Hepatocyte growth factor and its receptor cMet activate biological pathways necessary for repair and regeneration following kidney injury. Here, we evaluated the clinical role of urinary cMet as a prognostic biomarker in diabetic nephropathy (DN). A total of 218 patients with DN were enrolled in this study. We examined the association of urine cMet levels and long-term outcomes in patients with DN. The levels of urinary cMet were higher in patients with decreased renal function than in patients with relatively preserved renal function . A fully adjusted model revealed that a urinary cMet cutoff of 2.9 ng/mL was associated with a hazard ratio for end-stage renal disease of 2.33 . The addition of urinary cMet to serum creatinine and proteinuria provided the highest net reclassification improvement. We found that in primary cultured human glomerular endothelial cells, TGFβ treatment induced fibrosis, and the protein expression levels of collagen I, collagen IV, fibronectin, and αSMA were decreased after administration of an agonistic cMet antibody. In conclusion, elevated levels of urinary cMet at the time of initial diagnosis could predict renal outcomes in patients with DN. However, an adverse prognosis is not inevitable in patients with overt nephropathy. Some patients do not develop end-stage renal disease (ESRD) or die. Developing new ways to identify patients with a good prognosis from those with poor prognostic outcomes remains important for the management of individuals with diabetic nephropathy (DN).The presence and severity of chronic kidney disease (CKD) are the strongest predictors of adverse outcome in individuals with diabetes7. CKD nevertheless progresses in a notable percentage of patients with type 2 diabetes. Thus, an accurate test that complements current medical approaches in predicting CKD progression is necessary.Several recent articles have highlighted the potential importance of some molecules as biomarkers for progressive kidney disease9. The antifibrotic effect of HGF has been studied extensively in many animal models of CKD12.Hepatocyte growth factor (HGF) is a key cytokine that plays a critical role in the regulation of cell proliferation, wound healing and tissue fibrosis13. These pleiotropic events have generated profound interest regarding the potential therapeutic role of the HGF/Met pathway in animal models of kidney injury. Recent in vivo evidence also supports a role for the HGF/Met pathway as an antifibrogenic factor because injection of recombinant HGF protein or the HGF gene led to reduced renal myofibroblast activation and diminished tubulointerstitial fibrosis15.cMet, the tyrosine kinase receptor for HGF, has also been shown to be critical for cell survival via stimulation of an array of downstream signaling pathways, resulting in cell proliferation, spreading, migration, and inhibition of apoptosis16; moreover, HGF could predict all-cause mortality and cardiovascular mortality in the general population17. Several studies showed that urinary HGF is not only a prognostic biomarker in CKD19 but also useful in clinical risk prediction for recovery after acute kidney injury (AKI)21, especially in critically ill patients22 and kidney transplant recipients23.A recent study demonstrated that elevated serum HGF levels in the presence of protein-energy wasting increased all-cause mortality risk in ESRD patientsTo the best of our knowledge, there are currently no data regarding the prognostic role of urinary cMet in CKD, including CKD manifesting due to DN. In this study, we aimed to identify the association between soluble cMet levels in urine at the time of the initial DN diagnosis and clinical manifestations. We also evaluated the role of urinary soluble cMet in the progression of renal function and patient survival.2 as calculated by the CKD-EPI creatinine equation. The random urine protein creatinine ratio was 3.8 ± 4.6 g/g.The baseline characteristics of the study participants are shown in Table Urinary soluble cMet levels as measured by enzyme-linked immunosorbent assay (ELISA) were significantly higher in patients with CKD stage V than in patients with CKD I, II, III, or IV P < 0.001); this trend remained unchanged after urinary soluble cMet levels were adjusted for urinary creatinine  were purchased and employed. A series of two-fold dilutions were made using the calibrator provided in the kit starting from the highest concentration on the calibration curve (50 ng/ml). A total of 11 concentrations were prepared, and each concentration ranging from 0~50 ng/ml was run six times in a single assay to verify its accuracy and establish a coefficient of variation (CV) for precision. As shown in Supplementary Tables To measure the concentration of soluble cMet protein present in a human urine sample with high accuracy and precision, a commercially available soluble cMet ELISA kit was used. To confirm the validity of the kit for our human urine study, several parameters such as the lower limit of quantification (LLOQ), dilution linearity, and parallelism were analyzed based on the FDA’s draft guidance document Bioanalytical Method ValidationTo examine the matrix effect of human urine samples obtained from the patients with kidney disease, dilution linearity and parallelism were investigated at the laboratory scale (see Supplementary Table 2) compared with patients with relatively preserved renal function (≥30 ml/min/1.73 m2)  patients were diagnosed with ESRD, and 65 (29.7%) patients died. To investigate the association between the urinary soluble cMet concentration and ESRD or mortality, we performed a Kaplan-Meier survival analysis. The risk of ESRD Fig.  and all-The independent effect of soluble urinary cMet on renal and patient outcomes was examined by using multivariate Cox proportional hazard models Table . ElevateNext, we determined whether urine soluble cMet/Cr is a better indicator of ESRD than serum Cr and UPCR by comparing the receiving operating characteristic (ROC) curves (Delong test), integrated discrimination improvement (IDI) index and the category-free net reclassification improvement (cfNRI) index. The AUCs (95% CI) of Cr (model 1), Cr + UPCR (model 2) and Cr + UPCR + cMet model 3) were 0.858 (0.808–0.907), 0.813 (0.755–0.870), and 0.890 , respectively. The addition of urine soluble cMet/Cr to serum Cr or serum Cr + UPCR statistically improved the predictive value for ESRD and the composite outcome but not death  expressing cMet were abundant in the healthy glomerulus Fig. . Next, wing Fig. . During ing Fig. .Figure 4in vitro experiments. To assess the role of the HGF/Met pathway in kidney cell fibrosis, we treated primary cultured human GECs with TGFβ to induce fibrosis. Remarkably, the protein expression levels of collagen I, collagen IV, fibronectin and αSMA were upregulated after TGFβ treatment indicating that fibrosis was adequately induced. GECs treated with TGFβ and either cMet Ab  are correlated with renal function. More importantly, we report that increased urine soluble cMet levels are significantly associated with renal survival and mortality. Inclusion of urinary soluble cMet in the fully adjusted ESRD models improved their predictive power with three different statistical methods . Therefore, urine soluble cMet levels at diagnosis may serve as a novel biomarker to predict the renal progression in DN.25.More than 380 million people currently suffer from diabetes. The International Diabetes Federation estimated that this would rise to 592 million people within a generation. Despite the continuous increase in the prevalence of type 2 diabetes worldwide, there has been a gradual decline in mortality from macrovascular complications such as stroke and coronary heart disease owing to the advent of potent statins. However, this is not the case in DN because end-stage renal disease (ESRD) remains a global leading cause in individuals with DN, and its incidence is still rising. Notably, once nephropathy develops, approximately 20–40% of patients inevitably progress to ESRD. Therefore, we need biomarkers that would enable early risk stratification in DN26. Of the emerging candidate biomarkers, serum cystatin C4, fibroblast growth factor 23 (FGF23)7 and soluble tumor necrosis factor (TNF) receptors2 have provided the most promising data.In the past, persistent microalbuminuria was the most studied biomarker in DN. Both the presence and incremental changes in microalbuminuria have been shown to correlate with the development and progression of chronic kidney disease (CKD) in type 1 and type 2 diabetes9. Recombinant HGF treatment reversed fibrotic changes induced by streptozocin injection. rHGF also repressed TGFβ production, which plays a key role in renal fibrosis in glomerular mesangial cells even under hyperglycemic conditions. Moreover, intravenous administration of HGF not only attenuated urine protein excretion but also mitigated mesangial expansion, reduced collagen deposition, and prevented interstitial myofibroblast activation in type I27 and type II28 DN mouse models. A recent study demonstrated that HGF enhances β-cell proliferation and ameliorates hyperglycemia by maintaining compensatory hyperinsulinemia29. In the present study, administration of either agonistic cMet Ab or rHGF in primary cultured GECs downregulated the expression of fibronectin, collagen I, collagen IV and αSMA, which was consistent with the results of previous studies. Recently, the endothelial-to-mesenchymal transition (EndMT), in which endothelial cells acquire mesenchymal characteristics, has also been recognized as a crucial mechanism of renal fibrosis31, especially in diabetic nephropathy mouse models33. Therefore, regulation of endothelial dysfunction in the kidney might contribute to attenuate renal fibrosis and further experiments with DN mouse models to explore the efficacy of cMet agonistic Ab is needed in the future.The HGF/Met pathway has been shown to play a critical role in glomerular and tubulointerstitial fibrosis in a diabetic mouse model16, but the role of soluble cMet levels in predicting renal failure has not been studied in detail. In addition, there are no data regarding urinary soluble cMet levels. Previous studies have shown that soluble form of cMet could be generated via shedding and cleavage by γ-secretase: Sheddases or metalloproteinases cleave full-length cMet within its extracellular domain, resulting in different in a soluble extracellular N-terminal fragment (Met-NTF). We have demonstrated that cMet expression is increased in GECs, therefore it is reasonable to infer that soluble cMet in the urine is originated from the kidney35. Urine is a useful source of protein for biomarker analysis because it can be obtained by non-invasive methods. Therefore, using urine samples to identify novel biomarkers may have clinical implications. However, urine specimens demonstrate a high degree of variability in volume, protein concentration, total protein excreted, and pH and can degrade upon storage37. To account for these potential issues, we validated the cMet ELISA kit with several parameters such as the LLOQ, dilution linearity, and parallelism.Several studies have reported that serum HGF is associated with increased mortality in patients with CKDIn our study, urinary cMet/Cr levels at the time of DN diagnosis were significantly correlated with UPCR and negatively correlated with eGFR. However, cMet/Cr was predominantly increased in CKD stage V patients, but there was no difference in the other CKD stages. These results suggest that cMet filtration is simply increased as kidney function deteriorates. To confirm this hypothesis, we adjusted the cMet levels with the urinary creatinine levels.Compared with conventional prognostic markers such as serum creatinine and UPCR, cMet alone showed moderate utility as a biomarker in the ROC analysis. To improve the power of ESRD prediction, we evaluated the additive predictive performance of urine cMet/Cr at the time of DN diagnosis. Patients who developed ESRD had relatively high levels of urinary soluble cMet/Cr.38. In our cohort, 45% of participants developed ESRD during follow-up (n = 99 patients out of 218). This might be due to the relatively longer duration of the follow-up period (31.0 ± 22.4 months). Another strength is that this is the first article to show the prognostic effect of urinary soluble cMet, and we validated the ELISA kit for the LLOQ and the matrix effect of human urine samples.Modern improvements in diabetes care and cardiovascular survival have allowed many patients with diabetes to live long enough to require renal replacement. One key strength of this study is the large number of ESRD events in our cohort compared with previous studies of patients with DNHowever, the present study has some limitations. First, biopsy samples of DN were insufficient. Only some of the patients had biopsy-proven DN, so the time point of for the initial DN diagnosis might be not consistent. Since the diagnosis of DN is made by clinical judgment rather than histological examination, this problem is be a common limitation of DN studies. Nevertheless, this is the first article to prove the predictive effect of urinary soluble cMet in DN. Second, there were no data regarding the serum levels of soluble cMet. Identifying whether there exists any correlation between serum and urinary soluble cMet levels would be helpful, and studies addressing this are expected in the future.In conclusion, elevated levels of urinary soluble cMet at the time of initial diagnosis could predict renal outcome in patients with DN. The results suggest a critical role of cMet in renal fibrosis in CKD patients, especially those with DN.The present study was conducted with the approval of the Research Ethics Committee of the Seoul National University Boramae Medical Center. All procedures were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was obtained to human research participants for the use of blood, urine samples as well as tissue samples.In this study, 218 patients with clinically diagnosed DN were enrolled between January 2009 and December 2016. Among 218 patients, 20 patients had biopsy-proven disease. Patients under 18 years or who were planning to undergo kidney transplantation were excluded. Demographic and clinical characteristics at the time of diagnosis, including age, gender, body mass index (BMI), systolic and diastolic blood pressure, and comorbidities  were extracted from electronic medical records.Laboratory values, including complete blood cell counts, hemoglobin and serum levels of AST, ALT, albumin, urea, creatinine, calcium, phosphorus, cholesterol, glucose, and HbA1c, were collected. Urinary protein and creatinine levels were also collected, and the urinary protein-to-creatinine ratio (UPCR) was calculated.39.Laboratory values were determined using the following instrumentation: a Modular D2400 analyzer with ISE900 module  and a cobas 8000 modular analyzer . The eGFR was calculated by the CKD-EPI creatinine equationA soluble cMet ELISA  kit was obtained from Thermo Fisher Scientific Inc. . The ELISA was performed according to the manufacturer’s instructions. The LLOQ for soluble cMet was 0.78 ng/ml.The clinical outcomes assessed were ESRD, all-cause mortality and a composite of the two outcomes. ESRD was defined as an irreversible loss of renal function with the need to start dialysis or to undergo renal transplantation. The mortality data of patients who were lost to follow-up were obtained from the National Statistical Office database.After kidneys were fixed with paraformaldehyde (4%) and paraffin embedded, they were sliced into 4-μm sections and stained. The sections were then deparaffinized in xylene and rehydrated in a graded series of ethanol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase in methanol for 30 minutes at room temperature. Sections were microwaved for 30 minutes in an antigen unmasking solution for antigen retrieval. After the sections were incubated with 10% goat serum for 1 hour at room temperature, they were incubated at 4 °C with anti-Met (cMet) antibody  and anti-Met (cMet) (phospho Y1349) antibody (Abcam) overnight. Tissues were washed several times in phosphate-buffered saline (PBS) and incubated for 40 minutes with Alexa Fluor-conjugated secondary antibodies . 4′,6-Diamidino-2-phenylindole  was used to counterstain the nuclei. The negative control sections underwent the same treatment regimen except for the omission of primary antibodies. Sections were visualized using confocal microscopy with a Leica TCS SP8 STED CW .40. After 3 days of culture, cells were detached from the dishes with a 3 mM EDTA solution and a minimal amount of trypsin. Cells (2 × 105/well) were seeded on 8-well chamber slides with serum-free medium for 24 hours and then washed twice with PBS. Next, recombinant TGFβ  was added at a final concentration of 10 ng/ml except where otherwise indicated. Cells were incubated in the presence or absence of either recombinant HGF (10 and 20 mg/mL) or agonistic cMet antibody (250 and 500 ng/mL) for 48 hours.We isolated GECs from normal adjacent kidney tissue from patients with renal cell carcinoma according to guidelines approved by The Institutional Review Board of Seoul National University Hospital (IRB no. 1404-117-515). Kidneys were obtained from freshly sacrificed mice perfused through the heart with cold Hank’s balanced salt solution (HBSS), and glomeruli were prepared by a serial sieving method. Minced renal cortex tissue was serially passed through 160, 120 and 100 μm mesh stainless steel screens, and glomeruli were collected using a 75 μm mesh screen. The glomeruli, which contain small debris from the renal tubule, were suspended in HBSS , washed twice by brief centrifugation  and digested in 1.5 mg/ml collagenase  for 40 minutes at 37 °C with occasional vortexing. Undigested glomeruli were pelleted by brief centrifugation, and the supernatant containing a single-cell suspension of GECs was resuspended in growth medium  and plated on fibronectin-coated 35-mm dishes . Small colonies of GECs were observed within 1 week after plating. To remove contaminating mesangial and epithelial cells, brief trypsinization was performed until a culture with 98.1% endothelial cells was achieved. The cells were maintained in collagen-coated 100-mm culture dishes 4 cells/well) in medium supplemented with 10% FBS and left to grow into a confluent monolayer. To assay the basal cell migration ability of GECs, the confluent cell monolayer was serum-starved for 12 hours, after which a wound was scratched on the monolayer using a p20 pipette tip. The cell-free area was measured immediately (0 hours) and 30 hours after the scratch. To study the GEC response to cMet Ab and rHGF treatment, we cultured cells in medium supplemented with 10% FBS for 48 hours and then serum-starved the cells for 6 hours. The cells were subsequently treated with DMSO (control), medium containing cMet Ab (500 ng/mL), or rHGF (50 ng/mL). To visually assess viability, DAPI (Molecular Probes) was added, and cells were observed with a high-content imaging device  at different time points following treatment.GECs from the 2nd or 3rd passage were seeded into a 96-well plate . The kidney homogenate was centrifuged at 12,000 g for 30 min at 4 °C, and the protein concentration of the supernatant was determined by the Bradford method. Different amounts of extracted protein were separated by SDS-PAGE and transferred onto Immobilon-FL 0.4-μm polyvinylidene difluoride membranes (Millipore). Tris-buffered saline containing 0.1% Tween 20 was used as the wash buffer. After the membranes were treated with primary antibodies, anti-rabbit  and anti-mouse  secondary antibodies were used as appropriate. Detection of labeled proteins was performed with an enhanced chemiluminescence system . The band intensities were analyzed using a gel documentation system .GECs were lysed in RIPA buffer [50 mM Tris·HCl, pH 7.3; 150 mM NaCl; 0.1 mM EDTA; 1% (vol/vol) sodium deoxycholate; 1% (vol/vol) Triton X-100; 0.2% NaF; and 100 µM NaImmunoblotting was performed using primary antibodies against collagen-1 (Abcam), collagen-4 (Abcam), fibronectin , αSMA (Abcam), and β-actin .The patients were classified into two groups using either a urinary of cMet cutoff of 2.9 ng/ml or a cMet/Cr cutoff of 4.9 ng/mg, which were calculated with ROC curves. Categorical variables, which were expressed as frequencies and proportions, were compared using chi-square tests. After testing for normality, normally distributed continuous variables were expressed as the mean ± standard deviations and were compared using either Student’s t-test or one-way ANOVA test. The non-normally distributed variables were expressed as medians  and were compared using the Mann-Whitney U or Kruskal-Wallis test.To investigate the impact of urinary cMet or cMet/Cr levels on ESRD and mortality, Kaplan-Meier survival curves as stratified by the levels of urinary cMet or cMet/Cr were compared with log-rank tests.Cox proportional hazard models with time-fixed urinary cMet or cMet/Cr levels at the time of DN diagnosis were used for multivariate survival analyses. Significant covariates identified in the univariate analysis and clinically important covariates were included in the final multivariable-adjusted analysis, which was conducted in a backward stepwise manner. The adjusted covariates for each model were as follows: Model 1, age, sex, hypertension, Hb, albumin, AST, ALT, uric acid, cholesterol, Ca, and P; and Model 2, age, sex, hypertension, Hb, albumin, AST, ALT, uric acid, cholesterol, Ca, P, GFR, and PCR. HR, hazard ratio; CI, confidence interval.et al.41. Additionally, changes in the predictive accuracy of the models were evaluated by calculating the relative IDI and the cfNRI as described by Pencina et al.42.To examine the incremental prognostic value before and after inclusion of urinary soluble cMet levels to the traditional variables such as serum creatinine and the UPCR, we calculated the statistical significance of the difference between the areas under the two ROC curves (AUCs) with the method described by DeLong P value less than 0.05 indicated statistical significance.All statistical analyses were performed using SPSS version 22  and R . A Supplementary Information


Delayed neuropsychiatric sequelae (DNS) are serious complications of carbon monoxide (CO) poisoning that adversely affect poisoned patients' quality of life as well as socioeconomic status. This study aimed to determine clinical predictors of DNS in patients with CO poisoning. This retrospective study included all CO-poisoned patients admitted to the emergency department (ED) of Linkou Chang Gung Memorial Hospital in Taiwan from 1 January 2009 to 31 December 2015. The medical records of all patients with CO poisoning were carefully reviewed, and relevant data were abstracted into a standardised form. Univariate and multivariate logistic regression models were used to identify predictors of DNS after CO poisoning. Receiver operating characteristic (ROC) curve analysis was used to determine the ideal cut-off value for continuous variables that predict the development of DNS. A total of 760 patients with CO poisoning were identified during the study period. Among them, 466 were eligible for the analysis of predictors of DNS. In multivariate analysis, Glasgow Coma Scale <9 , transient loss of consciousness , longer duration from CO exposure to ED presentation , and corrected QT (QTc) prolongation  were found to be associated with a higher risk of DNS. The area under the ROC curve (AUC) for QTc interval measured within 6 h after exposure best predicted the development of DNS, with a result of 0.729 (95% CI 0.660–0.791). Moreover, the best cut-off value of the QTc interval was 471 ms, with a sensitivity of 53.3% and a specificity of 85.1%. We identified several potential predictors of DNS following CO poisoning. Among them, QTc prolongation found within 6 h after exposure is a novel predictor of DNS, which may be helpful in the future care of patients with CO poisoning. Carbon monoxide (CO) is a colourless, odourless, and highly toxic gas that is produced during incomplete carbon combustion. CO poisoning can occur following the inhalation of partially combusted charcoal briquettes, fuel gas, or oil in a poorly ventilated place. Domestic exposure can occur through incomplete combustion in gas-furnace water heaters, through inhalation of exhaust gas from automobiles, or in fire accidents , 2.http://dep.mohw.gov.tw/DOMHAOH/cp-332-8883-107.html).Since 2002, CO poisoning has been one of the leading causes of death by suicide in Taiwan. According to the National Suicide Database of Taiwan, the number of deaths by suicide using ‘gases in domestic use' or ‘other gases and vapours' has risen year-by-year, peaking in 2006. The number of deaths from burning charcoal has declined since 2012 after the government enacted regulation on the sale of charcoal (Most patients with CO poisoning present to the emergency department (ED) with nonspecific symptoms such as headache, dizziness, malaise, asthenia, nausea, vomiting, flu-like syndrome, transient loss of consciousness, altered mental status, chest tightness, palpitation, and dyspnoea. Life-threatening cardiac arrhythmia, seizures, and coma may also develop in cases of severe CO poisoning. In the absence of a clear medical history, the aforementioned manifestations may create diagnostic challenges for emergency physicians , 4.c oxidase and platelet haemoprotein. All the aforementioned mechanisms can induce a chain reaction of inflammation that increases the production of reactive oxygen species and induces oxidative stress, lipid peroxidation, and apoptosis [In addition to causing acute morbidity and mortality, CO poisoning can cause delayed neuropsychiatric sequelae (DNS) that occur days or weeks after initial complete clinical recovery from acute poisoning . The mecpoptosis –7.  [The incidence of DNS following CO poisoning varies widely from 3% to 46%, and the lag time ranges between 2 days and 6 weeks after exposure –11. Beca  . Thus, tStudies have revealed various clinical predictors of DNS following CO poisoning, including age >36 years, long exposure to CO, higher COHb levels (>25%), damage in the globus pallidus or white matter at initial ED presentation, Glasgow Coma Scale (GCS) score <9, seizures, systolic blood pressure <90 mmHg at ED presentation, elevated creatine phosphokinase concentration, leukocytosis, and a positive Babinski reflex , 8–14. H+ channel, which increases the late inward Na+ current, resulting in prolongation of the action potential and the associated intracellular Ca2+ transients, subsequently causing disruption of repolarisation and prolongation of the QT interval. However, no study has determined the relation between acute ECG alterations and DNS [In acute CO poisoning, numerous electrocardiogram (ECG) alterations, such as P-wave dispersion, corrected QT (QTc) dispersion, and QTc prolongation, have been reported , 15–18,  and DNS . To elucThe study protocol was approved by the Institutional Review Board of Chang Gung Medical Foundation (104-7628C) and was granted permission by the Medical Ethics Committee of Chang Gung Memorial Hospital.A retrospective medical record review was conducted for all CO-poisoned patients (ICD-9-CM code as 986) admitted to the ED of Linkou Chang Gung Memorial Hospital from 1 January 2009 to 31 December 2015. COHb levels were measured using an arterial blood gas analyser with a CO oximeter. Patients enrolled into this study were required to meet at least one of the following criteria: (1) a COHb level of >5% in nonsmokers and >10% in smokers at presentation and (2) delayed presentation with an unambiguous history of CO poisoning, such as being found in a confined space with burning charcoal, exposure to car or machine exhaust, or living in a place with a faulty water heater. The medical records of all patients were reviewed, and information on clinical symptoms and laboratory findings on admission to the ED, circumstances of exposure, treatment received, clinical outcomes, and other information available at the ED were abstracted.All patients were provided with 100% oxygen from a “nonrebreathing” facemask (NRM) as soon as CO poisoning was suspected at the ED or prehospital setting when attended by emergency services. Information on the following variables was collected on admission to the ED: age; sex; psychiatric history; referral institution; intentional or accidental exposure; CO exposure source; vital signs; Taiwan Triage and Acuity Scale (TTAS); concomitant use of tranquilisers; transient loss of consciousness; duration of loss of consciousness; duration from CO exposure to ED admission; evidence of myocardial injury (defined as electrocardiographic signs of ischaemia/arrhythmias and cardiac enzyme elevation); ECG; images such as brain computed tomography (CT); treatment modality (hyperbaric oxygen therapy(HBOT) at 2.5 atmosphere absolute for 90 minutes/session or 100% normobaric oxygen therapy through NRM); number of sessions of HBOT; duration from CO exposure to HBOT administration; and length of hospital and intensive care unit stay. The results of blood tests on ED visit and admission, including complete blood cell count, arterial blood gas analysis, COHb level, troponin I, creatine phosphokinase, and serum alcohol level, were also collected. Data on urine benzodiazepine and illicit drug screening were abstracted as well.ECG findings were further analysed to calculate the QTc interval. The QTc interval is the measured QT interval corrected for heart rate using Bazett's formula (QT/RRK). QTc prolongation has been defined as follows: longer than 440 ms in men and longer than 460 ms in women , 20.https://www.uhms.org/carbon-monoxide-poisoning/carbon-monoxide-poisoning.html).All CO-poisoned patients received 100% oxygen supplement through NRM upon ED arrival. Some patients received HBOT after consultation with an HBOT specialist following the management guideline of CO poisoning of the American Undersea and Hyperbaric Medical Society, 13th Edition  curve analysis. Finally, the proportion of QTc prolongation in patients with and without DNS stratified by duration from CO exposure to ECG recording was compared using Fisher's exact test. Data analysis was conducted using MedCalc Statistical Software version 13.1.2 . All odds ratios (ORs) were reported with relevant 95% confidence intervals (CIs). P < 0.05 was considered statistically significant.Demographic and clinical data of the patients with and without DNS were compared using Fisher's exact test for categorical variables or the Mann–Whitney  (Supplementary Table 1). A total of 294 patients were excluded because we were unable to confirm or exclude the diagnosis of DNS. Among the excluded patients, 19 were in a persistent vegetative state or unresponsive wakefulness; 12 died during ED admission; 3 were discharged from ED in a moribund status upon family's request; and contact was lost with 260 after discharge from the ED.A total of 760 patients with CO poisoning who met the inclusion criteria of this study were admitted to the ED of Linkou Chang Gung Memorial Hospital from 2009 to 2015. Among them, 466 patients were eligible for further DNS detectionTable 2).Among the 466 included patients, almost half were men , and the median age was 33 years (interquartile: 22–45 years). A total of 223 patients (47.9%) were transferred to our ED from another medical institution; 190 patients (40.8%) had been attempting suicide. Inappropriately ventilated gas from heating appliances was the most common cause of CO poisoning , followed by charcoal burning . Most patients presented with clear consciousness upon arrival, and 62  had poor consciousness with a GCS of <9. More than half had experienced transient loss of consciousness after CO poisoning , and most patients presented with loss of consciousness for <6 h . The median initial COHb level upon admission was 10.1% , and slightly over half of patients had leukocytosis . Laboratory test results revealed that 64 (18%) patients had myocardial injury. The median time from CO exposure to ED admission and to ECG recording was 4.0 and 5.1 h, respectively. A 12-lead ECG revealed that 42.1% of patients had QTc prolongation. A total of 279 (59.9%) received HBOT, with a median total of 3 sessions (P = 0.005); psychiatric history (P = 0.047); poor consciousness level at ED presentation and GCS <9 at admission (P < 0.001); higher TTAS (P = 0.014); intentional poisoning (P < 0.001); charcoal burning (P < 0.001); concomitant tranquiliser use (P = 0.015); transient loss of consciousness and longer duration of loss of consciousness (P < 0.001); longer duration from CO exposure to ED admission; and ECG revealing QTc prolongation. Laboratory test results revealed that the presence of myocardial injury (Trop-I > 0.5 ng/mL) and leukocytosis (WBC > 10.000/mL) was also associated with DNS  (Table 2).Among the 466 patients with adequate follow-up period to detect DNS, DNS diagnosis was confirmed in 62 (13.3%), with a median duration from CO exposure of 10 days (interquartile: 7–24 days). Univariate analysis identified the following risk factors for DNS: old age  (Table 3).Variables withTable 4 displays summaries of the ROC curves for the discrimination ability of QTc for DNS development stratified by duration from CO exposure to ECG recording. The result revealed that the discrimination ability was significant and acceptable for durations <2 h (AUC = 0.747), 2–3 h (AUC = 0.740), and 4–5 h (AUC = 0.743). The discrimination ability was poorer for durations of 6–7 h and ≥8 h.Figure 1 plots the ROC curve of QTc for discriminating DNS within 6 h from exposure to ECG recording. The relevant AUC was 0.729 , and the best cut-off point was 471 ms, with a sensitivity of 53.3% and a specificity of 85.1%, a positive predictive value of 23.5%, and a negative predictive value of 95.5%. Similarly, we found that the proportion of QTc prolongation in patients with and without DNS was statistically significant in periods <2 h , 2–3 h , and 4–5 h  from CO exposure to ECG recording .Neurological deficits and cognitive disorders impede the ability of patients to exercise self-care as well to participate in occupational and social activities; therefore, such conditions can affect the lives of not only patients but also their families . SeveralThis study aimed to identify predictors of DNS after CO poisoning, which may be of use to emergency physicians. Furthermore, to the best of our knowledge, this retrospective study enrolled the largest number of patients to date for any such study.The severity of CO poisoning is determined by the duration of CO exposure and the concentration of CO in the atmosphere, but not by the COHb level at admission , 21–23. In this study, we determined that GCS <9, transient loss of consciousness, longer duration from CO exposure to ED admission, and ECG revealing QTc prolongation were independent predictors of DNS in CO-poisoned patients . Among tA longer duration from CO exposure to ED admission indicates prolonged CO exposure or a longer interval from exposure cessation to hospital treatment. Longer exposure to CO is one of the determinants of severity, and a longer interval from CO exposure to ED admission implies a delay in treatment with any type of oxygen supplement such as normobaric oxygen or HBOT. in vitro studies, CO poisoning resulted in elevated NO levels in myocytes, causing S-nitrosylation of the Na+ channel and increasing the late component of the inward Na+ current. This subsequently resulted in prolongation of the action potential and the associated intracellular Ca2+ transients, leading to disruption of ventricular myocyte repolarisation and QT prolongation [Acute CO poisoning frequently affects the repolarisation of myocardium electrophysiology, resulting in abnormal ECG findings such as QTc prolongation and QTc dispersion , 19, 24.ongation . Studiesongation . HoweverQTc interval and its dispersion were dynamic parameters for CO poisoning in the present study. QTc prolongation was found at admission and resolved significantly between 6 and 24 h after admission . Our datThis study has several limitations. First of all, there has been a steady decline in the number of follow-up visits after discharging a patient from the ED, which has resulted in a decreased ability to detect DNS and might have adversely affected the statistical power of this study. Secondly, we adopted a symptom-based diagnostic criteria of DNS because of the lack of standard ones. Previous studies employed bundle battery of test , FolsteiDNS is the most serious complication after patient recovery from acute CO poisoning. DNS represents a challenge to emergency physicians because no established criteria exist for determining the risk of DNS in the ED setting. We conducted a retrospective study to identify independent predictors of DNS after CO poisoning. The results revealed that GCS <9, transient loss of consciousness, longer duration from CO exposure to ED presentation, and ECG revealing QTc prolongation are independent predictors for DNS of CO poisoning. Of these factors, ECG revealing QTc prolongation within 6 hours after CO exposure is first identified as a predictor of DNS. Early detection of CO-poisoned patients with a high DNS risk at the ED may help physicians plan better therapeutic strategies and arrange suitable follow-up treatment for the patients.


By applying Adeno-Cre to pancreatic duct cells in vitro, cells self-immortalized and established tumors in immunocompromised mice. When Adeno-Cre was administered to the main pancreatic duct in vivo, pigs developed extensive PDAC at the injection site hallmarked by excessive proliferation and desmoplastic stroma. This serves as the first large animal model of pancreatic carcinogenesis, and may allow for insight into new avenues of translational research not before possible in rodents.Although survival has improved in recent years, the prognosis of patients with advanced pancreatic ductal adenocarcinoma (PDAC) remains poor. Despite substantial differences in anatomy, physiology, genetics, and metabolism, the overwhelming majority of preclinical testing relies on transgenic mice. Hence, while mice have allowed for tremendous advances in cancer biology, they have been a poor predictor of drug performance/toxicity in the clinic. Given the greater similarity of  These respective technologies continue to provide tissue specific recombination and gene inducibility, while allowing for more clinically relevant models of non-syndromic cancers. By employing such model systems, mice have offered invaluable and unprecedented insight into the etiology of diseases that generally present in late stages, such as pancreatic ductal adenocarcinoma (PDAC)2. However, while mice have inarguably allowed for tremendous advances in cancer, they have considerable limitations as a disease model due to vastly different physiology3, and the conceptual leap from mouse to human has been a culprit in the failure of many promising preclinical therapies.Genetically modified mouse models have revolutionized cancer research, allowing for faithful and reproducible histotypes closely representing those observed in human patients. This is particularly true in recent years, largely attributed to the availability of mix-and-match systems such as Cre/lox and tTA/TREsus scrofa) is now emerging as a viable model for cancer and many other human diseases. While the mouse (Mus musculus) genome is approximately 14% smaller than that of humans (2.5 Gb compared to 2.9 Gb)4, the sus scrofa genome is more similar at 2.7 Gb6. Furthermore, the linkage conservation between humans and pigs is considerably more extensive than that between humans and mice7. As a result, pigs are more closely related to humans with respect to anatomy, physiology, and immunology8, and have the potential to more accurately represent a variety of human diseases, particularly those affecting the pancreas.The most obvious of these limitations is the mouse anatomy. The average human is roughly 2,500–3,000 times the size of an average mouse, severely limiting the use of mice for studies focused on surgical or radiologic interventions. Importantly, the domestic pig  has been used extensively and is considered a gold standard within the field. We sought to generate a similar histoype in sus scrofa pigs to provide a more physiologically and anatomically relevant model. We first generated pigs harboring analogous KRASG12D and TP53R167H mutations, which are observed in approximately 95% and 50% of human PDAC patients respectively12. After demonstrating the ability of these transgenes to induce adenocarcinomatous transformation of pig duct cells ex vivo, we administered an Adenoviral Cre Recombinase (Adeno-Cre) to the pig pancreas in vivo. Following refinement of the delivery system, these mutations successfully induced pancreatic neoplasms and cancer in the pig, providing the first documented large animal model of pancreatic carcinoma.Currently, the most widely used mouse model of pancreatic cancer consists of concurrent KRASG12D-TP53R167H pigs were generated as previously described13.LSL-KRASG12D-TP53R167H transgenic line. LSL-KRASG12D-TP53R167H pigs were dosed with 4 × 109 PFU of Ad5CMVCre-eGFP (Adeno-Cre), either directly into the body of the pancreas or into the pancreatic duct pushed toward the pancreas. For all cases the pancreas was accessed surgically through a ventral midline incision. Anesthesia was induced with TKX , intubated and maintained in a surgical plane of anesthesia with Isoflurane.All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee. All pigs used were of the LSL-KRASFollowing recovery, all animals were monitored for general health, weight and periodic blood chemistry evaluation. For euthanasia, each animal was sedated and euthanized with sodium pentobarbital overdose . All animals underwent gross and histopathologic evaluation. Tissues were then collected and subject to gross and histopathologic analysis.in vivo imaging, animals were sedated as described previously and 120 ml of the contrast agent Iohexal (Omnipaque) was delivered intravenously, allowing for visualization of potential pathologies by contrast computed tomography (CT). Scans were limited to the cranial abdomen.For 3 pieces in ice cold DMEM/F12 media with 20% animal serum complex, penicillin (100 units/mL), and streptomycin (100 μg/mL). Tissue was transferred to 50 ml conical tubes and washed with cold media and transferred to a sterile hood. Tissues were washed again and incubated with Collagenase Ia (1 mg/ml) and 0.25 mg/ml of trypsin inhibitor for five minutes at 37 °C. The Collagenase reaction was quenched with PBS with 20% animal serum complex and the cells washed. The reaction was similarly quenched with PBS with 20% animal serum complex and the cells washed in fresh culture media with 20% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL). The cells were then seeded on six well plates and allowed to adhere. Once the cells were seeded, they were infected with Adeno-Cre at a 200 to 500 MOI. The media was changed after six hours and cells maintained at 37 °C with 5% CO2.The pancreas from a two-month old female pig was collected and immediately cut into 1 mm6 PORC1 cells suspended in100 μl of Matrigel,  and injected subcutaneously into 3 SCID mice . Tumor growth data was monitored for up to 100 days. SQ tumors were routinely measured and overall health was assessed by weight and visual inspection. Mice were sacrificed by cervical dislocation, and tissues collected and subject to histopathology. All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee.5 × 10R167H and GAPDH primers . CAG-F: 5′-TCATATGCCAAGTACGCCCC-3′; CAG-R: 5′-CCCCATCGCTGCACAAAATA-3′; TP53-F: 5′-TGGCTCTCCTCAAGCGTATT-3′; TP53-R: 5′-ATTTTCATCCAGCCAGTTCG-3′. Bands displayed were run as shown and without modification.RNA was extracted using Trizol . Quantity/quality was evaluated by spectrophotometer  and integrity confirmed via gel electrophoresis. cDNA was then synthesized using the High-Capacity cDNA Reverse Transcription Kit per manufacturer specifications  and rtPCR was conducted with SYBR Green and commercially available TP53G12D-TP53R167H pigs or were euthanized and subjected to pathological examination the pancreas, small intestine, liver, bile duct, lymph nodes, spleen, omenteum, colon, thyroid, lung, heart, kindeys, diaphragm, esophagus, bladder, and stomach. Similarly, mice were sacrificed via cervical dislocation and xenograft tumors collected. Tissues were fixed in 10% formalin, paraffin-embedded, and sections at 4 μm interval were cut from each tissue, and stained with H&E, trichrome , or via immunohistochemistry (IHC)/immunofluorescence (IF).LSL-KRASG12D , PCNA , αSMA, Synaptophysin, Pancreatic amylase , Vimentin, E-Cadherin, mutant P53, and pERK  at 1:50-1:400 overnight at 4 °C. Slides were developed using an HRP-conjugated secondary antibody followed by DAB substrate/buffer (DAKO).For IHC, slides were deparaffinized and heated in a pressure cooker using DAKO retrieval buffer . Endogenous peroxidases were quenched in DAKO peroxidase block for 20 min. Tissues were blocked with 0.5% BSA in PBS for 30 min and exposed a primary antibodies against RASFor IF, slides were deparaffinized and heated in a pressure cooker using DAKO retrieval buffer . Endogenous peroxidases were quenched in DAKO peroxidase block for 20 min. Tissues were blocked with 0.5% BSA in PBS for 30 min and exposed a primary antibodies against CK19 , pPancreatic amylase, αSMA, PCNA, (Santa Cruz), Vimentin, or pERK  at 1:100 overnight at 4 °C. Slides were visualized using an Alexaflour-488 or 594 conjugated secondary (abcam).For cultured cells, cells were grown on chamber slides and fixed with ice-cold methanol at −20 °C for 10 minutes. Cells were blocked for 1 hour at room temperature with 0.5% BSA in PBS, and incubated with primary antibodies against CK19 (University of Iowa Hybridoma Bank), E-Cadherin, pERK,  or PCNA (Santa Cruz) at 1:50-100 overnight at 4 °C. Slides were visualized using an Alexaflour-488 or 594 conjugated secondary antibody (abcam).Cell or tissue lysates were lysed in 4% SDS buffer followed by needle homogenization. Equal amounts of protein (15–40 μg) were mixed with loading dye, boiled for 8 min, separated on a denaturing SDS–PAGE gel and transferred to a PVDF membrane. The membrane was blocked in 5% milk/TBS/0.1% Tween for 1 h and incubated with antibodies against pERK, ERK, Vimentin, , αSMA (abcam), β-Actin . The membrane was washed with TBS-0.1% Tween and then incubated with HRP conjugated secondary antibody (Santa Cruz Biotech) at room temperature for 1 h and rewashed. Protein bands were visualized by an enhanced chemiluminiscence method  and resolved digitally per the manufacturer’s specifications. For RAS activation assay, a standard kit was purchased and used per manufacturer specification (Thermo). All experiments were performed in triplicate unless otherwise specified.G12D antibody (New East Biosciences) 1:100 in PBS with 1% BSA over ice for 20 minutes. Cells were analyzed with a BD Fortessa. All flow plots correspond to size appropriate single cells and are representative of 100,000 events unless otherwise stated.Cultured pancreas cells were seeded into a round-bottom 96-well plate, washed in PBS, and stained with anti-RASAll experiments involving the use of mice were performed following protocols approved by the Institutional Animal Care and Use Committee at the University of Illinois. Patient slides and information was obtained in a de-identified fashion from the Northwestern University Pathcore, following local IRB approval. All patients offered informed consent for study participation. All methods were performed in accordance with the relevant guidelines and regulationsloxp-STOP-loxp (LSL)-KRASG12D-TP53R167H pigs as a potential model of PDAC, we first compared the microscopic anatomy of the human, murine, and porcine pancreas. Despite the described structural differences, we found that the gland histology was remarkably similar among the three species  or intraperitoneally (IP) into SCID mice (N = 7 per group). Mice with SQ injection developed large masses under the skin at each injection site  showed no evidence of gross tumor formation at the injection site or near the pancreas. Additionally, the hepatobilliary tract, spleen, liver, and regional lymph nodes appeared normal with no sign of lesions. The stomach was moderately filled with gas and fluid with a mild amount of hyper-attenuating material in the dependent region of the fundus, though there was no clear sign of overt abnormality Fig. . FurtherTumors were then fixed and sectioned, and compared directly to human PDAC samples. Upon histological evaluation of several geographically separate areas, porcine tumors consistently displayed morphological features consistent with human PDAC Fig. . In addiConsistent with a PDAC phenotype, porcine tumor sections were positive for both E-cadherin and CK19 Fig. . Though G12D and P53R172H proteins. These were accompanied by enhanced ERK activation as determined by western blotting  cassette resulted in a faithful recapitulation of the more common PanIN histotype17. Combining this model with an LSL-TP53R172H cassette resulted in a metastatic model of PDAC with histology closely resembling that observed in human patients10. These KPC mice have been favored by a number of investigators for this reason, and have provided a reliable and accurate model of human PDAC for over a decade. However, despite the success of the KPC mice, there are still the inherent limitations of the mouse anatomy and physiology that must be considered.The poor survival associated with pancreatic cancer stems largely from the fact that most patients initially present with highly advanced disease. As most of these patients are not candidates for surgical intervention and there is no effective pharmacological therapy, there is an urgent need to develop new therapeutic approaches. To do so, investigators have relied almost exclusively on genetically engineered mouse models. For example, the Pdx promoter has been used to drive Cre expression in the progenitor cells of the pancreas, which when crossed to a 18. In contrast, the mouse pancreas is soft and diffuse with three poorly defined lobes19. There are also pronounced differences of the ductal tree between the two species. In humans, the pancreatic acini are the predominant exocrine cell type and are organized into lobules that secrete to a small, intercalated duct. These intercalated ducts drain to a larger, interlobular duct that join to form the large, main pancreatic duct which empties to the duodenum adjacent to the common bile duct18. In mice, however, a large interlobular duct drains the three respective lobes. Ducts from the splenic and gastric lobes then merge with the common bile duct far more proximal to the duodenum than in humans18.Importantly, the mouse pancreas has significant micro and macroscopic differences to that of humans. The human pancreas is a firm organ that is segmented into three borderless, yet distinct parts: the head, body, and tail20, the mouse genome is substantially different from that of humans. Though much is shared, there is significant variance in transcriptional regulation and chromatin organization between the two species21. This is particularly true of genes involved in the inflammatory response22, indicating that mice may have limited utility in inflammation-associated diseases such as pancreatic cancer23. Another notable disparity between mouse and human genetics is the failure of mutations in Cystic Fibrosis Transmembrane Regulator (CFTR) gene to induce significant pancreatic disease as it does in humans24. These issues were resolved by modeling the disease in pigs, which lead to a more faithful recapitulation of the disease pathology and destruction of the exocrine pancreas26. We therefore attempted to recreate the success of the KPC model in the more genetically and anatomically similar sus scrofa domestic pig, in order to provide a more clinically relevant model of PDAC.In addition to these structural variances, there are several genetic factors that account for further differences in mouse and human carcinogenesis. Despite sharing a similar number of protein coding genesInitial attempts at intrapancreatic delivery of Adeno-Cre resulted in a mixed histotype. While the pigs developed PanIN lesions within 16 days, we also observed lethal smooth muscle derived leiomyosarcoma, likely due to the malignant transformation of vascular smooth muscle infected with Adeno-Cre. These two cancer types generally do not present simultaneously. Therefore, it was necessary to refine our approach in order to generate a clinically relevant PDAC histotype. By restricting the delivery of our Cre using an intraductal injection, we were able to induce locally invasive PDAC without the presence of an underlying sarcoma. Interestingly, this approach also resulted in a somewhat mixed histotype, this time consisting of PDAC as well as rare and geographically isolated regions of PNET.27. They are commonly aggressive tumors most frequently occurring in the head of the gland and management is often a combination of surgery and platinum-based chemotherapy. At the present time, this may be the most appropriate category for tumors induced by LSL-KRASG12D-TP53R167H pigs, though PDAC appeared to predominate, particularly in sections closest to the main pancreatic duct. Therefore, future work will employ improved exocrine targeting to exclude the neuroendocrine compartment.Mixed tumors and mixed adenoneuroendocine carcinomas (MANEC) of the gastrointestinal tract and pancreas have been well described by our group as well as many others28. Additionally, given the profound similarity between human and pig immune systems, these LSL-KRASG12D-TP53R167H pigs may provide an improved platform for preclinical immunotherapy. Given these advantages, several porcine models of cancer are emerging. These include a mutant APC porcine model of familial adenomatous polyposis (FAP), a heterozygous TP53 knockout model of spontaneous osteosarcomas, and a chemically induced hepatocellular carcinoma (HCC) model29. Similarly, our inducible LSL-KRASG12D-TP53R167H model has successfully generated soft tissue sarcomas and HCC30.Based on the size and anatomy of the pig, this model may allow for insight into surgical and interventional radiology techniques never before possible in rodents. Pigs and humans also have largely analogous Cytochrome P450 enzymes, allowing for better pre-clinical evaluation of drug metabolism and therapyG12D-TP53R167H pig to model a human-like pancreatic carcinoma both in vitro and in vivo (Summarized in Fig. By demonstrating sufficiency of the LSL-KRASSupplementary Information


The aim of this study is investigating the benefits and harms of neuromuscular blocking agents (NMBAs) in patients with acute respiratory distress syndrome (ARDS).We comprehensively searched PubMed, EMBASE, and Cochrane library for randomized controlled trials comparing NMBAs to any other comparator. We pooled data using relative risk (RR) for dichotomous outcomes and weighted mean difference (WMD) for continuous outcomes, with 95% confidence intervals. We assessed the quality of included studies using the Cochrane tool and levels of evidence using the GRADE method.n = 1557 patients) were eligible for analysis. The results showed NMBAs use was not associated with reduced 28 days mortality , 90 days mortality , and intensive care unit (ICU) mortality  in patients with ARDS. However, 21–28 days mortality was slightly lower in patients received NMBAs . Besides, NMBAs use could improve the PaO2/FiO2 ratio at 48 and 72 h, decrease plateau pressure and PEEP at 72 h. Additionally, NMBAs had no significant effects on days free of ventilation at day 28 , days not in ICU at day 28 , ICU-acquired weakness . Finally, NMBAs use was associated with a lower risk of barotrauma .Finally, six RCTs (In patients with respiratory distress syndrome, NMBAs may be beneficial in reverse refractory hypoxemia and may be associated with reduced short-term mortality and incidence of barotrauma. However, there is no significant effects of NMBAs on mid-term and long-term mortality, and further studies are required. Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by refractory acute hypoxemia . It is aThis study was performed and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines  independently performed literature search, any disagreement was resolved by discussion or consultation with a third author (MZ).PubMed, EMBASE and Cochrane library were searched from their inception to Jun 2019. There was no language limitation. Additional file Two authors  did study selection and data extraction. And disagreement was resolved by discussion or consultation with another author (SZ). Firstly, we excluded duplicated articles. Then, we excluded clearly non-relevant articles by screening titles and abstracts. Finally, we included eligible studies by reading the full-text of remaining studies. The following data were extracted: name of first author, publication year, country, sample size, characteristics of included patients, intervention strategies, control strategies, endpoints and other items necessary for quality evaluation. If necessary, we would contact the author of original articles for additional data.Patients: adult acute respiratory distress syndrome defined by each study.Intervention: neuromuscular blocking agents regardless of drug type, dose, or use duration.Control: none or placebo.Endpoints: the primary endpoints included 21 to 28 days mortality , ICU mortality  and 90 days mortality . The secondary endpoints included respiratory parameter such as PaO2/FIO2, plateau pressure (Pplat), and positive end-expiratory pressure (PEEP) at 24 h, 48 h, 72 h; days free of ventilation at day 28 (DFV); days not in ICU at day 28; incidence of biotrauma and ICU-acquired weakness.Two authors (SZ and XF) evaluated the qualities of all eligible studies using the Cochrane Risk of Bias Tool. The following domains were evaluated: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias. Each domain was classified as low risk of bias, unclear risk of bias and high risk of bias.2 statistic. P < 0.10 or I2 ≥ 50% indicated there was significant heterogeneity between studies and random effect model was used, otherwise, the fixed effect model would be used. If there was no significant heterogeneity, we would perform additional sensitivity analyses using random effects models to test the robustness of the results. A different number of studies were included in the various primary and secondary end-points analysis, detailed citations for included studies were shown in the different results. Publication bias would be assessed using Funnel plot and Egger test, if the number of included studies was over 10.Relative risks (RRs) and weighted mean differences (WMDs) with 95% confidence intervals (CIs) were used to estimate the pooled effect of dichotomous variables and continuous variables respectively. Heterogeneity between studies was assessed using the Q statistic and IIn order to minimize the risks of random errors resulting from sparse data during repetitive test, we performed trial sequence analysis (TSA) and calculated the optimal information size for the primary endpoints. In addition, we constructed the adjusted boundary line for favoring the NMBAs or controls to decide whether the meta-analysis could be terminated early. The optimal information size was calculated using α = 0.05 (two-sided), β = 0.20 (power 80%), the anticipated relative risk reduction, and the incidence in control arm.p value less than 0.05 was considered statistically significant. All analyses were performed in RevMan 5.3  software. TSA was perfomed using Trial Sequential Analysis v.0.9.5.10 beta .A https://gradepro.org/).GRADE  was used to evaluate the level of evidence. All patient center endpoints  were graded as high, moderate, low, and very low. This process was performed on GRADEpro GDT . But they had no significantly statistical effects on long-term mortality. Along with the higher risk of serious cardiovascular events among patients received NMBAs, we do not suggest routinely use of NMBAs in all patients with ARDS. Severe ARDS patients with patient–ventilator dyssynchronies, or who are vulnerable to ventilator-induced injury may benefit from NMBAs use. Thus, we think NMBAs could be used for improvement of oxygenation in patients with severe ARDS. Additionally, further studies should focus on the following major topics. Firstly, in recent years, different subgroups or phenotypes of ARDS have been pay attention to . PatientIn patients with respiratory distress syndrome, NMBAs may be beneficial in reverse refractory hypoxemia and may be associated with reduced short-term mortality and incidence of barotrauma. However, there is no significant effects of NMBAs on moderate and long-term mortality, and further studies are required according to insufficient evidence based on current research.Additional file 1: Table S1. Study search strategy . Table S2. Baseline characteristics of included trials. Table S3. Baseline respiratory parameters of included trials (mean ± SD). Table S4. Primary cause of ARDS, n (%). Table S5. Methodologic quality of included trials. Table S6. Effect of NMBAs on ICU-acquired weakness. Figure S1. PRISMA checklist. Figure S2. Risk of bias summary (each risk of bias item for each included study). Figure S3. Risk of bias graph . Figure S4. Forest plot for the mortality of 28 days estimated with random effect model. Figure S5. Trial sequential analysis of the NMBAs on 28 days mortality. Figure S6. Trial sequential analysis of the NMBAs on ICU mortality. Figure S7. Trial sequential analysis of the NMBAs on 90 days mortality. Figure S8. Forest plot of DFV at day 28 estimated with fixed effect model. Figure S9. Forest plot of days not in ICU at day 28 estimated with fixed effect model. Figure S10. Forest plot of barotrauma estimated with fixed effect model. Figure S11. Forest plot of ICU-acquired weakness estimated with fixed effect model. Figure S12. GRADE summary of findings.


Although medical requirements are urgent, no effective intervention has been proven for chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME). To facilitate the development of new therapeutics, we systematically reviewed the randomized controlled trials (RCTs) for CFS/ME to date.RCTs targeting CFS/ME were surveyed using two electronic databases, PubMed and the Cochrane library, through April 2019. We included only RCTs that targeted fatigue-related symptoms, and we analyzed the data in terms of the characteristics of the participants, case definitions, primary measurements, and interventions with overall outcomes.12U) and CoQ10 + NADH) and 5 non-pharmacological therapies . However, there was no definitely effective intervention with coherence and reproducibility.Among 513 potentially relevant articles, 55 RCTs met our inclusion criteria; these included 25 RCTs of 22 different pharmacological interventions, 28 RCTs of 18 non-pharmacological interventions and 2 RCTs of combined interventions. These studies accounted for a total of 6316 participants . CDC 1994 (Fukuda) criteria were mostly used for case definitions , and the primary measurement tools included the Checklist Individual Strength  and the 36-item Short Form health survey . Eight interventions showed statistical significance: 3 pharmacological :poly(CThis systematic review integrates the comprehensive features of previous RCTs for CFS/ME and reflects on their limitations and perspectives in the process of developing new interventions. Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a long-term debilitating illness characterized by medically unexplained, severe and disabling fatigue that persists at least 6 months and is not improved by rest, accompanied by post exertion malaise (PEM) and unrefreshing sleep . PatientTo date, diverse studies, including those of the immune system, metabolomics, endocrine system, gut microbiota and nervous system, have been conducted to determine the pathological mechanisms of CFS/ME . This ilIn addition, despite numerous approaches with various interventions, no definitively effective treatment has been approved for patients with CFS/ME . ThroughTo facilitate those tasks in the future, this systematic review aimed to integrate the features of the trials for CFS/ME conducted so far in terms of patient characteristics, case criteria, outcome measurements and interventions with overall results.http://www.ncbi.nlm.nih.gov/pubmed) and the Cochrane library (http://www.cochrane.org), through April 2019. The search terms used were encephalomyelitis, ME, chronic fatigue syndrome, CFS, ME/CFS, randomized controlled trial and clinical trial. The trial type was limited to RCTs, and all languages were included.A systematic literature survey was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines  using twArticles were screened according to the following inclusion criteria: (1) RCTs or randomized controlled crossover trials, (2) patients with CFS/ME as participants, (3) an evaluation of the efficacy of the intervention for CFS/ME treatment, and (4) fatigue-related primary measurement or main outcome. The exclusion criteria were as follows: (1) articles with no full text, (2) the number of participants was less than 45 , (3) studies without mention of the case definition or the characteristics of participants and (4) studies with a Jadad score less than 3 points.We extracted data on the number of participants, sex ratio, mean age, ME/CFS diagnostic case definition, intervention category, treatment period, dose, control and outcome measurement tool. We also obtained the outcome data with a statistical analysis of the treatment effectiveness compared to the control.To assess the quality of RCTs, the Jadad scale was used . The JadWe judged the intervention efficacies as ‘Significant’ or ‘Not significant’ based on the data presentations of the original articles. In general, ‘Significant’ meant that the intervention reached statistical significance  according to the primary measurement at the planned time point outcome assessment. We defined ‘partially significant’ for the following cases: (1) only part of the main outcomes was statistically significant, or (2) statistical significance was observed only at certain time points without a description of the fixed period for final assessment.This study basically does not need to apply statistical analysis. Regarding the number of participants, age and treatment period in two populations (adults and adolescents), data are presented as the mean and standard deviation (SD).From the PubMed and Cochran databases, a total of 513 articles were initially identified, and 55 articles ultimately met the inclusion criteria for this study Fig.  and 3.FiIn 55 RCTs, a total of 6316 participants  were enrolled. Fifty-four RCTs (98.2%) adapted at least one of the following CFS case definitions: CDC 1994 (Fukuda) criteria (42 RCTs), Oxford 1991 (Sharpe) criteria (12 RCTs), CDC 1988 (Holmes) criteria (3 RCTs), Lloyd 1988 criteria (2 RCTs), and Schluederberg 1992 (2 RCTs). There were 12 RCTs with two case definitions for inclusion criteria  was the most frequently used (36.4%), and others included the 36-item Short Form health survey , Sickness Impact Profile , Chalder Fatigue Scale (12.7%), Visual Analogue Scale  and Clinical Global Impression . There were 28 RCTs that used multiple primary measurements (Table 12U) [10 + NADH [A total of 22 different medications were evaluated by comparison with placebo in 25 RCTs . These medications included psychiatric drugs (n = 8), cortisol (n = 5), immunomodulators (n = 4), and mitochondrial modulators (n = 3). The mean treatment period was 10.8 ± 6.8 weeks . Three RCTs showed positive results with statistical significance: two with immunomodulators (Staphypan Berna [12U) ) and one0 + NADH   with 18 kinds of interventions, mainly CBT (n = 11), exercise (n = 5), and self-care (n = 5). The mean treatment period was 18.3 ± 9.0 weeks . Of the 11 CBT subcategories, 5 RCTs showed statistical effectiveness of CBT compared to the control , 49, 51.Two RCTs were conducted to assess the synergistic effects of 4 different interventions  + CBT). No synergistic efficacy was observed . An epidemiological feature of CFS/ME is the higher prevalence in women and even in adolescent populations   axis dysfunctional and psychiatric hypotheses were mainly proposed for the pathophysiology of CFS/ME . Accordi fatigue .Non-pharmacological interventions could be subgrouped into CBT, exercise such as GET, self-instruction with/without guidance, rehabilitation and acupuncture Table . In factRCTs of alternative medicines and self-therapies for CFS/ME have increased since the late 2000s. Regarding RCTs of alternative medicines such as acupuncture, qigong, and abdominal tuina , 64–66, To date, the overall results of RCTs have been more positive for non-pharmacological interventions than for medications Fig. . Among dOur review has some limitations. We searched literatures from PubMed and Cochrane library. Although these two databases are the major resources for scientific articles especially derived from RCTs, there would be a possibility of further information in other databases. In order to produce confident data, we excluded the too small-scale RCTs (< 45 participants), however this strategy also has a risk to loss any valuable information. In addition, only 9 of 55 RCTs had presented fragmentary data related to blood parameters. We could hardly find any practical indications due to very heterogenous parameters and no significant correlation with changes of fatigue symptoms. The identification of the blood-based biomarkers is necessary for diagnosis as well as classification of CFS/ME and should be applied to clinical trials in the future. Nevertheless, this is the first systematic review of RCTs targeting CFS/ME regardless of language, and this review shows the comprehensive features of CFS/ME. Our review offers fundamental information for future research on the pathophysiology of and new treatments for CFS/ME.This systematic review provides a comprehensive integration of previous RCTs for CFS/ME. Our data include characteristics of RCTs such as participants, case definitions, interventions and primary measurements. In addition, we found trends in the interventions used as well as in the overall results. Psychological treatments were predominant and had limitations curing CFS/ME. An exploration of the pathophysiology of CFS/ME and better development of treatments are needed.


The relationships among energy metabolic levels, behavioral and other physiological traits help to determine the trade-off of energy allocation between different traits and the evolution of life-history driven by natural selection. However, these relationships may be distinctive in selected animal taxa because of their unique traits. In the present study, the relationships among energy metabolic levels, behavioral defense strategies, and antioxidant capacity were explored in three freshwater turtle species with different shell morphologies, by assessing responses to attack, righting time, shell morphology, whole-organism metabolic rates, tissue metabolic enzyme activities and antioxidant levels.Chinemys reevesii, showed a passive defense strategy, relatively larger shells, a higher resting metabolic rate (RMR) and higher antioxidant levels compared to the snapping turtle, Chelydra serpentina, or the Chinese soft-shelled turtle, Pelodiscus sinensis. These latter two species both showed an active defense strategy, a higher factorial aerobic scope and better muscle anaerobic metabolic capacity but relatively smaller shells, lower RMR and antioxidant capacity.The Chinese three-keeled pond turtles, Our results indicate a negative relationship between RMR and activity levels in behavioral defense strategies along small-big shell continuum among the three turtle species. We also found a positive relationship between antioxidant capacity and energy metabolism but a negative one between antioxidant capacity and activity levels in defense strategies. The present study indicated a role of turtle shell in forming unique relationship between energy metabolic levels and behaviors in freshwater turtle taxa and a possible trade-off between the maintenance of physiological homeostasis and activity levels in energy allocation.The online version of this article (10.1186/s12983-019-0301-5) contains supplementary material, which is available to authorized users. Energetics and animal behaviors  are related to each other because both are associated with the slow-fast life-history continuum –3. Thus,The unique traits of freshwater turtles raise questions about their evolution, behaviors and physiology. They have peculiar characteristics as their unique shell  and theiChinemys reevesii that has a hard shell, the Chinese soft-shelled turtle Pelodiscus sinensis with a soft shell and the snapping turtle Chelydra serpentina with a small plastron [The aim of our study was to explore the relationships among energy metabolism, behavioral and physiological traits in freshwater turtles and how the shell participates in these relationships. We selected three freshwater turtles living in similar habitats for the study: the Chinese three-keeled pond turtle plastron –22. Amonplastron , 24. Theplastron . Direct plastron , 26. In P. sinenesis , were obtained from a turtle hatchery facility . C. reevesii  and C. serpentina  were obtained from an aquarium market . All turtles were raised in tanks (70 × 50 × 40 cm with water depth of about 6 cm) at 26 ± 1 °C  for at least 6 weeks. All turtles were given commercial feed daily. Photoperiod was kept at 12 L/12 D.Chinese soft-shelled turtles, n = 7). The other group, which was defined as the electrical stimulated group , experienced forced movement by electrical stimulation  [After acclimation, all turtles were assessed for (a) behavioral traits: righting time (RT) and response to attack (RTA), (b) morphological traits: carapace height/width ratio (H/W ratio), plastron/carapace flat area ratio (P/C ratio), and (c) metabolic traits: RMR, maximum metabolic rate (MMR), net aerobic scope (NAS) and factorial aerobic scope (FAS). Then turtles were transferred back to their original tanks for another two weeks. Afterwards, all individuals of each species were divided into two groups. One group was defined as the control group without treatment (austion) –29. AfteRighting time reflects the response to being overturned and the hiding time until the predator leaves , 30. TheMaximum carapace height and carapace straight width were measured with a digital caliper  to the nearest millimeter. According to the model of Domokos and Várkonyi , H/W ratRMR of each turtle was measured in air at 26 °C using an airtight chamber  and oxygen sensor . Turtles were fasted for 24 h and kept still in the chamber. Each individual was acclimated to the experimental environment for 30 min with the cover off. Then the cover was closed tightly, the survey was started and the change in oxygen percentage (%) over 30 min was recorded. To measure the MMR, turtles were forced to run for 5 min or until exhaustion in response to electrical stimulation . Then the turtle was quickly placed in the chamber to measure the change in oxygen percentage (%) over the following 30 min. The change in oxygen percentage inside the chamber during the initial 100 s was chosen to calculate the MMR. The air volume was measured by filling the chamber with water in the presence of the animal and then measuring the volume of water to the nearest milliliter. FAS (defined as the MMR/RMR ratio) and NAS (defined as difference between MMR and RMR) were calculated .2HPO4-7H2O, and 210 mg/L KH2PO4, pH 7.2) for biochemical assays. Reagent kits  were used to measure the activities of SDH, C3, CO, SOD, CAT, LDH, TAC and LA level according to the instruction manual for each kit.After sampling, tissues were homogenized in phosphate buffered solution . SDH activity was measured using a 2,6-dichlorophenolindophenol (DCPIP) reduction reaction. The DCPIP reduction speed was determined by the change in absorption value at 600 nm . C3 acti3+ was reduced to Fe2+ in the presence of antioxidants [2O2 decomposing in 1 g protein per second with the change of absorption measured at 405 nm. LDH activity was measured using the reverse reaction in which LDH catalyzes the conversion of lactate to pyruvate using NAD+ as the hydrogen donor. Then 2,4-dinitrophenylhydrazine was used to determine pyruvate product levels, monitored by absorption change at 440 nm. LA content was measured following the method of Buttery et al. [Portions of each tissue homogenate were centrifuged at 4500 g and 4 °C for 10 min to provide supernatants for antioxidant enzyme activity assays, LDH activity and LA content. TAC was measured by determining the absorption value at 520 nm when Feoxidants . SOD actoxidants . CAT acty et al. . LA was y et al. .w/v) and then homogenates were centrifuged at 20,000 g for 20 min. The supernatants were used for high performance liquid chromatography (HPLC) analysis with electrochemical detection  and a 5 μm analytical column . Injection volume was 20 μL, flow rate was 1 mL per min and ultraviolet detection was set as 243 nm.We employed the method described in Chen et al.  to measuP < 0.05 was set as the significance level. The correlations among different morphological and physiological parameters were analyzed using Pearson’s correlation analysis, while the correlations between behavioral parameters, which are discrete variables, and others were analyzed using Spearman’s correlation analysis.The hierarchical clustering algorithm was generated with all data according to a Euclidean method. The clustering analysis and the heatmap were conducted with R v. 3.5.0 (Development Core Team 2018). Then, all data were checked for normality by the Kolmogorov-Smirnov test and homogeneity of variance. For metabolic rates, a general linear model was employed to conduct interspecies comparation with mass as covariate. For enzyme activities and LA level, intraspecific differences between the control group and the treatment group (ESG) were assessed via a t-test. If no significant difference between the two groups was found, data of the two groups were combined for interspecific comparison. Interspecific variations in all parameters, except metabolic rates, were detected using one-way ANOVA followed by Tukey HSD post hoc test, or Kruskal-Wallis test followed by Mann-Whitney U post hoc test for data that did not fit the check for normality or variance homogeneity. P. sinensis and C. serpentina. The other one included all C. reevesii individuals. Detailed results were shown in Figs. Clustering results showed that the 45 turtles can be divided into two groups according to all parameters measured Fig. . One groC. reevesii spent the longest time whereas P. sinensis spent the shortest time in righting themselves , P/C ratio , liver index  and heart index  among the three turtle species .ies Fig. . P. sineina Fig. . Both H/C. reevesii showed the highest TAC among the three species  and CAT activity positively correlated with TAC in the heart . Furthermore, these parameters positively correlated with RMR  with a higher RMR of sis Fig. a. The hintenance  and linkntenance . In addiies Fig. b and c. ies Fig. , indicatC. reevesii was higher , indicating a greater energy cost to support bigger shells. The extra energy cost may represent both the energy required to grow and maintain the shell and the energy debt involved in the daily activity carrying the bigger shell. In fact, in the righting response, turtle species with a higher shell need less energy investment for their self-righting than the species with flat shell [The reason for the negative relationship between RMR and activity level in the behavioral defense strategies of these turtles might be their unique morphological trait, the shell. Our results indicated clear difference among the shell morphology of the three species and relationship among shell morphologies, behavioral traits and RMR. The shell of strategy , 54. Simstrategy . At the at shell , 56. Thu2−) generated in mitochondria in association with high rates of aerobic respiration among the three species . Our results provided evidence of the special negative correlation between energy metabolism, antioxidant capacity and behavioral activity levels in different defense strategies of freshwater turtles Additional file 2:Figure S1. Hepatic CO activity (A), cardiac C3 activity (B) and cardiac CO activity in three freshwater turtle species. Superscripts without common letters denote significant difference (P < 0.05). (TIF 775 kb)Additional file 3:Figure S2. Hepatic CAT activity (A), hepatic AA level (B) and cardiac SOD activity (C) in three freshwater turtle species. Superscripts without common letters denote significant difference (P < 0.05). (TIF 776 kb)Additional file 4:Figure S3. The correlation between parameters measured: hepatic SDH activity and RMR (A), hepatic C3 activity and RMR (B), hepatic SOD activity and hepatic SDH activity (C), hepatic SOD activity and hepatic C3 activity (D), hepatic SOD and hepatic CO activity (E), cardiac SOD activity and cardiac C3 activity (F). (TIF 870 kb)


To determine whether lung involvement is related to microvascular perturbations, nailfold videocapillaroscopy (NVC) was performed in patients with systemic sclerosis (SSc).A cross-sectional study was consecutively accomplished in 152 SSc patients. NVC, a pulmonary function test and echocardiography were undergone within a 3-month period. Finally, 134 patients with at least eight NVC (200× magnification) images were selected for quantitative and qualitative examinations.p = 0.005) and higher median of neoangiogenesis . A higher quantity of neoangiogenesis capillaries was found in patients with pulmonary arterial hypertension . Multivariate linear regression analysis established a correlation between neoangiogenesis and decreased forced vital capacity (FVC) (p < 0.001): for each capillary with neoangiogenesis visualized on average per 1 mm, FVC was 7.3% reduced. In qualitative NVC, a late pattern as defined by Cutolo was also associated with lower FVC (p = 0.018). The number of giant capillaries was associated with reduced diffusion capacity of the lung for carbon monoxide (DLCO) (p = 0.016); for each giant capillary per 1 mm, DLCO was 11.8% diminished.Patients with interstitial lung disease presented lower median capillary density (4.86/mm vs 5.88/mm, A good correlation was observed between distinctive quantitative and qualitative NVC features with lung functional parameters such as FVC and DLCO. It is suggested that vasculopathy could play a role in SSc lung involvement. Systemic sclerosis (SSc) is a connective tissue disease characterized by microvascular damage. It seems that endothelial activation induces the release of inflammatory mediators, platelet activation and inflammatory cell recruitment –3. SubseThe lung is one of the most frequently jeopardized organs in SSc; thus interstitial lung disease (ILD) affects around 55–65% of patients subjected to high-resolution computed tomography (HRCT) , 8. As aCapillary microscopy was initially used as a qualitative technique to describe microvascular abnormalities in SSc and other autoimmune-related diseases. Maricq et al.  defined The hypothesis of the present study was that microcirculation damage leads the pathogenesis of the main manifestations in SSc such as ILD and PAH. The main aim of the work was to analyse NVC alterations using a quantitative method and to investigate their correlation with the pulmonary function test (PFT).A cross-sectional study was carried out on SSc patients from the Vall d’Hebron Hospital cohort. All patients fulfilled LeRoy et al.’s SSc classification criteria , and accCutaneous subsets were defined as previously , accordiInterstitial lung disease was defined as radiologic evidence of interstitial findings on HRCT examined by an expert thorax radiologist . PAH wasMusculoskeletal disease included arthritis, tendon fiction rubs, joint contractures and myositis. Gastrointestinal SSc disease was established if any of the following were present: oesophageal dysmotility, gastric antral vascular ectasia, gastric or bowel dysmotility, intestinal bacterial overgrowth, intestinal pseudo-obstruction or anal sphincter dysfunction.Antinuclear antibodies (ANA) were evaluated by indirect immunofluorescence (IIF) assay using HEp-cell line 2. Anticentromere antibodies (ACA) were described by IIF, and anti-topoisomerase I antibodies were determined by enzyme-linked immunosorbent assay. A commercial line blot assay was performed to detect anticentromere proteins A and B, anti-RNA pol III and anti-PM/Scl antibodies (EUROLINE Systemic Sclerosis (Nucleoli) Profile (IgG), Euroimmun, Germany) according to the manufacturer’s instructions.Complete PFTs including the percentage of the predicted forced vital capacity (FVC) and the percentage of the predicted diffusion capacity of the lung for carbon monoxide (DLCO) were accomplished using MasterLab equipment , following the ATS/ERS recommendations . EchocarNailfolds were examined using an Optilia Digital Videocapillaroscope  with 200× lens magnification and an LED lamp by the same operator (AS-O) who was blinded to clinical patients’ conditions. Each patient was acclimatized for at least 20 min at room temperature of 20–24 °C before the examination. NVC was conducted with a contact adapter and immersion oil dropper, studying the middle nailfold from the second to fifth fingers of both hands. Optipix Lite software (Optilia Instruments AB) was used for visualization of consecutive 1-mm-wide images of first-line capillaries. Two images were taken on each finger, although when a finger was not possible to evaluate additional pictures were taken on other fingers. Patients with fewer than eight images were excluded from the study. Quantitative and qualitative analyses of the images were performed blind to clinical data by investigator AS-O. Inter-observer reliability and intra-observer reliability were also explored with a calculated sample size of 45 patients. This sample was randomly selected and blindly assessed once by investigator AG-DC to calculate the inter-observer reproducibility. Afterwards, to estimate the intra-observer repeatability, a second round was independently performed by investigators AS-O and AG-DC with a minimal time interval of 2 weeks between both measurements.Quantitative NVC was carried out according to previous definitions as follows: capillary density, number of capillaries measured in the distal row following the 90° method ; enlargeThe mean of each capillaroscopic feature was calculated from the sum of consecutive images for each digit. Subsequently, the average values from eight fingers were added together and divided by the number of studied digits. The resulting value indicated the number of this capillaroscopic feature adjusted by each millimetre of the nailfold.Qualitative NVC analysis of images was performed following capillaroscopy patterns described by Cutolo et al.  .U test was used. Bonferroni correction was applied in multiple comparisons. Associations between NVC and clinical features, PFTs and echocardiography variables were analysed using Spearman’s correlation.For the descriptive statistics, qualitative data were expressed as the mean and standard deviation (SD) after approving the normal distribution test; and non-normal qualitative variables were described as the median and interquartile range (IQR). Normal distribution was assessed using the Kolmogorov–Smirnov test. To assess whether there were statistically significant differences between groups of patients, the Mann–Whitney In order to analyse inter-observer and intra-observer reliability, a sample size of 45 patients was calculated, based on two observers (AS-O and AG-DC), with a power of 80% to detect that an intra-class coefficient (ICC) of 0.7 was significantly higher than 0.45 , 31. Thep < 0.05 was considered significant. Statistical analysis was conducted with SPSS 20.0 for Windows .Multiple linear regression tests were performed to determine the association with FVC and DLCO. The multivariate models for FVC included NVC features, age at NVC, time elapsed from first symptom to NVC, dcSSc subset, gender, interstitial findings on HRCT, different autoantibodies and DU. For DLCO models, FVC, PAH confirmed by RHC, TRV ≥ 2.9 m/s or other echo PH signs also were introduced. n = 113, 84.3%) and in the lcSSc subset . More than 90% of the population fulfilled the 2013 ACR/EULAR classification criteria. ACA were the most common antibodies in 49 (36.6%) patients, followed by anti-topoisomerase I antibodies in 31 (23.1%) patients. Considering major organ involvement, digestive involvement was present in 110 (82.1%) patients, cardiac disease in 103 (76.9%) patients, ILD in 58 (43.3%) patients, musculoskeletal involvement in 40 (29.9%) patients and PAH in 11 (8.2%) patients.Demographic characteristics of the 134 SSc patients are presented in Table The distribution of different cardiac manifestations was 76.9% of patients presenting cardiac conduction abnormalities, 57.5% mitral regurgitation (with no CVRF), 47% diastolic dysfunction (with no CVRF), 13.4% pericardial effusion, 8.2% microvascular ischemic heart disease (with no CVRF), 3.7% LVEF < 50% and 3% of patients macrovascular ischemic heart disease (with no CVRF). Among all patients, there were 19 (14.1%) with overlap features as follows: nine (6.7%) patients had dermatomyositis, seven (5.2%) patients had Sjögren syndrome and the other three (2.2%) patients had rheumatoid arthritis.PFTs and echocardiography findings are presented in Table p = 0.005). Moreover, the number of capillaries with neoangiogenesis was higher in ILD patients . Focusing on PAH, patients with this manifestation also showed greater frequency of capillaries with neoangiogenesis compared with patients with no PAH . All prior comparisons remained statistically significant after Bonferroni correction (p < 0.008).A total of 2186 images were analysed from 134 patients, with a mean of 16.3 (± 5.0) images per subject and the median (IQR) of images for each finger was 2 (1–3). The median (IQR) of capillary density adjusted by each millimetre of nailfold was 5.44/mm (4.33–6.74) (Table p = 0.006). In addition, a late pattern showed lower FVC and DLCO values compared with normal, early and active patterns  was more prevalent in patients with ILD  and the quantity of microhaemorrhages . The time elapsed from first symptom to NVC was negatively associated with the number of microhaemorrhages  and positively with the number of capillaries with neoangiogenesis . Higher capillary density was positively correlated with the % DLCO  and negatively with the RVSP . The number of capillaries with neoangiogenesis was inversely associated with % FVC  and % DLCO  and was positively associated with RVSP .Age at NVC was inversely correlated with the number of enlarged capillaries (κ of 0.83 (0.66–1.0), even when each pattern was analysed separately, with the only exception of an early pattern which had a substantial reproducibility with Cohen’s κ of 0.73 (0.42–1.0).The inter-observer reproducibility for all quantitative NVC features was almost perfect with an ICC higher than 0.80 Table , except Regarding intra-observer repeatability, similar results were obtained for quantitative and qualitative variables, with an increment of neoangiogenesis repeatability up to 0.81 (0.56–0.90).To elucidate which NVC findings were more strongly associated with pulmonary parameters, a multivariate analysis was conducted using % FVC as a dependent variable. Table A lower % DLCO value was associated with a reduced number of enlarged capillaries and an increased number of giant capillaries, along with ILD on HRCT, lower % FVC, PAH confirmed by RHC, TRV ≥ 2.9 m/s or other echo PH signs on echocardiography. In this way, for each enlarged capillary on average per 1 mm the DLCO would be 7.8% higher; however, for each giant capillary it would be diminished 11.8%.Multivariate analysis showed an association between late pattern and lower % FVC values. FVC would be diminished 14.3% in a patient with late NVC pattern features Table . None ofThis cross-sectional study in an SSc population indicated that the presence of capillaries with neoangiogenesis in NVC was related to lower percentage FVC values independently of other baseline characteristics. According to our results, a patient with one capillary with neoangiogenesis per millimetre on average in NVC would have 7.3% lower FVC percentage regardless of other clinical or capillaroscopic conditions. Concerning qualitative NVC assessments, a late pattern was also associated with lower FVC. We also found a substantial or almost perfect inter-observer and intra-observer reliability of all NVC features.The median capillary density was similar to that reported previously, but our study included a higher significant number of evaluated images per patient , 34. PriWe found that SSc patients with ILD had a lower capillary density and a higher number of capillaries with neoangiogenesis. These findings support the prior semi-quantitative NVC study, which identified higher mean avascular scores in patients with ground-glass opacities . SimilarRecently, Avouac et al.  publisheFurthermore, regarding qualitative NVC studies, the progression from normal/early/active to a late pattern was related to the development of new DU, lung vascular progression, progression of skin fibrosis and worsening on the Medsger severity scale . AccordiConsequently, it appears that endothelial damage mediates both lung fibrosis and peripheral microvascular changes visualized in NVC. SSc patients with more extensive ILD may also show higher nailfold capillary perturbations, ineffective angiogenesis and vasculogenesis, which reflects a higher frequency of bushy or bizarre capillaries which are denominated as neoangiogenesis . In factRecently, a qualitative NVC study has noted the predictive role of NVC and specific autoantibodies in cardiopulmonary involvement . SpecifiHofstee et al.  found thLimitations of this study included the cross-sectional study, with no follow-up data, which did not allow us to infer whether capillaroscopic findings were linked to a specific outcome of pulmonary disease. However, although an association was observed between microvascular features and functional respiratory parameters, this fact does not prove a causal relationship between them. We cannot exclude that both phenomena would be influenced by an unknown parameter that may lead a parallel change in the first two. Notwithstanding, the present study also had strengths, because all patients had undergone echocardiography and PFTs close to the NVC examination. The images were analysed blind of clinical data by the researchers. For the statistical analysis, patients were excluded with fewer than eight explored fields, either due to technical difficulties or digital amputations. NVC explorations after autologous stem cell transplantation had demonstrated an improvement in vascular damage . ConsequThis study describes a lower capillary density and a higher number of capillaries with neoangiogenesis in patients with ILD, and demonstrates a quantitative and qualitative relation between specific NVC abnormalities and lung function tests (both % FVC and % DLCO parameters). It seems that NVC findings are linked to a wide spectrum of clinical variables in SSc, which emphasizes the crucial role of microcirculation damage in this medical condition, especially within the lung.