but not with fluid creep.Previous studies reported that the median daily amount of fluid creep was ≥ 600 mL, constituting approximately 35% of the total intravenous-fluid volume in critically ill patients , 4. The Sodium intake was reported to be 83–238 mEq/day in critically ill patients . NotablySeveral randomized controlled trials comparing balanced crystalloids with normal saline demonstrated that the use of balanced crystalloids reduced hyperchloremia and major adverse kidney events, which was a composite of death, new renal replacement therapy, or persistent renal dysfunction , 37. FurResuscitation fluid restriction has been assessed in septic shock patients –15. AlthTo the best of our knowledge, no studies have assessed fluid creep according to oxygenation in mechanically ventilated adult patients. Our study suggests that fluid creep is a major source of fluid volume, sodium, and chloride in these patients. Furthermore, the amount of fluid creep did not decrease during the 7 days of ICU admission in patients with severe hypoxemia. Fluid creep may not be restricted enough even in those patients who commonly receive conservative fluid management.This study has certain limitations. First, enteral intake data were not collected. However, enteral intake may be difficult to reduce for sufficient nutrition therapy. Our study provides adequate information to form a basis for further investigation of fluid restriction. Second, output data were not collected. Fluid balance is important in fluid therapy. However, considering the few differences in patient severity, mortality, and intravenous-fluid volume compared with previous studies , 3, the Fluid creep was the major source of fluid volume, sodium, and chloride among patients who underwent respiratory support, and the burdens due to fluid creep were prolonged in those with severe hypoxemia. Hypoxemia was associated with a significant reduction in the total fluid intake, but not with fluid creep. Since fluid creep was predominantly crystalloids containing sodium and chloride, these burdens can be decreased by the use of 5% dextrose. Interventional studies are required to assess the feasibility of further fluid restriction, including fluid creep, in these patients.Additional file 1: Table S1. Daily intravenous fluid intake according to fluid type. Table S2. Daily sodium intake according to fluid type. Table S3. Daily chloride intake according to fluid type. Table S4. Estimates of the effects of covariates on the fluid volume, sodium, and chloride in the multiple linear regression models.


If considering all cases with CE tumour lesions, the performance dropped significantly (median Dice = 0.40). Volume agreement was excellent with CCCs of 0.997 (CE tumour volumes) and 0.922 (NE volumes). The findings have implications for the application of the HD-GLIO algorithm in the routine radiological workflow where small contrast-enhancing tumours will constitute a considerable share of the follow-up cases. Our study underlines that independent validations on clinical datasets are key to asserting the robustness of deep learning algorithms.In the context of brain tumour response assessment, deep learning-based three-dimensional (3D) tumour segmentation has shown potential to enter the routine radiological workflow. The purpose of the present study was to perform an external evaluation of a state-of-the-art deep learning 3D brain tumour segmentation algorithm (HD-GLIO) on an independent cohort of consecutive, post-operative patients. For 66 consecutive magnetic resonance imaging examinations, we compared delineations of contrast-enhancing (CE) tumour lesions and non-enhancing T2/FLAIR hyperintense abnormality (NE) lesions by the HD-GLIO algorithm and radiologists using Dice similarity coefficients (Dice). Volume agreement was assessed using concordance correlation coefficients (CCCs) and Bland–Altman plots. The algorithm performed very well regarding the segmentation of NE volumes (median Dice = 0.79) and CE tumour volumes larger than 1.0 cm Patients with primary malignant brain tumours have some of the poorest prognoses of all cancer patients even after extensive surgery, radio-, and chemo-therapy ,2,3. To Tremendous progress has been made in order to automate the task of 3D brain tumour delineation by use of deep learning (DL) algorithms as demonstrated by the contributions to the annual Brain Tumor Segmentation (BraTS) Challenges . Yet, brThe aim of this study was to undertake an evaluation of the HD-GLIO brain tumour segmentation algorithm on an external dataset from a consecutive patient cohort that underwent routine MRI for primary brain tumour response evaluation using ground-truth delineations by independent radiologists. Thus, the study addresses the important issue of brain tumour segmentation of patients having undergone surgery and aims to provide evidence for the robustness and generalisability of a state-of-the-art segmentation algorithm.The performance of the HD-GLIO algorithm was tested on a retrospective dataset consisting of consecutive adult patients (aged ≥ 18 years) that underwent brain tumour follow-up MRI during the period 2 July to 16 August 2020. Data were collected on General Electric Sigma (1.5 Tesla) and Premier (3.0 Tesla) MR scanners with standard anatomical sequences . The data are from a single time-point providing one MRI examination per patient. Other than MRI examinations missing these sequences, there were no exclusion criteria.The non-brain-extracted images were registered to the brain-extracted cT1-w sequence.The images were Z-normalised (zero mean and unit variance) based on the voxel values within the brain mask.The voxel values were rescaled to integers and converted from NifTI to DICOM format for compatibility with Mirada.The image processing pipeline is illustrated in The delineations were exported from Mirada as files in DICOM-RT Structure Set (RTSS) format and then converted to NifTI binary masks corresponding to the HD-GLIO output format using a custom script.The image processing was performed on a PC  running the Ubuntu 20.04 desktop operating system. Processing from original DICOM images to HD-GLIO segmentation had a duration of approximately 10 min per subject.Reference or ground truth image segmentation of CE tumour lesions and NE lesions followed definitions provided by Kickingereder et al.   and a board-certified neuroradiologist (JC), consensus delineation was achieved and used for the analysis. A senior neuroradiologist (VL) with 23 years of experience was consulted for a final decision in cases that proved particularly difficult to segment.A and R refer to the algorithm and radiologist, respectively.We assessed the agreement between CE and NE delineations generated by HD-GLIO and corresponding radiologist delineations using Dice similarity coefficients and Bland–Altman plots, and volume agreement was assessed using concordance correlation coefficients (CCCs). The Dice similarity coefficient (Dice) is defined as in :DiceA,R=Unless explicitly stated, reported volumes represent the union volumes of the radiologist and HD-GLIO delineations providing a neutral reporting of the delineations in case of disagreement.After statistical analysis, we inspected the delineations by HD-GLIO and radiologists in every case and lesion-wise. We studied the disagreements and summarised in which situations they occurred.Sixty-six consecutive MRI examinations of individual patients were retrospectively collected as described in the previous section. Six scans were excluded due to missing sequences; thus, N = 60 scans were included in this study. Patient characteristics are summarised in 3 to 103 cm3. The case presenting with a CE tumour volume of 103 cm3 accounted for 44% of the total CE tumour volume across all patients. 18 cases presented with CE tumour volumes of less than 1.00 cm3.Contrast-enhancing tumour lesions were delineated in 31 of the 60 patients by HD-GLIO or radiologists. Size (union volume) ranged from 0.011 cm3  and together represent 2.59 cm3 or 1.11% of the total CE tumour volume across all patients.In 29 of the 60 patients, the HD-GLIO algorithm and the radiologists agreed that there were no CE tumour lesions. In 19 of the 60 patients, the HD-GLIO algorithm and the radiologists agreed on the presence of CE tumour lesions. In the remaining 12 of the 60 patients, only HD-GLIO (seven patients) or only the radiologists (five patients) identified CE tumour lesions. The CE tumour volumes of those 12 cases were all less than 1.0 cm3. For CE tumour volumes less than 1.0 cm3, the median Dice similarity coefficient was 0.00 . Dice similarity coefficients are shown in 3 ± 2.81 cm3.Tumour volume estimates are shown in 3 and 213 cm3 and with a median of 28.1 cm3. Dice similarity coefficients are shown in NE lesions were delineated in all 60 cases by both HD-GLIO and radiologists, with NE volumes (union volume) ranging between 1.34 cmWe inspected every patient with CE tumour lesion delineation (31 patients) and categorised the differences between delineations by HD-GLIO and radiologists lesion-wise. In 5 of 31 patients, HD-GLIO and radiologists agreed on the presence of all CE tumour lesions, and there were mostly minor disagreements on the spatial extent of the individual lesions.In 15 patients, one or more CE tumour lesions were not detected by HD-GLIO. All the lesions were small and could be categorised as follows: punctate (occurred in five patients), weakly and diffusely contrast-enhancing (occurred in four patients), with inhomogeneous intensities on the T1-w (occurred in three patients) or as being present in scarred regions (occurred in three patients). In nine patients, HD-GLIO delineated one or more contrast-enhancing structures that were not CE tumour lesions. The lesions were mostly small and could be categorised as follows: dural thickening or leptomeningeal enhancement (occurred in three patients), scarred tissue (occurred in three patients), plexus choroideus (occurred in two patients) or as a vessel junction (occurred in one patient). We likewise inspected the NE lesion delineation in all 60 patients. In general, there was agreement between HD-GLIO and radiologists on the locations of most NE lesions as reflected by the median Dice similarity coefficient of 0.79. However, the following types of repeatedly occurring disagreements were noticed (more than one type of disagreement is possible in each patient): In 19 patients, HD-GLIO missed NE lesioned tissue with relatively lower signal intensities compared to other regions of NE lesioned tissue in the same patient. This was seen as decreased size of the individual NE lesion delineations (occurred in eight patients) but also altogether missed NE lesions (occurred in seven patients). In 18 patients, periventricular regions of NE lesions were delineated only by HD-GLIO (6 patients) or radiologists (12 patients). The impression was that the thicker the periventricular NE lesion, the likelier HD-GLIO was to delineate it. In 6 of 15 patients with NE lesions in subcortical structures, the brainstem or cerebellum, HD-GLIO did not delineate the NE lesions altogether. 3 (median Dice = 0.86). However, if considering all cases with CE tumour volume, the performance dropped significantly (median Dice = 0.40). The findings have implications for the application of the algorithm in the routine radiological workflow where small CE tumours will constitute a considerable share of the follow-up cases.In this study, we evaluated the performance of the HD-GLIO algorithm for volumetric segmentation of brain cancer on a consecutive cohort of 66 patients that underwent routine brain tumour follow-up MRI. The algorithm performed very well on NE volumes (with median Dice = 0.79) and CE tumour volumes larger than 1.0 cm3 are, thus, close to Kickingereder et al. [The HD-GLIO algorithm was presented in the study by Kickingereder et al.  and testr et al. . However3) and 0.63 (volume < 3 cm3); i.e., as in our study, the algorithm’s performance was greatly reduced on small CE tumour volumes. In summary, previous studies on adults with external evaluation considered the preoperative setting [Only a very limited number of previous studies have evaluated deep learning brain tumour segmentation on external datasets using delineations by independent radiologists as ground truth. Drai et al.  evaluate setting ,32,33 anThe patient population of the present study included patients with recent surgery and the majority had no or only small CE tumour volumes. The patient population thus differs from the larger of two test datasets used by Kickingereder et al., the multi-centre EORTC-26101 dataset, where all patients had confirmed first progression of glioblastoma after standard chemo-radiotherapy. Except for Drai et al. , the extThe current study has some limitations. The dataset included MRI examinations of 60 consecutive patients out of which the radiologists identified CE tumour volumes in 24 patients. Yet, the dataset provided a sufficient number of complex cases to demonstrate features that caused disagreement between HD-GLIO and radiologist ground truth delineations. Regarding the ground-truth segmentation, our radiologists delineated CE tumour lesions and NE lesions mainly using a thresholding brush tool. The delineations employed by Kickingereder et al. were carried out using a semi-automatic approach in the segmentation tool itk-SNAP. Here, tumour voxels are classified in a machine-learning step followed by segmentation with active contours . Though In perspective, deep learning-based 3D segmentation of brain tumours has the potential to improve radiological response monitoring and may reduce radiologist workload. The performance of the HD-GLIO algorithm by Kickingereder et al. clearly indicates that automatic volumetry will be the future of brain tumour response monitoring. The results also indicate that it may be beneficial to train the algorithm further, possibly on a dataset acquired with a focus on including patients with small contrast-enhancing lesions. The techniques behind the algorithms transform well between different domains and radiographic modalities ,35,36. A3. However, the performance dropped for the small contrast-enhancing tumour volumes often encountered in treatment response monitoring.In this study, we evaluated the performance of the HD-GLIO algorithm for volumetric segmentation of brain cancer on a consecutive cohort of patients that underwent routine brain tumour follow-up MRI using independent radiologist ground truth segmentations. The algorithm performed well on the delineation of non-enhancing T2/FLAIR hyperintense abnormality volumes and contrast-enhancing tumour volumes larger than 1.0 cm


After the second vaccination, we observed an at least twofold increase in ELISpot responses towards gE peptides in 22 out of 29 patients (76%). Male sex, good kidney function, early time point after transplantation, and treatment with tacrolimus or mycophenolate were correlated significantly with higher VZV-specific cellular immunity, whereas diabetes mellitus was correlated with impaired responses. Thus, our data indicate that vaccination with Shingrix™ significantly augmented cellular, VZV gE-specific immunity in KTX recipients, which was dependent on several covariates.Solid organ transplant recipients have an up to ninefold higher risk of varicella–zoster virus (VZV) reactivation than the general population. Due to lifelong immunosuppressive therapy, vaccination against VZV may be less effective in kidney transplant (KTX) recipients. In the current study, twelve female and 17 male KTX recipients were vaccinated twice with the adjuvanted, recombinant zoster vaccine Shingrix™, which contains the VZV glycoprotein E (gE). Cellular immunity against various VZV antigens was analyzed with interferon-gamma ELISpot. We observed the strongest vaccination-induced changes after stimulation with a gE peptide pool. One month after the second vaccination, median responses were 8.0-fold higher than the responses prior to vaccination ( Varicella–zoster virus (VZV) is a member of the herpesvirus family that causes varicella/chickenpox after primary infection and zoster/shingles after reactivation. Viral DNA persists in neurons of the dorsal root and cranial nerve ganglia, where it can remain quiescent for decades . As all TM, GlaxoSmithKline Biologicals S.A., Rixensart, Belgien) is recommended, especially from the age of 60 and for individuals with immunodeficiency [TM could reduce the risk of contracting shingles during one’s lifetime in the general population from 33% to 3% [TM and 130 who received a placebo, a study by Vink et al. [In Germany, the United States, and many other countries, a live attenuated vaccine is licensed, and its use is recommended for vaccination against primary infection ,9. Moreoficiency . This reficiency . Previou3% to 3% . Moreove3% to 3% . Considek et al.  reportedTM, in which VZV-specific cellular immunity was monitored at four time points prior to and post vaccination. We stimulated the patient cells with peptides of glycoprotein E (gE), the most abundant and immune-dominant glycoprotein of VZV [In the present study, we report on 29 KTX recipients who were vaccinated twice with Shingrixn of VZV , with a n of VZV . MoreoveTM. The participants in this observational study were recruited at the University Hospital Essen (Germany) in August 2020 according to the inclusion and exclusion criteria outlined in 2). All patients reported previous chickenpox, and eight reported shingles. Ten patients were grafted with a living donor and 19 with a deceased donor. The patients were tested at the times of the first and second vaccination and approximately one and four months after the second vaccination. The median interval between the transplantation and first vaccination was 7.2 years, and that between the two vaccinations was 71 days.Our prospective single-center study includes 29 KTX recipients who were tested longitudinally before and after vaccination with ShingrixIn parallel, in August 2020, we included four age-matched, healthy controls . All volunteers reported previous chickenpox, and the female reported previous shingles. According to the current recommendations , healthyThe study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of the University Hospital Essen, Germany . Informed consent was obtained from all subjects involved in the study.TM contains 50 µg of the adjuvanted, recombinant VZV gE antigen produced in immortalized ovarian cells of the Chinese hamster (CHO cells) [Quillaja saponaria Molina plant extract, fraction 21 (QS-21), and 50 µg 3-O-desacyl-4′-monophosphoryl lipid A (MPL) from Salmonella minnesota. ShingrixTM is licensed for the prevention of shingles and postherpetic neuralgia in adults ≥50 years of age [The subunit vaccine ShingrixO cells) . It is as of age . Vaccina® ELISpot kit, Mikrogen GmbH, Neuried, Germany; formerly Lophius Biosciences GmbH, Regensburg, Germany). Cultures of 200,000 freshly isolated PBMCs were incubated without and with VZV antigens in 150 µL of AIMV medium  at 37 °C for 19 h. Stimulation with the T-cell mitogen phytohemagglutinin  served as positive control. Colorimetric detection of cytokine-secreting cells was performed according to the manufacturer’s instructions. Spot numbers were analyzed with an ELISpot plate reader . VZV-specific spots were determined as stimulated minus non-stimulated (background) values (spot increment). Of note, the negative controls reached a median value of 0, a mean of 0.11 spots, and a standard deviation of 0.61 spots. The positive control with PHA indicated that all results included in this study were valid .Nine milliliters of heparinized blood was collected, and peripheral blood mononuclear cells (PBMCs) were separated through Ficoll gradient centrifugation. Numbers of PBMCs were determined with an automated hematology analyzer . To assess VZV-specific cellular immunity, we performed IFN-γ ELISpot assays while using a peptide pool and two protein antigens as stimuli. In parallel experiments, we applied a gE peptide pool , a native VZV glycoprotein , and a whole native VZV antigen . The gE peptide pool contained 153 peptides derived from a peptide scan (15-mers with 11 aa overlap) through the envelope protein (Swiss-Prot ID: P09259) of the VZV strain Dumas. For the production of the two native antigens, VZV glycoprotein, and whole VZV antigen, HEL 299 cells were infected with the VZV strain Ellen. After cultivation, the antigens were isolated through lectin affinity chromatography or ultra-centrifugation through a sucrose cushion, respectively. The production of IFN-γ was determined using pre-coated ELISpot plates and a standardized detection system , interval between transplantation and first vaccination, interval between first and second vaccination, and interval between second vaccination and blood sampling as numerical variables. Moreover, sex, first vs. second kidney transplantation, living vs. deceased donor, diabetes mellitus, hypertension, coronary heart disease, previous malignant tumor, chronic obstructive pulmonary disease, previous cytomegalovirus, herpes simplex virus or VZV infection (chickenpox or shingles), previous antiviral treatment , immunosuppressive therapy , and allograft rejections  were considered as categorical, dichotomous variables (yes/no).U-test. Correlation analyses of numerical variables were performed by Spearman test (two-tailed). The impact of categorical variables was also analyzed with the Mann–Whitney test. The impact of clinical variables on ELISpot responses was furthermore tested with multivariate analysis . If not otherwise stated, median values are indicated. Results were considered significant at p < 0.05.Data were analyzed using GraphPad Prism 8.4.2.679  or IBM SPSS Statistics version 25 . The calculation of the sample size was performed with the program G*Power 3.1.9.4  using thp = 0.0006) and 4.8-fold higher than the responses after the first vaccination (p = 0.0007). However, at month 4 vs. 1 after the second vaccination, ELISpot responses already declined significantly (p = 0.01) (p = 0.03) and 1.9-fold higher than the responses after the first vaccination (p = 0.01).In 29 KTX patients vaccinated with Shingrix™ , we foll = 0.01) a. The re = 0.01) b. After  = 0.01) c. One mop < 0.05) for stimulation with the gE peptide pool at month 4 after the second vaccination  fulfilled this criterion, which we used to assess the response rate for cell-mediated immunity. The respective number for the native glycoprotein was 6 out of 29 (21%), and for the whole VZV antigen, it was 17 out of 29 (59%).r = 0.42 and p = 0.02) and to the native glycoprotein of VZV (r = 0.41 and p = 0.03), i.e., patients with a better kidney function showed higher VZV (gE)-specific ELISpot responses at baseline  and to the whole VZV antigen (r = −0.42 and p = 0.02)  c,d. Thusp = 0.03)   . Diabetictively) .p = 0.02) and for the whole VZV antigen at month 1 and month 4  , for the whole VZV antigen after the first vaccination (p = 0.01), and for all three VZV antigens at month 4 after the second vaccination .Patients treated with tacrolimus had stronger ELISpot responses after the second vaccination, reaching significance for the gE peptide pool at month 1 (ctively) . Patientctively) . The resThe remaining clinical parameters had no significant influence on VZV (gE)-specific cellular immunity. However, age tended to correlate negatively with ELISpot responses prior to and post vaccination, i.e., older patients had slightly lower ELISpot responses.2 = 54.0). Immunity towards the native glycoprotein at month 4 was similarly affected by eGFR, the interval to transplantation, and mycophenolate (χ2 = 39.7–44.4), and, to a lesser extent, by sex (χ2 = 28.4) and diabetes mellitus (χ2 = 22.9). Finally, immunity towards the whole VZV antigen at month 4 was especially affected by diabetes mellitus (χ2 = 937.3), followed by sex (χ2 = 58.9) and interval to transplantation (χ2 = 29.8).The correlation of the clinical parameters with significant results with the univariate analysis was further examined by using multivariate analysis . The VZVThe Spearman analysis in 29 KTX recipients showed that the ELISpot responses to the different VZV antigens and at the different time points were positively correlated, i.e., immunity to one VZV antigen was predictive of a response to the other two antigens, and data at the different time points were also correlated .TM could significantly increase VZV (gE)-specific cellular immunity in KTX recipients, which was detected after in vitro stimulation with a gE peptide pool and a whole VZV antigen. However, as compared to the healthy controls, the cellular responses were lower, as expected. A comparative analysis of various VZV antigens showed that vaccination-induced changes in VZV-specific immunity were most pronounced after stimulation with the gE peptide pool, where we observed an 8.0-fold increase after the second vaccination compared to the baseline. Similar results were observed in a cohort of hematopoietic stem cell transplant recipients, where the gE peptide pool was also most suitable for measuring VZV (gE)-specific vaccination responses [TM contains recombinant gE, the most abundant and immune-dominant glycoprotein expressed on the surface of VZV-infected cells [The current data indicate that vaccination with two shots of Shingrixesponses . As the ed cells , this fied cells . Previoued cells . In addied cells  and quaned cells . Similared cells . This grThere are already data on T cell immunity after vaccination with Shingrix™ in a cohort of 32 kidney transplant recipients . HoweverMoreover, vaccination with Shingrix™ had no effect on allograft function as defined by serum creatinine , which cThe interval between transplantation and testing and ELISpot results showed a negative correlation, i.e., sooner after transplantation, cellular immune responses were higher. This observation was not expected at first glance. Especially within the first years after transplantation, reactivation of herpesviruses is common , and it p < 0.0001), which could be explained by stronger VZV-specific T cell immunity. Several studies showed sex-dependent immune responses—for example, various concentrations of cytokines or vaccine antibodies [In addition, we could identify male sex as a factor correlated with increased VZV-specific immunity. Consistently with that finding, the previous literature indicated that the incidence of shingles also differed between males and females . The anntibodies ,34,35,36tibodies  or antibtibodies ,37. Howetibodies . It is, The correlation of diabetes mellitus with impaired cellular responses was expected because hyperglycemia in diabetes is thought to cause dysfunction of the immune response, which fails to control the spread of invading pathogens and makes diabetic subjects more susceptible to infections . We obseIn KTX recipients, vaccination with the adjuvanted, recombinant vaccine Shingrix™, which contains the VZV gE, led to a significant increase in in vitro cellular responses, especially towards VZV gE. This is the first study assessing vaccination efficacy in this setting with ELISpot, an assay that measures active secretion of IFN-γ upon stimulation with VZV antigens. However, as compared to age-matched controls, cellular immune responses after vaccination were weaker in kidney transplant recipients. Furthermore, we could identify sex, kidney function, time point after transplantation, immunosuppressive drugs, and diabetes mellitus as covariates of VZV (gE)-specific cellular vaccination responses; these have not yet been reported.


Vascular calcification is characterized as the deposition of hydroxyapatite mineral in the form of calcium-phosphate complexes in the vasculature. Transdifferentiation between vascular smooth muscle cells (VSMCs) and osteoblast-like cells is considered essential in the progression of vascular calcification. The pathophysiological mechanisms underlying vascular calcification and VSMC osteogenic differentiation remain to be fully elucidated, and the development of novel therapies is required. In the present study, PCR and western blot analysis were conducted to quantify the mRNA and protein expression levels of calcification-associated markers  and adropin in VSMCs and rat vascular tissues. The calcification of VSMCs was assessed using alizarin red staining. Moreover, adropin expression levels in VSMCs were analyzed using immunofluorescence. Lentiviral transfection and small interfering RNA were used for overexpression and knockdown of adropin in VSMCs, respectively. The results demonstrated that adropin alleviated vascular calcification in vivo. Moreover, adropin also inhibited osteogenic differentiation and the calcification of VSMCs in vitro. Notably, results of the present study revealed that the tyrosine protein kinase JAK2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway played a key role in the aforementioned inhibition. In conclusion, the results of the present study demonstrated that adropin inhibited VSMC osteogenic differentiation to alleviate vascular calcification via the JAK2/STAT3 signaling pathway. Vascular calcification is defined as the deposition of calcium phosphate mineral in the cardiovascular system, mainly in blood vessels, myocardium, and cardiac valves . PatholoAdropin is a highly conserved peptide hormone , which is mainly secreted from the liver. Results of a previous study demonstrated that adropin may play a key role in the regulation of the vascular endothelium . Moreoveβ-glycerophosphate-induced VSMC calcification via JAK2/STAT3 signaling [Protein kinase JAK2 (JAK2) is an upstream kinase in the signal transducer and activator of transcription 3 (STAT3) phosphorylation pathways . Lu et aignaling . He et aignaling . These sIn the present study, we hypothesized that adropin treatment may exert a positive effect in the treatment of vascular calcification and the JAK2/STAT3 signaling pathway may be involved. The inhibitory impact of adropin on vascular calcification was investigated in an animal model and in osteogenic differentiated VSMCs in the present study. The findings of the present study may provide a novel theoretical basis for further understanding the molecular mechanisms associated with the protective effects of adropin in vascular calcification. They may help us to develop new methods and medicine based on adropin and JAK2/STAT3 signaling to treat vascular calcification diseases, such as atherosclerotic disease, hypertension, diabetes, and chronic kidney diseases.2 at 37°C. The medium was changed every 2 days.The human VSMC line was obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. 1640 medium  supplemented with 20% fetal bovine serum  was used to culture VSMCs. VSMCs were seeded into T-75 flasks and incubated in a humidified incubator supplied with 5% COβ-Glycerophosphate and 1 M CaCl2) was used for osteogenic differentiation in VSMCs. VSMCs were cultured in osteogenic differentiation medium for 8-10 days to obtain an osteoblast-like phenotype. The culture medium was changed every day.Osteogenic differentiation medium  was used to visualize target proteins. The primary and secondary antibodies used are listed in Supplementary Table QuickBlock™ immunostaining blocking reagent (Beyotime Institute of Biotechnology) was used to block nonspecific binding sites, and 3% HAlizarin red staining was performed using the Alizarin Red Staining kit  to determine the extent of matrix calcification. The protocol was performed in accordance with the manufacturer's instructions. Briefly, cultured VSMCs were fixed with 4% paraformaldehyde. A total of 1 ml staining reagent was added to every well of a 6-well plate for 60 min. Subsequently, the staining reagent was washed off using ice-cold PBS. All images were captured using an optical microscope (Zeiss AG), and all results are presented as the relative integrated optical density IOD (sum/area sum).ALP activity was measured using an ALP Color Development kit (Beyotime Institute of Biotechnology). Briefly, VSMCs were incubated with ALP-labeled antibodies and washed 3-5 times for 4-6 min each time. Subsequently, each solution was washed off. Following the last wash, a BCIP/nitrotetrazolium chloride blue (NBT) dye working solution was added, and the sample was incubated at room temperature for 30 min. All results are presented as the relative IOD (sum/area sum).μm PVDF membrane . Subsequently, specific primary antibodies were used for overnight incubation at 4°C. The following day, the membranes were washed 3-5 times and incubated with the secondary antibodies at room temperature for 1.5 h. Proteins were visualized using chemiluminescence reagents . Band intensities were quantified using Image-Pro Plus 6.0 , and GAPDH was used for normalization. The primary antibodies and secondary antibodies used for western blot analysis are detailed in Supplementary Table Radioimmunoprecipitation assay (RIPA) buffer  was used to extract proteins from VSMCs or rat vascular tissues. The amount and quality of the protein were checked by bicinchoninic acid assay (BCA)  in a Synergy H1 Microplate Reader . Diluted protein was incubated for 5 min at 100°C, and then equal amounts of protein were loaded on a 12% gel from a TGX Stain-Free FastCast Acrylamide Kit  and finally transferred onto a 0.45 ΔΔCT- method, and GAPDH was used for normalization. The primers used for RT-qPCR are listed in Supplementary Table Briefly, total RNA from VSMCs or rat vascular tissues was extracted using an DNA/RNA/Protein Extract kit , and cDNA was synthesized using a reverse-transcribed kit  on a Mastercycler X50 (Eppendorf). Then, qPCR was performed using ChamQ SYBR Color qPCR Master Mix (Vazyme) in a ViiA 7 system . The relative expression levels of target genes were calculated using the 22 for 6 h, the medium was replaced with DMEM  containing 10% FBS . Following transfection, the cells were harvested after 48 h for RNA extraction.To inhibit the adropin expression, cultured VSMCs at 70-80% confluency were transfected with a specific siRNA (100 nM) or scrambled siRNA (100 nM) using Lipofectamine® 3000 . Genomeditech  assisted in the design and production of siRNA. Following incubation at 37°C with 5% COThe adropin overexpression was carried out using a lentiviral vector. Negative control vectors and adropin lentiviral vectors were synthesized by GeneChem, Inc. Briefly, VSMCs were cultured into 6-well plates and transfected with the lentivirus vector using Lipofectamine® 3000  according to the manufacturer's instructions. The medium was changed every 6 h, and VSMCs were cultured for 72 h at 37°C.SD rats  were obtained from the Shanghai Model Organisms Center. All rats were housed in an environment with a temperature of 23 ± 1°C, a humidity of 50 ± 10%, and a light/dark cycle of 12/12 h. Rats were given access to standard rat food and water ad libitum. Animal handling and treatment were performed in compliance with Chinese national guidelines. Ethical approval for all experimental protocols was issued by the Animal Care Committee of The Second Affiliated Hospital of Soochow University .To establish the vascular calcification model, rats were injected with vitamin D3  and simultaneously administrated with nicotine (25 mg/kg in 3 ml peanut oil) at 9 : 00 a.m. on the first day. The nicotine administration was repeated at 5 : 00 p.m. on the same day, and the modeling continued for 8 weeks. Rats in the control group received an injection of saline and oral gavage of peanut oil. Rats in the adropin group were administered 50 mg/kg adropin intragastrically  each day. After 8 weeks of treatment, all rats were injected with ketamine /medetomidine  and euthanized by cervical dislocation. The vascular tissues were collected for subsequent analyses.p < 0.05 was considered to indicate a statistically significant difference.Statistical analysis was performed using SPSS 21.0 Software (IBM Corp.). One-way ANOVA followed by a Bonferroni post hoc test was performed to examine the quantitative data. Data are presented as the mean ± standard deviation. The expression levels of adropin were analyzed in the progression of vascular calcification. A C57/BL6 rat model was developed with vascular calcification for in vivo investigation, and VSMCs were stimulated for osteogenic differentiation for in vivo investigation. As shown in Figures As adropin was expressed at low levels in osteogenic differentiated VSMCs, the impact of adropin in the calcification of VSMCs was subsequently explored. Adropin was silenced using siRNA and overexpressed using plasmid transfection in VSMCs Figures . SubsequAs the JAK2/STAT3 signaling pathway may regulate VSMC calcification in vitro , 11, wheThe calcification of VSMCs was visualized using alizarin red staining. Compared with the control group, the alizarin red positive area was significantly increased in osteogenic differentiated VSMCs and VSMCs with adropin expression knocked down . HoweverTo elucidate the inhibitory impact of adropin in the JAK2/STAT3 signaling pathway, entry of STAT3 into the nucleus was evaluated in each group of VSMCs. Results of the present study demonstrated that the protein expression levels of STAT3 in the nucleus were decreased in osteogenic differentiated VSMCs following adropin overexpression Figures . HoweverThe role of the JAK2/STAT3 signaling pathway in osteogenic differentiation was investigated following modulation of the adropin expression. Notably, the results of the present study demonstrated that the mRNA expression levels of calcification-associated markers  in VSMCs were decreased following adropin knockdown or treatment with AG490 ) in vascular tissues were significantly decreased following treatment with adropin , Runx2, and  Figures . This re Figures . Moreove Figures  and 2. IVascular calcification is characterized by the deposition of hydroxyapatite mineral in the form of calcium-phosphate complexes in the vasculature . At presAdropin was initially identified in 2008, as a protein encoded by the energy homeostasis associated gene . In receOur research also found that adropin was downregulated in in vivo and in vitro calcification models, which caught our interest, as some undetected mechanisms might regulate the content of adropin in cells or animal models. Some upstream of adropin had already been reported recently. For example, Li and Xie found that HDAC11-mediated histone deacetylation may inhibit the level of adropin to suppress atherosclerosis . In the α proteins and cell cycle-associated proteins, and the hyperproliferation of VSMCs [Previous studies reported that the JAK2/STAT3 axis regulated the cellular metabolism of VSMCs. For example, Han et al. demonstrated that cytokine-induced apoptosis inhibitor 1 accelerated the vascular remodeling of VSMCs via JAK2/STAT3 in vitro . Hossainof VSMCs . Based oof VSMCs . Wang etof VSMCs . STAT3 iof VSMCs . For futIn conclusion, to the best of our knowledge, the present study was the first to demonstrate the specific therapeutic mechanism of adropin in vascular calcification. Notably, the results of the present study demonstrated that adropin alleviated vascular calcification in vivo. Moreover, adropin may also inhibit the osteogenic differentiation and calcification of VSMCs in vitro, and the JAK2/STAT3 signaling pathway may play a key role in this process. Thus, these findings may be beneficial for further elucidating the molecular mechanism associated with the protective effects of adropin in vascular calcification.


Tumours are complex systems with dynamic interactions between tumour cells, non-tumour cells, and extracellular components that comprise the tumour microenvironment (TME). The majority of TME’s cells are cancer-associated fibroblasts (CAFs), which are crucial in extracellular matrix (ECM) construction, tumour metabolism, immunology, adaptive chemoresistance, and tumour cell motility. CAF subtypes have been identified based on the expression of protein markers. CAFs may act as promoters or suppressors in tumour cells depending on a variety of factors, including cancer stage. Indeed, CAFs have been shown to promote tumour growth, survival and spread, and secretome changes, but they can also slow tumourigenesis at an early stage through mechanisms that are still poorly understood. Stromal–cancer interactions are governed by a variety of soluble factors that determine the outcome of the tumourigenic process. Cancer cells release factors that enhance the ability of fibroblasts to secrete multiple tumour-promoting chemokines, acting on malignant cells to promote proliferation, migration, and invasion. This crosstalk between CAFs and tumour cells has given new prominence to the stromal cells, from being considered as mere physical support to becoming key players in the tumour process. Here, we focus on the concept of cancer as a non-healing wound and the relevance of chronic inflammation to tumour initiation. In addition, we review CAFs heterogeneous origins and markers together with the potential therapeutic implications of CAFs “re-education” and/or targeting tumour progression inhibition. According to the American Cancer Society, in 2022 there will be an estimated number of 1.9 million new cancer cases diagnosed and 609,360 cancer deaths in the United States. According to these data, cancer is, and will continue to be, one of the worst diseases in the world. Numerous people continue to pass away from cancer every year, despite major recent improvements in our understanding of the molecular and genetic causes of the disease [According to the “seed and soil” hypothesis, a suitable host microenvironment (the soil) is necessary for the best growth of tumour cells (the seed) . Many reIt is becoming more and more obvious that every tumour is different in some way, and that the way a tumour interacts with its surroundings has effects that appear to vary between tumour kinds and even between individuals with the same tumour type. There are still many unsolved issues about the connection between CAFs and the tumour, despite intriguing findings showing that CAFs depletion causes delayed tumour growth and enhanced response to treatment . Do theyDormant or resting fibroblasts are detected in areas of interstitial stroma between functional parenchymal layers in adult and well-differentiated tissues. They are described as thin, elongated cells with frontal and posterior extensions and fusiform shapes. From a molecular point of view, quiescent fibroblasts remain poorly defined ,9. In re2 and N2, respectively, and can easily react with other molecules in a cell) produced by activated inflammatory cells cause deoxyribonucleic acid (DNA) damage and genomic instability [Tumour stroma, in fact, shares many similarities with non-healing wounds, including inflammation, tissue formation, and remodelling . In facttability ,21. Furttability . Inflammtability ,24. Moretability ,26,27. Itability ,29. The tability ,31.The cancer cell secretome, which includes factors such as TGF-β, platelet-derived growth factor (PDGF), and fibroblast growth factor 2 (FGF2), recruits stromal cells during tumourgenesis ,49 Tabl. The mosSignals from other cells in the TME, as well as tumour cells, may instruct CAF function ; for exaFurthermore, the fibrotic environment offers benefits for optimal tumour development, such as the formation of a physical barrier to immune surveillance, which allows for tumour growth . CAFs alAlthough all fibroblasts present in a tumour are defined as CAFs, they come from diverse cell sources . We haveMany studies support the idea that mesenchymal stem cells (MSCs) are recruited and induced by the developing tumour to enhance proliferation and to acquire a CAFs-like phenotype . MSCs maA recent study found that loss of E-cadherin in tumour buds increased VIM expression and CAFs activation, all of which are signs of EMT and are associated with more aggressive tumours . CAFs, oWe can agree that a clear correlation exists between tumour initiation, fibroblast corruption, and tumour progression, so identifying CAFs markers could help to determine the stage of tumour development and the initiation of the pre-metastatic niche . HoweverIn the first place, we identify αSMA, a cytoskeleton protein associated with TGF-β production, with a highly contractile phenotype , which iAnother highly accepted marker is the fibroblast-specific protein 1 (FSP-1 or S100A4). This marker is also not only limited to CAFs but can be found in epithelial cells undergoing EMT ,171 and Further, Vimentin (VIM), a type III intermediate filament protein that maintains cell integrity and is involved in cell migration, motility, and adhesion ,175, whiTenascin-C (TN-C), a member of ECM glycoproteins, is markedly up-regulated in pathologic tissues that are undergoing remodelling, such as those that are suffering inflammation, wound healing, or tumour progression ,178. In Periostin (POSTN), a matricellular N-glycoprotein, is implicated in the EMT process, and is crucial for the collagen fibrillogenesis process ,185 and The tissue inhibitor of metalloproteinase-1 (TIMP-1) has been shown to co-express in fibroblasts with α-SMA, and thus is a candidate to become a CAFs marker . In factOsteopontin (OPN), a chemokine-like phosphorylated glycoprotein released intracellularly, is typically elevated in a variety of human malignancies . It is eThe next candidate is FAPα, a type II integral membrane serine protease of the prolyloligopeptidase family. FAPα has been reported to be overexpressed in CAFs in many types of carcinomas, including colorectal, ovarian, breast, bladder, and lung carcinomas ,194. FAPPlatelet-derived growth factor receptors (PDGFR) are cell surface tyrosine kinase receptors for members of the PDGF family. PDGFRα and PDGFRβ are high molecular weight membrane proteins (around 180 kD) which dimerize non-covalently upon ligand binding. PDGFRα is a marker for fibroblasts in some tissues like skin , whereasSDF-1 is a homeostatic chemokine that is also known as CXCL12 . FibroblFinally, Prostaglandin E2 (PGE2), known to be produced by immune cells, is also produced by malignant stromal cells like CAFs, and it promotes angiogenesis, induces metastasis, and increases tumour cell survival and proliferation ,203,204.Normal fibroblasts appear to have a double effect on cancer cells. Fibroblasts act against malignant progression as early as during tumourigenesis ,209, butInterestingly, a recent study found that CAFs are capable of reverting lung cancer spheroids from a dysplastic to a hyperplastic state by knocking out Sox2 activity. Therefore, CAFs are capable of modifying intrinsic oncogenic characteristics and influencing tumour development . SLITs aInjecting a teratoma cell line into an early mouse embryo demonstrated that healthy cells have tumour suppressor properties; the resulting postnatal animals were derived from both host cells and transplanted tumour cells, but the mouse presented normal development with no sign of specific features of tumour cells ,214. DorIn solid tumours, not only do CAFs remain persistent and autocrine activated, but they also stimulate neoplastic cells in a paracrine manner ,219, accCAFs increase the secretion of MMPs as observed in studies of head and neck squamous cell carcinoma (HNSCC), highlighting their crucial role in cancer progression and early stages of tumour growth . For exaThe role of tumour stromal fibroblasts in carcinogenesis was studied using an immunodeficient mouse tumour xenograft model in which tumour cells were implanted alongside CAFs, homologous fibroblasts extracted from non-cancerous tissue from the same individual, or normal fibroblasts extracted from healthy donors . TumoursIn addition, CAFs support invasion and metastasis by modelling the ECM , increasInterestingly, the key role of CAFs as chaperons in metastasis is becoming more widely accepted. CAFs facilitate intravasation and extravasation and escort and sustain neoplastic cells within the blood stream on their way to metastatic sites ,236,237 Furthermore, major mediators of breast cancer metastasis to the lung include metastasis-associated fibroblasts (MAF) expressing FSP-1, TN-C, and VEGF-A . The actFinally, CAFs’ support to CSCs has also been proven. Chen et al. recently found that CAFs, through IGF-II and IGF1-R intercommunication, can improve lung cancer root plasticity through mechanisms dependent on Nanog , a transCAFs are beginning to take the lead rather than play a supporting role in immune modulation in recent years. The CAFs secretome may vary dynamically during the evolution of cancer by supporting tumour immunity at various stages of the tumour, but it does not contribute to a state of prolonged activation of the defensive level ,244.CAFs can affect the host immune response both directly and indirectly, according to Knops et al.’s (2020) study. By sustaining an abnormal pro-tumour inflammatory milieu, they can hinder or abolish the cytotoxic actions of T and NK cells ,246. In The subtype known as CAFs-S1, which is identified by the expression of FAPα, αSMA, PDGFR, and CD29 markers, was found to be associated with the recruitment, retention, and differentiation of Treg cells, enhancing their capacity to inhibit CD4+CD25+ T-cell proliferation in vitro. This was revealed by a FACS-based analysis of CAFs in human breast tumours. Additionally, it was able to prolong T-cell life and trigger their differentiation into regulatory lymphocytes with the CD4+CD25+FOXP3+ phenotype . This prMore recently, it has been observed that CAFs can lead to activation of CD4+ T cells and suppression of CD8+ T cells . TargetiCAFs-derived CXCL14 affects macrophage recruitment to the tumour  and shunIn a mouse model of transplantable colorectal carcinoma (CRC), it was found that fibroblasts expressing high levels of FAPα recruited myeloid cells via CCL2, which resulted in resistance to anti-PD-1 immune checkpoint therapy that was eliminated by targeting the FAPα. This is significant because it indicates that myeloid cell recruitment by CAFs is also associated with resistance to therapy. These results were confirmed in tissue samples from human CRCs, where the presence of fibroblasts with high FAPα+ concentration was positively connected with myeloid cell infiltration and negatively correlated with T-cell infiltration ,259. SimTumour stroma is not only implicated in all stages of cancer development and its immune resistance, but it can also function as an interstitial barrier, preventing effective drug delivery and promoting radioresistance ,266. FibCAFs’ immunological regulation, proangiogenic effects, and metabolic reprogramming might promote cancer cell survival and make it easier for the disease to evade the effects of therapy. For the first time, it was discovered in 2009 that breast cancer patients with high stromal gene expression did not respond well to neoadjuvant chemotherapy because of an elevated intratumour interstitial fluid pressure that subtly reduced antineoplastic medication uptake . NotablyAdditionally, CAFs’ released soluble components have been linked to resistance to anticancer treatment therapy. One example is the HGF, which has been linked to prostate cancer and is thought to play a critical role in CAF-mediated resistance to therapy with tyrosine kinase inhibitors or EGFR receptors ,81 Figu. ColorecThe idea that there are several factors involved in chemo/radiotherapy resistance that go beyond the tumour cells is on the rise. The physical barrier implied by the TME, with not only the proteins that form the stroma but with the interactions of the different cells that play a role on it, is a key example in which CAFs are protagonists. Thus, a detailed and personalized characterization of CAFs and stromal profiles is essential for the detection of novel targets to optimize therapeutic strategies while maintaining the survival of healthy cells and preventing the emergence of resistance . Since tRecently, new therapeutic strategies aimed at “re-educating” CAFs into a non-tumour associated status have been developed . The useOther approaches consist of the use of TGF-β inhibitors. The dual role of TGF-β in tumour progression is clear, acting as a suppressor at early stages and as a promotor later on. This functional duality could be demonstrated by comparing the effect of TGF-β signalling pathways in tumour cells of transgenic mice where it suppressed initial tumourigenesis but promoted the final metastatic process . DetaileRecent research has examined the use of miRNAs as a potential method to deactivate CAFs ,117 Fig. MiR-19aOn the other hand, miR-145, an inhibitor of the TGF-β pathway, was upregulated in both CAFs produced by TGF-β signalling and CAFs from primary oral cancer, but not in healthy fibroblasts. Prior to TGF-β therapy, normal oral fibroblasts were transfected with miR-145, which inhibited their activation to the CAFs phenotype. After being converted to CAFs by TGF-β, they were transfected with miR-145, and this resulted in the restoration of their quiescent phenotype, indicating that normal fibroblasts may already have a built-in negative feedback loop to guard against CAF conversion . AnotherAnother therapeutic approach has been focusing on therapies to inhibit paracrine signalling of CAFs, for example, inhibitors of the HGF/MET signalling pathway, hedgehog, or angiotensin II receptor showed a reduction in the expression of CAFs and ECM content, thus enhancing drug delivery and inhibiting tumour growth and metastasis  Figure . HGF is The stromal tumour feedback requires the overexpression of PDGF, as was already mentioned. Additionally, as shown by gene targeting experiments, this signalling also supports angiogenesis in an autocrine and paracrine manner ,71, and Inhibiting the mTOR pathway in pancreatic CAFs with the somatostatin analogue SOM230 results in reduced secretion of IL-6, which stimulates chemoresistance, mediated by CAFs, and potentiated pancreatic tumour drug sensitivity . Other tOn the other hand, more drastic strategies have been proposed with the aim of eliminating or diminishing CAFs population. In this regard, FAPα inhibitors have shown promising preclinical results in reducing tumour progression and promoting an antitumour immune response . In xenoGPR77 is an additional cell surface marker that has been researched in pre-clinical models. It has been demonstrated that this protein, along with CD10, can be used to pinpoint the fibroblast subpopulation that results in chemoresistance. It has been demonstrated that blocking GPR77 with a neutralizing antibody lowers the number of CSCs in tumours and improves the response to chemotherapy  Figure . FurtherThese findings suggest that rather than eliminating CAFs entirely, effective anti-CAFs medicines for cancer would likely need to re-educate them towards a NAF phenotype or decrease CAFs number. Another factor to consider is the heterogeneity of CAFs subpopulations and, hence, current studies should aim at differentiating CAFs subtypes in order to therapeutically affect pro-tumoural CAFs and support antitumour CAFs. Notably, Olive et al. investigated whether co-delivery of the synthetic experimental medication IPI-926 , which sFurthermore, an enzymatic degradation of the ECM to decompress the tumour and to normalize vasculature has been proposed to promote a more efficient chemotherapeutic delivery to cancer cells in solid tumours. In this respect, hyaluronic acid (HA) degradation or anti-angiogenic therapies are under consideration. Examples include, among others, the angiotensin inhibitor losartan  and enzyMMPs derived from CAFs have also demonstrated their importance in inducing tumour growth, metastasis, and angiogenesis in animal model experiments . TherefoCAFs reprogramming, as well as short-term inhibition of CAFs-predominant signalling pathways, have been recognized as better strategies than complete ablation or chronic inhibition. Indeed, several studies have found that strategies aimed at CAF ablation are ineffective ,302.Finally, the diverse therapeutic approaches emerging from basic and applied research are promising and encouraging. Unfortunately, because of the variability in the role of CAFs in each tumour, as well as their specific tissue source, they must be targeted at each tumour histotype. As a result, any future therapy targeting CAFs will be more effective after extensive research on their taxonomy and a precise and detailed understanding of the stroma–tumour relationship.Quiescent fibroblasts expand upon activation in response to stimuli released by damaged organs and emerging inflammation, generating growth factors and ECMs to autoregulate their expansion as well as to regulate inflammation and immunity. For decades, activated fibroblasts were thought to be cancer cells’ accomplices in promoting tumour growth. However, fibroblasts appear to act as involuntary regulators of tumour growth, either positively or negatively, depending on the origin of the fibroblast on one hand, and on the other hand, the context in which it is found, such as the stage of tumour progression .To make things worse, CAFs exhibit remarkable heterogeneity and plasticity, allowing CAFs from the same tumour to arise through different pathways, have different phenotypes, and have functionally different effects on tumour cells and the microenvironment. Nonetheless, it is clear that controlling, reducing, or re-educating the CAFs subpopulation within the tumour is critical to improving current treatments. CAFs are appealing drug targets for a variety of reasons, including: (i) they are genetically more stable than neoplastic cells, making them less prone to developing resistant phenotypes due to high mutation rates and clonal selection; (ii) they have epigenetic differences from normal stromal cells; (iii) they are responsible for the structure of the tumour ECM; and, finally, (iv) CAFs support neoplastic cells throughout the disease spectrum, from pre-invasive lesions to the metastatic focus. Thus, by targeting CAFs, different stages of the disease, as well as numerous pathophysiological processes such as angiogenesis, EMT, and immune evasion, could be influenced.All in all, the true role of tumour stroma in the invasion and metastasis of cancerous lesions remains unclear. In fact, experimental findings have supported both a protective and a permissive role against tumour development depending on the stage of tumour progression. Therefore, it is more than possible that fibroblasts are both “heroes” and “villains”, and it is necessary to investigate, in depth, the different factors involved in these interactions during the different tumour stages and to determine what causes fibroblasts to switch from one team to another and at what time.


Pigmented dermatofibromasarcoma protuberans (DFSP) is a rare variant of DFSP which is an intermediate-grade tumour due to its tendency for local reccurence. Morphology of this variant impose a differential diagnosis that is must know for a pathologist. We describe two cases that presented with pigmented papulo-nodular form involving back and lower limb in middle aged to an elderly female. Histopathology coupled with immunohistochemistry yielded daignosis of Bedner tumour. A surgical histopathologist needs to have knowledge of all differential of pigmented neoplasm of skin just to ensure not to skip this rare entity. It is a slowly growing, locally aggressive tumour which shows frequent recurrences. Dermatofibrosarcoma protuberans comprises 1% of skin malignancies and usually occurs in 3 infancy . It accoPatients’ information: patient-1 is a 34-year-old female, presented with pigmented nodular swelling in the left lower limb for the last 6 years. Patient-2 is a 70 year-old-female presented with the dermatology outpatient department (OPD). The lesions were in form of multiple tender nodules which were firm to soft in consistency. The patient also gave a history of excision of a similar nodule 1 year back. However, no histopathology report could be found. There is no family history of similar lesions in both patients.Clinical findings: patient-1 showed a papulo-nodular, pigmented, firm, tender swelling with restricted mobility  (ry (IHC) . A panelry (IHC) .Therapeutic interventions: as a measure of therapeutic intervention a wide local excision/excisional biopsy with one c.m normal margins was done in both patients.Follow-up and outcome: both patients were discharged postoperative day one with no complications. Patients were called for follow-up after one week.Patients’ perspective: patients were satisfied with the treatment given and thankful to the treating clinician.Informed consent: written consent was obtained from both patients.et al. reported six dermoscopic features: delicate pigment network (87%), vessels (80%), structure less light-brown areas (73%), shiny white streaks (67%), pink background coloration (67%) and structureless hypopigmented or depigmented areas (60%) [Dermatofibrosarcoma protuberans is a dermal tumour which also infiltrates into the deeper subcutaneous tissue, fascia, muscle and sometimes even bone . Initialas (60%) . In our et al. [Microscopically, DFSP presents as a single well-circumscribed proliferation of spindle cells with mild nuclear pleomorphism and low mitotic count. The tumour cells are arranged in short irregular fascicles and storiform patterns similar to classical dermatofibrosarcoma but with scattered melanosome-containing dendritic cells. Certain unusual morphological features like prominent meningothelial- like whorls have also been reported in a case of Bednar tumour by Wang et al. .Other rare variants of DFSP include myxoid DFSP contains abundant myxoid stroma. This type of tumour is uncommon, presents a diagnostic challenge and is important to recognize in order to prevent both under- and over-treatment. Giant cell fibroblastoma, referred to as juvenile DFSP affects children and adolescents, and is characterized by giant cells in the tumour . It appeA high degree of clinical suspicion is required for diagnosing it at an early stage. Hence to overcome the usual delay in diagnosing this entity, Bednar tumour should be considered as a differential diagnosis in any long-standing, slowly growing pigmented skin tumour or nodular lesion, which does not spontaneously resolve. Bednar tumor may sometimes undergo fibrosarcomatous transformation with rare examples of pulmonary metastasis . Bednar Bednar tumour needs to be recognized by the practising pathologist to differentiate from other cutaneous spindle cell neoplasm and close follow-up is always needed due to its frequent recurrence.


Whether early fluid accumulation is a risk factor for adverse renal outcomes in septic intensive care unit (ICU) patients remains uncertain. We assessed the association between cumulative fluid balance and major adverse kidney events within 30 days (MAKE30), a composite of death, dialysis, or sustained renal dysfunction, in such patients.We performed a multicenter, retrospective observational study in 1834 septic patients admitted to five ICUs in three hospitals in Stockholm, Sweden. We used logistic regression analysis to assess the association between cumulative fluid balance during the first two days in ICU and subsequent risk of MAKE30, adjusted for demographic factors, comorbidities, baseline creatinine, illness severity variables, haemodynamic characteristics, chloride exposure and nephrotoxic drug exposure. We assessed the strength of significant exposure variables using a relative importance analysis.Overall, 519 (28.3%) patients developed MAKE30. Median (IQR) cumulative fluid balance was 5.3 (2.8–8.1) l in the MAKE30 group and 4.1 (1.9–6.8) l in the no MAKE30 group, with non-resuscitation fluids contributing to approximately half of total fluid input in each group. The adjusted odds ratio for MAKE30 was 1.05 (95% CI 1.02–1.09) per litre cumulative fluid balance. On relative importance analysis, the strongest factors regarding MAKE30 were, in decreasing order, baseline creatinine, cumulative fluid balance, and age. In the secondary outcome analysis, the adjusted odds ratio for dialysis or sustained renal dysfunction was 1.06 (95% CI 1.01–1.11) per litre cumulative fluid balance. On separate sensitivity analyses, lower urine output and early acute kidney injury, respectively, were independently associated with MAKE30, whereas higher fluid input was not.In ICU patients with sepsis, a higher cumulative fluid balance after 2 days in ICU was associated with subsequent development of major adverse kidney events within 30 days, including death, renal replacement requirement, or persistent renal dysfunction.The online version contains supplementary material available at 10.1186/s13613-022-01040-6. Fluid resuscitation is cornerstone treatment of haemodynamic instability during the early phase of sepsis . ConventExperimental data propose a possible link between fluid accumulation and renal parenchymal oedema, which may induce congestion and impaired glomerular filtration rate –10. ThisIn addition to fluid volume accumulation, fluid composition and adjunct ICU therapies may contribute to adverse renal outcomes. For example, data from large randomized trials suggest that fluid resuscitation with hydroxyethyl starch , 6 or chSince, greater illness severity is associated with the administration of larger fluid volumes, more intense cardiovascular support, and greater use of potentially nephrotoxic drugs, the relative importance of fluid accumulation with respect to adverse renal outcomes is an important clinical question that remains uncertain. Accordingly, we conducted an observational multi-centre study to assess the independent association between early fluid accumulation and major adverse kidney events, a composite of death, dialysis, or sustained renal dysfunction, within 30 days (MAKE30) in ICU patients with sepsis. In addition, we aimed to assess the relative importance of fluid accumulation and other potential factors for MAKE30 development. We hypothesized that the degree of fluid accumulation after 2 days in ICU would be associated with subsequent MAKE30 after controlling for illness severity, level of cardiovascular support, and nephrotoxic drug use in such patients.The study was approved by the Stockholm Ethical Review Board  with a waiver of informed consent.We conducted a multicenter, retrospective cohort study of patients admitted to five ICUs in three hospitals in the Stockholm Region between June 2005 and August 2018. We included adult (16 years or older) patients admitted with a diagnosis of sepsis  severe sepsis (ICD-10 code R65.1) or septic shock (ICD-10 code R57.2) on their first ICU admission. We excluded patients with end-stage renal disease and patients who died, who were discharged or who received renal replacement therapy within the first two days in ICU. We also excluded patients without data on fluid balance or creatinine.We collected data from the electronic ICU patient data management system Centricity Critical Care  and the hospital electronic health record system Take Care . From the Centricity Critical Care data warehouse, we included demographic data, admission diagnoses and comorbidity data (ICD-10 codes), information on administered intravenous fluids, enteral and parenteral nutrition, blood products and drugs (including continuous drug infusions), total daily fluid input and output, arterial blood gas data, laboratory data, timing of invasive mechanical ventilation and renal replacement therapy initiation, and invasive mean arterial pressure (MAP) data (recorded every second minute on average). From the Take Care data warehouse, we included information on pre-admission and post-discharge creatinine and death date.Primary exposure was the cumulative fluid balance  during the first two days in ICU (exposure period). We also included baseline exposure variables  and the following variables collected during the exposure period: variables reflecting illness severity , exposure to potentially nephrotoxic drugs , level of systemic inflammation (C-reactive protein level), and other variables potentially associated with both the primary exposure and outcome . Definition and categorization of exposure variables are presented in Additional file The primary outcome was major adverse kidney events occurring after the exposure period until 30 days after ICU admission (MAKE30). MAKE30 was defined as the composite of death, initiation of renal replacement therapy, or sustained renal dysfunction (a last inpatient plasma creatinine level [within 30 days] ≥ 200% of the baseline level) , 20. We The secondary outcome was the composite of renal replacement therapy initiation or sustained renal dysfunction.p-values were reported. We considered all exposure variables presented in Additional file p value less than 0.05 was considered statistically significant.The association between the log-odds for suffering MAKE30 and the cumulative fluid balance during the exposure period was estimated using univariable and multivariable logistic regression analysis. The robust sandwich estimator was used to estimate standard errors and two-sided A total of 4033 patients were admitted to ICU with a diagnosis of sepsis (according to the ICD-10 coding system) between June 2005 and August 2018. We excluded 2199 patients who were discharged or received renal replacement therapy within the first 2 days in ICU, had end-stage renal disease, or had missing creatinine or fluid balance data. Therefore, we included 1834 patients . Total urine output was lower in the MAKE30 group whereas total fluid input was similar in the two groups (Table p < 0.001). Patients with MAKE30 were more likely to receive a noradrenaline infusion rate above 0.20 µg/kg/min (p < 0.001) and to receive inotropic support (p = 0.005), vancomycin (p = 0.03), furosemide (p = 0.006), and/or invasive mechanical ventilation (p = 0.002). In addition, highest blood lactate and bilirubin levels were higher, whereas lowest platelet count was lower in the MAKE30 group  patients met the secondary outcome event (102 survivors and 64 non-survivors) and 1668 (90.9%) were event free (1315 survivors and 353 non-survivors) , defined as a composite of death, dialysis, or sustained renal dysfunction. This finding was independent of baseline patient characteristics, degree of illness severity, cardiovascular support, duration of MAP below 65 mmHg, chloride exposure, nephrotoxic drug exposure, and invasive ventilatory support. On relative importance analysis of independent variables, the strongest factors regarding MAKE30 were baseline creatinine, cumulative fluid balance, and age. The second main finding of the study is that the administration of non-resuscitation fluids significantly contributes to the early fluid input and cumulative fluid balance in septic ICU patients. The third main finding of the study is that the urine output component of the cumulative fluid balance rather than the fluid input component is responsible for the association with MAKE30.Our findings are to some extent, but not entirely, in line with previous studies. Observational data suggest a positive relationship between greater fluid accumulation in the ICU and death among sepsis patients –25, but This lack of robust evidence underpins the debate on whether fluid accumulation per se contributes to adverse outcomes or is simply a marker of the degree of illness severity. Assessing the effect of fluid accumulation on renal outcomes is particularly challenging as AKI in itself is a major contributor to fluid gain (risk of reverse causation). The potential problem of death as competing risk for adverse renal events should also be acknowledged. In the present study, we set out to minimize these issues by (a) assessing an exposure period (first 2 days in ICU) to allow sufficient fluid accumulation before the outcome was allowed to occur; (b) considering potentially important patient-related, treatment-related, and illness-severity-related confounders in the analysis; (c) including death in the composite outcome (MAKE30) as recommended by consensus statements . Our anaDuring the exposure period, approximately half of the total fluid input came from non-resuscitation fluids, a finding consistent with observations from other cohorts. For example, in a recent study including 1229 sepsis patients from one ICU in Belgium, non-resuscitation fluid administration was responsible for more than 50% of the cumulative fluid balance after 48 ICU hours . FinallyImportantly, the higher cumulative fluid balance in our MAKE30 group was a consequence of lower urine output rather than greater fluid input. Moreover, in our adjusted sensitivity analysis, lower urine output was independently associated with higher odds of MAKE30, whereas the OR for fluid input remained insignificant. Low urine output is a physiological consequence of pending AKI, which in itself is linked to MAKE30  and/or non-resuscitation fluids  and/or efforts to increase fluid removal.Our study has strengths. We studied a previously unexplored association between early fluid accumulation and MAKE30 in adult ICU patients with sepsis. We used data from five ICUs, from three large university hospitals, involving almost 2000 patients, which provides a degree of external validity for applying our findings to similar settings. In addition, as to present the strengths of independent associations between exposure variables and outcomes (using adjusted odds ratios), we also provide estimates of the importance of exposure variables. Finally, our findings were consistent in sensitivity analyses lending robustness to our findings.Our study has limitations. First, it is an observational study and can only describe associations. Second, we used ICD-diagnoses of sepsis rather than Sepsis-3 criteria. Our approach likely underestimated the true sepsis incidence. In fact, recent Swedish data indicate that only one-third of septic ICU patients received the sepsis diagnosis at discharge . Third, In adult patients with sepsis admitted to five ICUs in Sweden, a higher cumulative fluid balance after two days in ICU was independently associated with major adverse kidney events within 30 days, including death, renal replacement requirement, or persistent renal dysfunction.Additional file 1: Detailed list of exposure variables; Filtering and processing of mean arterial pressure data; Description of Variable Importance analysis.Additional file 2:Figure S1. Cumulative fluid balance by admission year, Figure S2. Restricted cubic spline curves, Table S1. Backwards selection logistic regression adjusted for early AKI, Table S2. Backwards selection logistic regression adjusted for fluid input and urine output, Table S3. Forward selection logistic regression (association with MAKE30), Table S4. Backwards selection logistic regression , Table S5. Forward selection logistic regression .