P=0.018) but not with improvement in primary physical illness, sex, age or study duration. The findings indicate a potentially causal role for cytokines in depression and that cytokine modulators may be novel drugs for depression in chronically inflamed subjects. The field now requires RCTs of cytokine modulators using depression as the primary outcome in subjects with high inflammation who are free of other physical illnesses.Inflammatory cytokines are commonly elevated in acute depression and are associated with resistance to monoaminergic treatment. To examine the potential role of cytokines in the pathogenesis and treatment of depression, we carried out a systematic review and meta-analysis of antidepressant activity of anti-cytokine treatment using clinical trials of chronic inflammatory conditions where depressive symptoms were measured as a secondary outcome. Systematic search of the PubMed, EMBASE, PsycINFO and Cochrane databases, search of reference lists and conference abstracts, followed by study selection process yielded 20 clinical trials. Random effect meta-analysis of seven randomised controlled trials (RCTs) involving 2370 participants showed a significant antidepressant effect of anti-cytokine treatment compared with placebo (standardised mean difference (SMD)=0.40, 95% confidence interval (CI), 0.22–0.59). Anti-tumour necrosis factor drugs were most commonly studied (five RCTs); SMD=0.33 . Separate meta-analyses of two RCTs of adjunctive treatment with anti-cytokine therapy and eight non-randomised and/or non-placebo studies yielded similar small-to-medium effect estimates favouring anti-cytokine therapy; SMD=0.19  and 0.51 , respectively. Adalimumab, etanercept, infliximab and tocilizumab all showed statistically significant improvements in depressive symptoms. Meta-regression exploring predictors of response found that the antidepressant effect was associated with baseline symptom severity ( Cytokine-mediated communication between the immune system and the brain has been implicated in the pathogenesis of depression.15 of non-steroidal anti-inflammatory drugs (NSAIDs), given as sole treatment or as adjunct to antidepressants, indicates that they may be more effective than placebo in treating depression , 0.08–0.45),16 although there are limitations of individual studies included such as attrition. Although these results point to an inflammatory component to depression, further examination of the evidence regarding cytokine-modulating drugs may help to elucidate the relevance of inflammation for pathogenesis and treatment of depression specifically. NSAIDs are broad-spectrum anti-inflammatory agents that also act on other targets, such as glucocorticoid receptors.17 These receptors are, themselves, relevant for depression pathophysiology,18 so the extent to which the antidepressant effect of NSAIDs is due solely to anti-inflammatory action is unclear. Some NSAIDs such as cyclooxygenase-2 inhibitors increase risk of cardiovascular disease,19 a known comorbidity for depression, so their use in depression may be problematic. Besides, many trials of anti-inflammatory drugs for depression are based on people with chronic physical illness16 but it is unclear whether the improvement in depression is due to the improvement in physical illness.A meta-analysis of randomised controlled trials (RCTs)20 Supported by a large number of RCTs, cytokine modulators are well established treatments for chronic inflammatory conditions such as rheumatoid arthritis21 and psoriasis.22 Many of these clinical trials have also reported on secondary psychosocial outcome measures including depressive symptoms, which could be utilised to address important questions regarding potential usefulness of anti-cytokine treatment for depression. In addition to quantifying the antidepressant effect of anti-cytokine treatment, examination of the relationship between the improvement in depressive symptoms and that in physical symptoms could elucidate the role of inflammation in depression specifically. Previous RCTs indicate baseline severity of depression moderates the antidepressant effects of monoaminergic drugs23 and psychotherapy;24 it not clear whether this is also the case for anti-cytokine treatment.Cytokine modulators, which include monoclonal antibodies and cytokine inhibitors, constitute a more pure anti-inflammatory class because they target-specific cytokine pathways. Recently, a proof-of-concept RCT of infliximab, a tumour necrosis factor alpha (TNF-α) specific monoclonal antibody, has reported improvements in patients with treatment-resistant depression characterised by high inflammation at baseline.We report a systematic review and meta-analysis of secondary data from clinical trials of anti-cytokine treatment in chronic inflammatory conditions to address the following key outstanding questions: (1) does blocking-specific inflammatory cytokine pathways lead to improvement in depressive symptoms; (2) what is the relationship between the antidepressant effect and the improvement in physical illness; (3) is the antidepressant effect related to baseline severity of depressive symptoms; (4) is there any sex or age difference of the antidepressant effect. Answers to these questions would provide important clues on whether inflammatory cytokines have a causal role in depression, and whether cytokine modulators may be useful for treating depression.A systematic search of the PubMed, Embase, PsycINFO and Cochrane databases was carried out for all clinical trials of cytokine modulators involving human subjects published in the English language until 5th April 2016, where depression or depressive symptoms were reported as an outcome. The search terms included indexing terms as well as wildcards to maximise return: ‘ AND (depression OR depressive symptom)’. We also searched reference lists of retrieved articles and conference abstracts, and wrote to key authors in the field for unpublished data.25 The literature search and study selection were carried out by NK and GMK; any differences were resolved by discussion.Clinical trials of specific cytokine inhibitors or monoclonal antibodies against specific inflammatory cytokines that measured depressive symptoms using a recognised tool were included. In order to obtain a comprehensive overview of the literature, we included all clinical trial designs, that is, RCTs, double-blind or open-label, as well as non-randomised studies . Studies that examined cytokine modulators as an adjunct to interferon therapy, used monoclonal antibodies as a method for detecting leukocyte subtypes, or examined monoclonal antibodies against targets other than an inflammatory cytokine  were excluded.27 values were extracted using software that has been shown to be a reliable method of data extraction for meta-analyses.28 RCTs were quality assessed using the CONSORT criteria;29 scores reflect the percentage of CONSORT items the study adhered to. We aligned depression scales so higher scores reflect higher symptom severity by multiplying scores from reversed scales by -1. For studies that included multiple intervention arms based on different dosages of the same drug, we pooled means and s.d.s of these groups to calculate a single outcome measure. Here, the pooled s.d. (sdpooled) equalled the square root of (sd1^2 × (n1−1)+sd2^2 × (n2−1))/((n1−1)+(n2−1)). Effect sizes were calculated as standardised mean difference (SMD) for continuous outcomes according to the formula 1Group1 × Outcome2Group2)/(Outcome2Group1 × Outcome1Group2). Then odds ratios were transformed to SMD by dividing the natural logarithm of the odds ratios by 1.81.30Data were extracted from published articles or obtained by directly contacting the authors if relevant data were not reported. When authors could not be reached but data were available in the paper in graph format (two RCTs),31 In addition, we calculated SMD using change in depression severity score from baseline to end of trial in each group to examine the robustness of original results . Sensitivity analyses were also carried out after excluding specific RCTs from meta-analysis. For other trials, SMD was calculated as difference in depression severity score from baseline to end of trial, as no control group was present. All studies were weighted using an inverse-variance method so studies with larger samples were given higher weight. When possible, pooled effect estimates were calculated for specific drugs. Heterogeneity between studies was investigated by calculating the Cochrane’s heterogeneity statistic Q and the I2 statistic that represents the fraction of variation between studies attributable to heterogeneity.32 Publication bias was assessed for each group of studies by visual inspection of funnel plots including Egger’s test,33 and the trim and fill method.34 We analysed the RCTs of adjunctive treatment with anti-cytokine therapy separately because these studies compared combination of anti-cytokine drug and a disease-modifying anti-rheumatic drug (DMARD) with DMARD alone. One RCT35 administered adalimumab to all participants for 4 weeks before randomising them to placebo or adalimumab for subsequent 52 weeks, so we also included data from the first 4 weeks in the meta-analysis of ‘other trials’. Data from two studies37 that randomised individuals to different dosage regimens of the same drug but lacked a placebo group, and a non-randomised study38 were also included in this category.Based on study methodology, three types of studies were combined separately using random effect meta-analysis: RCTs of anti-cytokine drug vs placebo; RCTs of adjunctive treatment with anti-cytokine therapy (anti-cytokine drug plus active treatment vs active treatment); and other trials (non-randomised and/or non-placebo studies). For RCTs effect estimates were calculated as SMD using depression severity scores in treatment and placebo groups at the end of trial. SMD is an appropriate measure of effect estimate when studies assess the same variable  but measure it in a variety of ways.43 that were used to calculate effect sizes (SMD) of baseline symptom severity for the sample compared with the general population. All analyses were performed in R using the metaphor package for meta-analysis and meta-regression;44 analysis codes are available on request.All secondary analyses were based on placebo-controlled RCTs only as they represent the highest quality of scientific evidence of efficacy. Meta-regressions were performed to examine the relationship between the effect estimates for depression and (1) the effect estimates for primary physical outcome; (2) baseline severity of depressive symptoms; (3) sex ; and (4) mean age of sample. We quantified baseline depression symptom severity in a standardised manner rather than simply using mean scores because the studies used different scales to measure depression. We identified general population distributions for each scaleThe literature search retrieved 350 potentially relevant articles of which 20 studies were finally included in the review see .47 involving 1309 subjects treated with anti-cytokine drugs and 1061 subjects treated with placebo, showed significant improvement in depressive symptoms with anti-cytokine treatment compared to placebo; SMD=0.40  . Anti-TNF drugs were most commonly studied (five RCTs),46 which as a group showed significant antidepressant effect; SMD=0.33; 95% CI, 0.06–0.60 . Regarding specific drugs, based on two trials each, the effect estimate for adalimumab46  and that for etanercept27  were similar  but there was evidence of heterogeneity among the etanercept trials .Meta-analysis of seven randomised, double-blind, placebo-controlled trials,22–0.59) . There w.06–0.60 . There w similar . There w47 of anti-cytokine drugs vs placebo based on change in depression severity scores from baseline to end of trial yielded very similar results to that of the original analysis; SMD=0.37  .Sensitivity analysis of seven RCTs,50 that used anti-cytokine drugs as adjunctive treatment, two provided enough data for meta-analysis (50 Both of these studies compared combination of etanercept and a DMARD (n=544) with DMARD alone (n=405). Meta-analysis showed a small effect of etanercept plus DMARD on depressive symptoms compared with DMARD alone . There was no evidence of heterogeneity between studies .Of three trialsanalysis .49, 50 B57 eight provided enough data for meta-analysis involving 1744 patients with data at baseline and follow-up . There was evidence of significant heterogeneity among these studies . Anti-TNF drugs were most commonly studied (six studies), which as a group showed significant antidepressant effect  . Regarding specific drugs, adalimumab, infliximab and tocilizumab all showed statistically significant improvements in depressive symptoms. Meta-analytic effect estimates for adalimumab: SMD=0.67 ; infliximab: SMD=0.66 ; and tocilizumab: SMD=0.31 . There was no evidence of significant heterogeneity in any of these meta-analyses .Out of 10 non-randomised and/or non-placebo studies,ollow-up .35, 36, 47 found no evidence for an association between improvement in depressive symptoms and that in primary physical illness outcome measure at the end of trial   . The stu47 showed an association between antidepressant effect of anti-cytokine treatment and baseline severity of depressive symptoms . However, the association became non-significant after excluding the RCT by Raison et al.20 . We explored the association by excluding the RCT by Raison et al.20 because baseline depression severity was much higher in this study (SMD of baseline depression 6.36) compared with the other RCTs (0.08–1.91).Meta-regression of seven RCTs47 did not reach significance . Re-analysis after excluding the study by Raison et al.,20 an outlier in which only 33% of subjects were male, attenuated the association further . Similarly, there was no association between SMD for depressive symptoms and mean age of sample .There was no strong evidence for a sex difference in the antidepressant effect of anti-cytokine treatment as the association between improvements in depressive symptoms and the percentage of male subjects in the sample, based on meta-regression of seven RCTs,et al. .Duration of follow-up in the RCTs of anti-cytokine drug vs placebo was similar (12–16 weeks) except the study by Loftus et al. . Meta-re47 excluding two studies by Tyring et al.27 from which data were extracted using software did not change results substantially; SMD=0.37  . Unlike the other RCTs, the trial by Raison et al.20 was based on cases of treatment-resistant depression without any significant physical comorbidity. Meta-analysis of RCTs47 excluding this trial yielded a slightly larger effect estimate; SMD=0.46  . The RCT by Loftus et al.35 was an outlier in terms of follow-up length  .Sensitivity analysis of RCTsngth see . Meta-anP>0.05). Similarly, trim and fill analyses did not show any evidence of funnel plot asymmetry for RCTs. There were too few studies to allow assessment of publication bias among RCTs of adjunctive treatment with anti-cytokine therapy. For the other trials  Egger’s test for funnel plot asymmetry (P>0.05) and visual inspection of the funnel plot did not suggest any evidence for publication bias .Visual inspection of funnel plot of effect estimates from seven RCTs suggested that there was no evidence of publication bias , we have examined RCTs of adjunctive treatment with anti-cytokine therapy, and non-randomised studies offering a comprehensive update of the literature.Findings from this large systematic review of 20 studies including meta-analyses of 16 studies totalling 5063 participants indicate that anti-cytokine treatment improves depressive symptoms. We observed significant results favouring cytokine modulators over respective control groups with effect estimates of 0.40 for RCTs of anti-cytokine treatment vs placebo, 0.19 for RCTs of adjunctive treatment with anti-cytokine therapy, and 0.51 for other (non-randomised and/or non-placebo) studies. Regarding predictors of response, additional analyses based on RCTs indicated that the antidepressant effect was associated with severity of depressive symptoms at baseline, but not with improvement in physical illness , sex and age of participants, or study duration. Sensitivity analyses using a different method to calculate SMD or exclusion of specific RCTs did not alter results substantially suggesting that the findings are robust. The results provide important clues regarding the role of inflammatory cytokines in depression and the potential for cytokine modulators as treatments for depression. Anti-cytokine drugs seem to offer treatment effects in the range of small-to-moderate effect sizes, which is comparable to estimates observed for common antidepressants.et al.20 report continuous measures of depression, that is, symptom severity scores, not a categorical diagnosis of depression. To calculate effect size we have used depression severity scores for treatment and placebo groups at the end of trial for all RCTs including one by Raison et al. We also used change in depression scores from baseline to end of trial. The lack of an association between the improvement in depressive symptoms and that in primary physical illness points to a causal role for inflammatory cytokines in depression, suggesting that the mood improvement is not simply an artefact of feeling physically better after anti-cytokine treatment. Examination of the time course of effect in individual studies may provide some clues regarding the underlying mechanism of antidepressant effect of anti-cytokine treatment. The RCT by Tyring et al.27 reported scores for fatigue and depression during the course of 12-week treatment with etanercept vs placebo for psoriasis. Compared with placebo, etanercept led to improvements in fatigue by 2 weeks into the study and in depressive symptoms by week 4. In this study improvement in depressive symptoms was associated with improvement in fatigue but not (strongly) with improvement in psoriasis, which is in line with our meta-regression finding. Neurovegetative symptoms such as fatigue, which develop rapidly following immune activation in humans are attributed to actions of inflammatory cytokines on the brain.61 Therefore, it is possible that the antidepressant effect of anti-cytokine treatment is mediated by improvements in cytokine-induced neurovegetative symptoms. This hypothesis needs testing in future studies.Studies included in this review except one by Raison et al.20 included in this meta-analysis showed beneficial effects of infliximab for treatment-resistant depression cases only in those with elevated serum CRP levels at baseline. Treatment-resistant depression cases with elevated inflammatory marker levels may be ideal candidates for RCTs of cytokine modulators for a number of reasons. About a third of all depressed patients are antidepressant resistant62 and about a third of all depressed patients have elevated serum CRP (>3 mg l−1).63 This may not be a coincidence. Indeed, activation of the inflammatory system as reflected by elevated serum inflammatory marker concentrations predicts poor antidepressant response,14 and treatment-resistant patients continue to show elevated cytokine levels.65 Inflammation and consequent activation of the tryptophan-metabolising enzyme indoleamine 2,3-dioxygenase is thought to underlie persistent symptoms despite antidepressant therapy in depressed patients who are ‘inflamed’.66 Inflammatory cytokines such as interferon-γ, TNF-α and IL-6 can induce indoleamine 2,3-dioxygenase.68 In mice, blocking TNF-α with etanercept67 or IL-6 with a monoclonal antibody69 have been reported to prevent depression-like behaviour following exposures to an inflammatory stimulus or stress. Therefore, cytokine-modulating therapy is likely to be beneficial for a subset of depressed patients, specifically those with evidence of inflammation.In future, RCTs of anti-cytokine treatment using depression as the primary outcome in subjects with high inflammation who are free of other physical illnesses are needed. Although further studies are currently underway , results from the RCT by Raison 23 This is possibly because depression severity was relatively low in all of the RCTs except the one by Raison et al.,20 which included cases of treatment-resistant depression. It is well known that baseline symptom severity is positively correlated with antidepressant treatment effect, so demonstration of a beneficial effect of anti-cytokine treatment in samples with relatively low depression scores indicates strong antidepressant properties of these drugs. There was no evidence for an association between antidepressant effect of cytokine modulators and sex or age of participants. Similar to depression, autoimmune chronic inflammatory conditions such as rheumatoid arthritis are more common in women than men (3:1),70 but sex does not influence response to anti-TNF therapy in patients with established rheumatoid arthritis,71 which is in line with our findings. A possible explanation for the lack of an association with age could be limited variability in age among the included studies (<10 years).Regarding specific types of studies, meta-analysis of two RCTs of anti-cytokine drugs as adjuncts to DMARD treatment showed a smaller effect size than that of the placebo-controlled randomised trials, which might be due to potential antidepressant effects of drugs used as comparison treatment. On the other hand, the pooled effect size for the non-randomised and/or non-placebo studies was larger than that for RCTs probably because of the lack of a comparison group to account for placebo effect. The association between baseline severity of depressive symptoms and response to anti-cytokine treatment was markedly reduced after excluding an outlier although an association between initial severity and antidepressant response is well known.27 However, the software used to extract data has been reported to be a reliable method for data extraction for meta-analysis,28 so any variation from original data would be too small to have any meaningful impact on the pooled effect estimate. Because a physical illness was the primary outcome in all but one studies included in this review, we were not able to comment on potential side-effects of cytokine-modulating treatment in depressed individuals specifically. Administration of monoclonal antibodies carries the risk of immune reactions such as acute anaphylaxis as well as various target-specific adverse effects including increased risk of infections, cancer and autoimmune disease.72 Infliximab and placebo groups in the study by Raison et al.20 were similar in terms of adverse events (except increased urinary leukocyte esterase in the placebo group) and no serious side-effects were observed in either group. Nevertheless, further studies regarding safety and tolerability of cytokine modulators in depressed individuals are needed. There was evidence for significant heterogeneity in the meta-analysis of RCTs and that of other trials. Heterogeneity is likely to be driven by heterogeneity of effect size because all studies included in these analyses except one showed effect in the same direction, that is, anti-cytokine treatment improves depressive symptoms  helped to demonstrate the relevance of inflammation, particularly inflammatory cytokines, for the pathogenesis and treatment of depression. A potential limitation is the use of data extracted from published graphs due to lack of response from authors of two studies.toms see . HeterogIn conclusion, this systematic review and meta-analysis provides an up-to-date summary of the existing literature on antidepressant effect of anti-cytokine treatment. The findings show robust improvements in depressive symptoms after anti-cytokine therapy  with a small-to-moderate size effect. These results suggest inflammatory cytokines may have a key role in the pathogenesis of depression and that anti-cytokine drugs may be effective for some patients with depression, particularly treatment-resistant cases characterised by increased inflammation. The field now needs RCTs of anti-cytokine treatment using such patients, which would pave the way for novel, effective and personalised treatment for depression and could reduce the burden presented by such a serious and multifactorial illness.


Late gadolinium enhanced cardiovascular magnetic resonance (LGE-CMR) has excellent specificity, sensitivity and diagnostic accuracy for differentiating between ischemic cardiomyopathy (ICM) and non-ischemic dilated cardiomyopathy (NICM). CMR first-pass myocardial perfusion imaging (perfusion-CMR) may also play role in distinguishing heart failure of ischemic and non-ischemic origins, although the utility of additional of stress perfusion imaging in such patients is unclear. The aim of this retrospective study was to assess whether the addition of adenosine stress perfusion imaging to LGE-CMR is of incremental value for differentiating ICM and NICM in patients with severe left ventricular systolic dysfunction (LVSD) of uncertain etiology.We retrospectively identified 100 consecutive adult patients (median age 69 years (IQR 59–73)) with severe LVSD (mean LV EF 26.6 ± 7.0%) referred for perfusion-CMR to establish the underlying etiology of heart failure. The cause of heart failure was first determined on examination of CMR cine and LGE images in isolation. Subsequent examination of complete adenosine stress perfusion-CMR studies  was performed to identify whether this altered the initial diagnosis.On LGE-CMR, 38 patients were diagnosed with ICM, 46 with NICM and 16 with dual pathology. With perfusion-CMR, there were 39 ICM, 44 NICM and 17 dual pathology diagnoses. There was excellent agreement in diagnoses between LGE-CMR and perfusion-CMR . The addition of adenosine stress perfusion images to LGE-CMR altered the diagnosis in only two of the 100 patients.The addition of adenosine stress perfusion-CMR to cine and LGE-CMR provides minimal incremental diagnostic yield for determining the etiology of heart failure in patients with severe LVSD. Identifying the etiology of heart failure has important management and prognostic implications . TherapeLate gadolinium enhanced CMR (LGE-CMR) has been shown to be non-inferior (and indeed may be superior) to coronary angiography in differentiating heart failure due to coronary artery disease (CAD) from NICM , 9. SubeCMR first-pass myocardial perfusion imaging (perfusion-CMR) has been shown in several large studies to have excellent sensitivity and specificity for detection of CAD and may thus play a role in distinguishing heart failure of ischemic and non-ischemic origins , 15. HowAdenosine stress perfusion-CMR imaging adds time (approximately 15 min added time for the perfusion-CMR protocol) and expense to the CMR protocol, due to the need for additional electrocardiograms, adenosine preparations, infusion pumps and lines, as well as physician-supervision. Undesirable symptoms and potentially serious complications may occur with adenosine infusion, albeit infrequently –19. ThesIn our regional cardiac centre, over 1000 clinical perfusion-CMR studies are performed annually. Approximately 10% of these are to identify the cause of heart failure, and despite a lack of evidence of additional benefit, stress perfusion is usually requested and performed.The aim of this retrospective study was to assess whether the addition of adenosine stress perfusion imaging to LGE-CMR is of incremental value for differentiating ICM and NICM in patients with severe LVSD of uncertain etiology.We retrospectively identified 100 consecutive adult patients with severe LVSD on echocardiography, referred for perfusion-CMR to ascertain the underlying etiology of heart failure. Patients were scanned between April 2015 and March 2016. Severe LVSD was defined as a left ventricular ejection fraction (LV EF) ≤35% and was confirmed by CMR volumetric and functional analysis prior to inclusion in the study. Participants’ past medical history, medications, electrocardiographic data, resting pulse and blood pressure were recorded at the time of the CMR.The study was approved as a clinical audit and ethics approval was deemed unnecessary.Clinical perfusion-CMR studies were undertaken on either a 1.5 T  or 3 T scanner . Cardiac volumes and function were performed using standard CMR techniques as previously described by our group . For strScans were anonymized and sent to a separate workstation for analysis which was performed blinded to all patient details, by two specialists in CMR (JRA and JK) as recommended by expert consensus . No clinCoronary angiograms were examined in those patients in the cohort who underwent angiography on clinical grounds. Images were analyzed blinded to patient details and CMR image interpretation results by an experienced cardiologist (GPM). Coronary artery disease was pre-defined as being present if a coronary artery stenosis of ≥50% the luminal diameter of the artery was observed and was noted as severe if stenosis severity was >70%.Normality was assessed using Kolmogorov-Smirnov tests, histograms, and Q-Q plots. Continuous data were expressed as mean ± standard deviation, if normally distributed. Non-parametric variables were expressed as median and interquartile range. One-way analysis of variance (ANOVA) was used to compare normal data and the Independent-Samples Kruskal-Wallis test for non-normal data between groups. The Kappa statistic was used as a measure of agreement between the components of the CMR scans with and without stress perfusion. Statistical analysis was undertaken using SPSS version 20.0 software.The study group consisted of 100 patients referred for clinical stress perfusion-CMR to investigate the etiology of severe LVSD. Demographic characteristics and CMR volumetric and functional data are shown in Table n = 15, 94%) of patients with dual pathology had a combination of hyperenhancement patterns .Image quality was excellent or good in 98 and poor in 2 studies. Thirty-eight patients were classified as having ischemic, 46 non-ischemic and 16 dual pathology as the cause of LVSD (Table p=<0.001)  patients with infarction and in 1 patient with no LGE. There was excellent agreement in diagnoses between LGE-CMR and perfusion-CMR  Table . The per1) Table , but not1) Table . The dia1) Table . Subsequ1) Table  and the n = 30, 93.8%) of these showed significant CAD. Both patients who underwent angiography that did not reveal significant CAD had LGE-CMR and perfusion-CMR diagnoses of NICM. LGE-CMR had a sensitivity of 87% for predicting significant CAD in subjects with severe LVSD, identifying ICM or dual pathology in 26/30 patients with significant CAD on angiography. Sensitivity of perfusion-CMR for predicting significant CAD was 90% . Only three cases of CAD identified by angiography were not detected by either LGE-CMR or perfusion-CMR . Perfusion-CMR did, however, identify reversible ischemia in 43% of patients with a non-stress-CMR diagnosis of ICM/dual pathology. Whilst this did not alter the diagnosis in these patients it may have implications on clinical management. Generally both LGE-CMR and perfusion-CMR had excellent sensitivity for detection of CAD. Importantly these analyses were made entirely blinded to patient details and medical history, which in clinical practice would ordinarily guide risk stratification and decision-making and raise suspicion of CAD.Our study demonstrates that stress perfusion-CMR is of minimal incremental benefit in diagnosing the cause of severe LVSD. It is therefore contentious whether stress testing should be routinely performed in this context. Patients with severe LVSD attributed to previous infarction on LGE-CMR with a likelihood of functional recovery often undergo coronary angiography and revascularisation. However the role of revascularization in patients with ICM in the absence of symptoms is controversial. In the STITCH trial, there was no significant difference in outcomes when patients with heart failure and coronary artery disease underwent surgical revascularization versus medical therapy alone . In suchThe present study challenges the incremental role of perfusion-CMR over LGE-CMR for diagnosing the etiology of heart failure in severe LVSD. Clinical guidelines do not specifically recommend perfusion-CMR for this purpose , 5. HoweThe retrospective and single-centre design limits the strength of this study, as does the relatively small sample size. Coronary angiography, as the reference standard for CAD, was not performed on all subjects in the cohort to exclude CAD. Computed tomography coronary angiography can be used to reliably exclude the presence of CAD but will be difficult to interpret in high risk patients with coronary calcium and does not provide prognostic information related to LGE .Adenosine stress perfusion-CMR is of minimal additional benefit to cine and LGE-CMR for determining the etiology of heart failure in patients with severe LVSD. Prospective studies are required to define the role of perfusion-CMR in heart failure and identify those patients most likely to benefit from the addition of perfusion imaging to LGE-CMR.


P = 0.001), disease-specific survival (DSS) (P < 0.001), and disease-free survival (DFS) (P = 0.003) than patients with poorly differentiated GSCC. Moreover, matched-pair analysis indicated that increased differentiation was associated with a significantly reduced risk of overall death , death owing to disease , and disease recurrence , and these risks were reduced approximately 4-fold, 3.7-fold, and 9-fold, respectively, after adjustment for cancer-associated variables. Survival differed significantly between the well-differentiated and poorly differentiated GSCC patients after adjustment for cancer prognosis-associated variables. Thus, identifying potential differences in the molecular characteristics between these two groups of patients would help to further stratify these patients and ensure appropriate individualized treatment decisions. Basing treatment strategies on the level of differentiation may improve survival.To compare survival outcomes between patients with poorly differentiated versus well-differentiated glottic squamous cell carcinoma (GSCC). Fifty-five patients with well-differentiated newly diagnosed GSCC were pair-matched to 55 patients with poorly differentiated GSCC according to age, sex, year of diagnosis, overall stage, treatment , smoking, and alcohol use. Survival analysis was performed using Kaplan-Meier estimates, and matched-pair survival was estimated using the Cox proportional hazards regression model. Patients with well-differentiated GSCC had significantly better overall survival (OS) ( Laryngeal squamous cell carcinoma (LSCC) is one of the most common head and neck cancers, and the incidence rate of LSCC ranks second among carcinomas of the respiratory system, after lung cancer –3. LSCC Studies regarding GSCC incidence, mortality, and prognosis often consider it as one entity, and historically, clinicians used TNM stage and smoking status as prognostic markers to guide treatment decisions for GSCC patients. However, the prognosis of these patients differs significantly between those with well-differentiated and poorly differentiated GSCC. For example, in our clinics, we have found that some patients with early TNM stage and poorly differentiated GSCC have a poor prognosis, whereas other patients with advanced TNM stage and well-differentiated GSCC have a good prognosis. Thus, patients with the same stage of GSCC may have different degrees of differentiation. It is our postulation that well-differentiated and poorly differentiated GSCC tumors may have different genetic components and thus different susceptibility to similar treatment, but it is not known whether these differences affect the responses of GSCC tumors to treatment.Although both differentiation and stage are known to affect tumor aggressiveness, the effect of differentiation on the prognosis of GSCC patients at different stages is unclear. Furthermore, the prognosis of patients with GSCC is known to depend on a number of other clinical features, lifestyle factors, and epidemiologic variables, and these prognostic factors may further confound the survival analysis. Matched-pair analysis allows for the removal of many confounding factors and a more accurate comparison of survival. Thus, we were the first to perform a matched-pair analysis of patients with well-differentiated and poorly differentiated GSCC and compare survival to determine whether differentiation has any effect on the survival of GSCC patients at different stages. This study mainly aimed to provide new information to help make decisions about which treatment should be used to improve the individualized treatment of GSCC patients.2 laser microlaryngoscopic resection. Neck dissection included selective neck dissection, modified neck dissection, and radical neck dissection. A minority of patients (23.6%) in each group received postoperative radiotherapy or chemotherapy on the basis of their pathologic results.Table vs 68.8% for 3-year OS and 90.2% vs 50.9% for 5-year OS), and the patients with early disease had better 3-year and 5-year OS rates than the patients with advanced disease (96.3% vs 70.6% for 3-year OS and 92.9% vs 52.2% for 5-year OS). Similar differences were found for DSS and DFS between these two groups (data not shown). However, the study was designed to avoid selection biases by comparing groups tightly matched on the major prognostic, demographic, and treatment variables. Although this study was designed to compare whether patients with well-differentiated GSCC had significantly better survival than those with poorly differentiated GSCC, such analysis was seriously confounded by disease stage. We found that patients with well-differentiated GSCC and early disease stage had the best survival, and patients with poorly differentiated GSCC and advanced disease stage had the worst survival. However, patients with late disease stage and well-differentiated disease had significantly better survival than those with early disease stage and poorly differentiated disease. The 3-year and 5-year OS rates for patients with well-differentiated and stage I/II disease, well-differentiated and stage III/IV disease, poorly differentiated and stage I/II disease, and poorly differentiated and stage III/IV disease were 100% and 100%, 91.1% and 83.5%, 84.7% and 61.2%, and 41.2% and 33.0%, respectively.As expected given the different degree of differentiation and disease stage, the patients with well-differentiated GSCC had better 3-year and 5-year OS rates than the patients with poorly differentiated GSCC (93.5% P = 0.013). Patient outcomes for the two groups are listed in Table P = 0.001, P < 0.001, and P = 0.003, respectively). Figure P < 0.001).Follow-up time ranged from 4.2 months to 170.6 months, with an average of 62.2 months  for well-differentiated patients and 47.1 months  for poorly differentiated patients. The follow-up time differed significantly between the two groups (P = .00). Multivariable analysis was performed for further adjustment with factors that significantly affect prognosis. The well-differentiated patients had an approximately 75% reduced risk of overall death compared with the poorly differentiated patients after multivariate adjustment . Multivariate analysis was performed, fully adjusting for other important confounders, and the risk of death owing to disease was reduced approximately 75% in the well-differentiated patients , and this risk among well-differentiated patients was reduced approximately 90% compared with the poorly differentiated patients after further adjustment for other important prognostic confounders  [In a study of 1252 cases of LSCC, the 5-year control rates of poorly differentiated and well-differentiated LSCC were 44% and 76%, respectively ( < 0.01) . Poorly  < 0.01) . AlthougWe found that among patients who didn't receive postoperative chemoradiation, patients in the poorly differentiated group had worse prognosis than those in the well-differentiated group. Postoperative chemoradiation could play an important role in the improvement of prognosis. Hence, surgery alone might not be suitable for GSCC patients with poorly differentiated tumors, even those with early-stage disease, and chemotherapy should be considered or even preferred.Several factors, including age, sex, TNM staging, surgical margins, and lymphatic metastasis, may confound the prognosis of GSCC –12. The Our findings suggest that well-differentiated and poorly differentiated GSCC could have different landscapes of genetic and epigenetic changes. It is therefore biologically reasonable to find differences in their clinical behavior, as molecular changes may result in more aggressive disease in poorly differentiated GSCC and lead to a worse prognosis.At present, our treatment approaches have not substantively changed, but our findings suggest that more targeted therapeutic methods may help improve outcomes and optimize the patients’ quality of life. Some GSCC patients who present with more advanced disease and well-differentiated tumors have better survival than patients with early stage and poorly differentiated tumors. The current American Joint Commission on Cancer (AJCC)/Union for International Cancer Control (UICC) TNM staging system for GSCC was developed for differentiation-unrelated GSCC. A new staging system is needed to adequately predict outcomes of patients with differentiation-related GSCC.Several limitations may exist in this study. It is likely that selection biases affecting either the patients referred to our institution or the patients recruited to this study could exist. We did not compare the outcomes of patients treated with surgery versus chemoradiation alone, and a prospective study is necessary to analyze different treatments for patients with poorly differentiated GSCC. Furthermore, GSCC patients were identified for enrollment without a strictly defined screening or follow-up regimen, and some patients had short follow-up times. Longer follow-up of these patients could help clinicians monitor the patients’ condition. Finally, we did not collect some other important data on smoking, such as intensity and duration for pack years smoked as well as family cancer history, while we are collecting such data and continuing to recruit such patients for our future larger studies.In conclusion, GSCC patients with poorly differentiated tumors had worse OS, DSS, and DFS compared with those with well-differentiated tumors and equivalent age, sex, year of diagnosis, disease stage, smoking and alcohol use, and treatment in our hospital. These results are substantively confounded by disease stage. Although our findings suggest that the poorly and well-differentiated GSCC patients may have more fundamental differences, further studies are required to elucidate the molecular differences between these two groups and the effect of disease differentiation on survival. Therefore, more attention should be paid to the treatment of poorly differentiated GSCC. To improve survival and quality of life, when assessing the prognosis of patients with different tumor stages, the impact of differentiation is critical to making treatment decisions, particularly for poorly differentiated GSCC patients. Although confirmation of our findings in other patient populations is needed, we propose consideration of new GSCC categories as an alternative to the traditional GSCC categories.We reviewed the medical records of more than 1000 consecutive patients with newly diagnosed, previously untreated GSCC who were treated at Beijing Tongren Hospital  from September 1997 to December 2013. All subjects signed an informed consent form; and the study protocol was approved by the institutional review board of Tongren Hospital. Only incident cases of pathologically confirmed poorly differentiated primary GSCC without distant metastasis or localized resectable lesions were enrolled. The exclusion criteria were as follows: (1) supraglottic or subglottic laryngeal carcinoma; (2) moderately or well-differentiated laryngeal cancer; (3) non-squamous cell carcinoma; (4) secondary onset; (5) recurrent disease; and (6) no initial treatment or only palliative care, or initial treatment at a different hospital.Fifty-five patients with poorly differentiated GSCC were available for matching. We used the patient chart database, which contains epidemiologic and clinical information  to establish our matched-pair analysis. Well-differentiated GSCC was defined as tumor containing less cellular atypia and more keratin pearls, whereas poorly differentiated GSCC was defined as containing more cellular atypia and mitotic count, without keratin pearls.vs 2011–2014), treatment received , disease stage (stage I/II vs stage III/IV), smoking, and alcohol use. Fifty-five pairs of poorly differentiated and well-differentiated GSCC cases were matched. A control group of well-differentiated GSCC patients was selected at the same time. The matched-pair data were followed at a 1: 1 ratio. The pairing criteria were as follows: (1) age difference ≤ 5 years; (2) same sex; (3) diagnosis time < 1 year; (4) same TNM stage, clinical stage, therapeutic strategy  surgery as primary choice. All pathological diagnoses based on biopsies were confirmed by two pathologists.After providing informed consent, all patients participating in this study had their demographic and exposure information retrospectively retrieved from their chart records. “Ever smokers” were those subjects who had smoked more than 100 cigarettes in their lifetime, whereas “never smokers” had smoked 100 or fewer. “Ever drinkers” were those individuals who drank alcoholic beverages at least once a week for more than 1 year, and the others were “never drinkers.” The patients’ medical records were also reviewed for primary site, clinical stage, treatment, differentiation, and outcome events. From this cohort, each poorly differentiated GSCC patient was matched with one well-differentiated GSCC patient. The matching variables were age (within ± 5 years), sex, year of diagnosis  was used for the statistical analyses. The primary outcome events of this study were overall deaths, deaths owing to disease, and recurrence. Time to recurrence was defined from the date of the end of treatment to the date of last follow-up or clinical detection of recurrent cancer . Participants who were recurrence free or lost to follow-up were censored. Overall survival (OS) was defined as the time from first appointment to death from any cause or date of last follow-up. Disease-specific survival (DSS) was defined as the time from first appointment to death from disease or date of last follow-up. For both OS and DSS calculations, participants who were alive at the end of the study period or lost to follow-up were censored.The differences in disease-free survival (DFS), DSS, and OS were compared between the well-differentiated and poorly differentiated GSCC patient groups using Kaplan-Meier estimates and the log-rank test for equality of survival curves. Matched survival analysis was completed using the Cox proportional hazards regression model. The assumption of proportionality was tested and met for the Cox proportional hazards analysis. Matching was accounted for in the Cox proportional hazards model by including a matching variable, which accounted for the matching in the analysis based on age, sex, year of diagnosis, treatment , disease stage, smoking, and alcohol use. Those factors, which were significantly different, were used as variables in the Cox proportional hazards model. The relative risk was obtained for each type of event between the well-differentiated and poorly differentiated GSCC patients. The power of the study to detect the relative risk obtained was based on the model of Dupont and Lummer . Given o


Bone marrow mononuclear cell (BM-MNC) therapy in ST-elevation acute myocardial infarction (STEMI) has no biological inclusion criteria. Here, we analyzed 63 biomarkers and cytokines in baseline plasma samples from 77 STEMI patients treated with BM-MNCs in the TIME and Late-TIME trials as well as 61 STEMI patients treated with placebo. Response to cell therapy was defined by changes in left ventricular ejection fraction, systolic/diastolic volumes, and wall motion indexes. We investigated the clinical value of circulating proteins in outcome prediction using significance testing, partial least squares discriminant analysis, and receiver operating characteristic (ROC) analysis. Responders had higher biomarker levels (76–94% elevated) than non-responders. Several biomarkers had values that differed significantly (P < 0.05) between responders and non-responders including stem cell factor, platelet-derived growth factor, and interleukin-15. We then used these lead candidates for ROC analysis and found multiple biomarkers with values areas under the curve >0.70 including interleukin 15. These biomarkers were not involved in the placebo-treated subjects suggesting that they may have predictive power. We conclude that plasma profiling after STEMI may help identify patients with a greater likelihood of response to cell-based treatment. Prospective trials are needed to assess the predictive value of the circulating biomarkers. One current hypothesis is that its efficacy is limited because of poor engraftment into the heart after delivery and rapid cell death4. Schemes to increase cell survival and engraftment include, among many other, support scaffolds5 and co-delivery of pro-survival agents7. However, these approaches do not address a deeper issue: within any patient population that there is likely a wide variation in the effectiveness of stem cells. Current inclusion criteria for ST-elevation myocardial infarction (STEMI) cell therapy trials8 largely use cardiac function indexes such as left ventricular ejection fraction (LVEF). Yet, these indices only reflect the extent of cardiac damage after reperfusion and provide guideline for subsequent medical therapy. They offer no molecular level information to better identify patients who would benefit from cell therapy. In addition to current selection criteria, circulating proteins might have additional value in predicting individual response to cell therapy. Such biomarkers and cytokines might indicate a robust microenvironment capable of promoting cell survival and functional recovery. Just as biomarkers for graft-versus-host disease following allogeneic hematopoietic cell transplantation have the promise to predict response10, we hypothesized that this approach might identify positive responders in cell-based therapy for STEMI patients.Cell-based cardiac therapy has produced encouraging but inconsistent results in treating ischemic heart disease11, which are known to have a role in myocardial regeneration. Similarly, biomarkers involved in cell growth 12, repair 12, adhesion 13, and inflammation (interleukins and chemokines) might predict the outcome of cell therapy.Candidate biomarkers include circulating proteins such as stem cell factor (SCF), stromal cell derived factor (SDF1), and colony stimulating factors6) or placebo. The data were obtained from both the TIME14 and LateTIME trials15 conducted by the Cardiovascular Cell Therapy Research Network. While these trials did not show an aggregate benefit to therapy relative to placebo controls, there were “responders”, i.e., patients in whom cardiac function improved, within both the cell-treated and placebo-treated cohorts. Our goal here was to profile the serum of responders and non-responders in both cohorts and identify the differences in biomarker profiles, which correlated positively with a response to cell therapy.In this study, we measured 63 circulating proteins in baseline serum samples obtained from reperfused STEMI patients (N = 138) who were then treated with autologous BM-MNCs  and Late-TIME trial , which were conducted between July 2008 and November 2011. Both studies assessed left ventricle function at baseline and six months after intracoronary delivery of BM-MNCs after STEMI. BM-MNC administration was performed 3 and 7 days (TIME) or 14–21 days (Late-TIME) after reperfusion. The primary inclusion criterion was a left ventricle ejection fraction (LVEF) <45% as assessed by echocardiography. Specimens in TIME were collected within a week of the myocardial infarction. Specimens in LateTIME were in general 2–3 weeks after the infarction. Patients with previous bypass surgery or prior STEMI with residual left ventricular dysfunction were excluded. Blood samples were collected after reperfusion but prior to administration of cells in tubes containing EDTA as an anticoagulant. Blood samples were centrifuged and plasma was decanted and stored at −80 °C. All samples were collected after subjects provided written informed consent. This study received Institutional Review Board approval by the University of Texas IRB. All patients provided written informed consent following broad discussions of the risks, benefits, and alternatives of the TIME and LateTIME trials. All methods were performed in accordance with the relevant guidelines and regulations and the Declaration of Helsinki.16.Patients were classified as either responders or non-responders based on the original outcome data. For example, a patient with a positive ΔEF was considered a responder as was a patient with a decreased ΔEDVI. In addition to the five metrics above, we also included a subset of high ΔEF responders. These were patients with a ΔEF of >5 (absolute increase in ΔEF >5). Next, for each biomarker, a mean and two-sided student’s t-test were calculated using the raw mean fluorescent intensity (MFI) data. This MFI approach has utility when samples have low levels of biomarker because interpretation with a sigmoidal calibration curve can suppress subtle differencesWe compared the mean biomarker levels between the cell-treated and placebo-treated cohort, and calculated P values  to determine the significance of these changes. These significant biomarkers as well as biomarkers with mean values of more than 2.0-fold different between responders and non-responders were characterized with receiver operating characteristic (ROC) curve; an area under the curve (AUC) and an associated P value was calculated with GraphPad Prism. P values < 0.05 were considered statistically significant.17. We assumed that the number of latent factors was not significantly different from the model with the minimum predicted residuals sum of squares (PRESS) value18. These latent variables were then used in the discrimination analysis instead of the original individual predictors (biomarkers).PLS-DA analysis used SAS software, version 9.3, and JMP Genomics, version 6.0 . Samples were adjusted for age, sex, and BMI. Using PLS-DA, we assessed the relation between circulating biomarkers and absolute or relative changes in ejection fraction (EF), end diastolic volume index (EDVI), end systolic volume index (ESVI), infarct zone wall motion (INZ), or border zone wall motion (BDZ) analyzed as continuous variables. In categorical analysis, we identified a pattern of cytokines associated with response to stem cell therapy (responders versus non-responders). PLS-DA creates several linear combinations (latent factors) of the log-transformed predictors to maximize the covariance between the predictors and the outcome variables19. In both continuous and categorical analysis, cytokines with a VIP >1.5 were considered influential. The distributions of all cytokines were analyzed by transformation to the common logarithm. The VIP score was calculated as the sum of the squared correlations between the latent factors and the biomarker, weighted for the percentage of variation explained by the latent factors in the model. Finally, V-plots were constructed to illustrate the importance of each biomarker in the model  in relation to the coefficient size of each biomarker (y axis). These V plots illustrate the contribution of all biomarkers to the model used to explain outcome as well as their effect on the outcome.The importance of each cytokine in the construction of the latent variables is assessed from the variables importance for projection (VIP) scores as proposed by WoldWe also performed NRI analysis in the cell-treated patients to determine whether the selected biomarkers better reclassified the treated patients by response. The default classifier consisted of age and sex, whereas the new classifier additionally included the latent factor(s) derived from the informative biomarkers and related to the treatment response 14 and Late-TIME15 manuscripts. The majority of the participants were male (73%), white (78%), and hyperlipidemic (70%). Table Detailed descriptions of the trial participants are available in the TIME21. The inset and caption of Fig. We validated the assays using standard curves and calibration samples for every biomarker according to standard Luminex protocols Fig. 16,20,21.Next, the mean of the 63 biomarkers measured in the baseline samples were compared between the responders and non-responders using the raw data (MFI). We used these mean values to compare the fold-change in biomarker levels between responders and non-responders Fig.  in both The fold-change in the ΔEF cohort is measured by averaging the biomarker value of responders normalized to the average biomarker value of non-responders in the treated cohort Fig. . Assumin). Multiple biomarkers had AUC values over 0.60.In many of the highest fold-changes  in continuous and categorical PLS-DA analysis are presented in Supplementary Table .5 in conPLS-DA analysis was repeated in placebo patients  for dEF, dINZ, dBDZ and the triple responders, indicating a strong difference in the proportion of patients reclassified by the set of biomarkers selected by PLS between the two sets of non-responder & responder. In short, it thus might be clinically relevant to assess this set of biomarkers for prediction of response to SC treatment.The NRI is only statistically significant for dEF Table , but the22, the potential clinical role of the baseline measurement of these cytokines in cell therapy for heart disease may enable a personalized approach to predict responders.We identified significant differences in biomarker concentrations between cell therapy responders and non-responders that changed as a function of the different outcome metrics. This finding was attributed to the multiple metrics, which were used to classify different patient response. One consistent finding, however, was the up-regulation of all biomarkers in the responders compared to the non-responders. While increased levels of cytokines predict worse outcomes in hematopoietic cell therapy23, it is possible that elevated levels of cytokines attracted circulating endogenous stem cells, activated endogenous resident stem cells24 or initiated self-repair response to complement the cell therapy. Thus, patients with an overall elevated pool of these biomarkers (at least during this baseline measurement) might be more receptive to endogenous repair.Myocardial infarction results in an inflammatory cascade as the body removes dead cells and matrix debris from the necrotic region while also activating repair mechanisms. Thus, we suspected that the patients in the TIME trial would have higher cytokine levels because they were enrolled at an earlier time point after the onset of STEMI. However, of the 63 biomarkers, 36 were higher in the Late-TIME patients and 27 were higher in the TIME subjects. Only two—IL1RA and IL12P40—showed a significant difference between the two trials. While prolonged periods of inflammation produced worse outcomes and decreased cardiac function26 did not have a pronounced effect in this study. The most frequent biomarkers were SCF and IL15, which were implicated in all analysis methods. SCF had an AUC of 0.63 (ROC P = 0.049) and IL15 had an AUC of 0.70. VCAM-1 was implicated with simple t-testing but ROC analysis did not confirm this finding. Others have suggested that VCAM-1 was a poor predictor of heart disease outcomes27. Eotaxin, an eosinophil chemotactic protein, was implicated in multiple outcome metrics28. PDGFBB and MIP1 were implicated in stem cell migration and their elevated levels in the responder suggested recruitment of endogenous stem cells30. A previous study of MCP1, RANTES, and MIP1a showed that the patients with severe left ventricular dysfunction had elevated levels of these markers one month after STEMI31.Different biomarkers were implicated in different analysis methods and different outcome metrics. The previously reported factors implicated in cardiac repair, e.g., VEGF, SDF1, and GSCF32.The JAK/STAT pathway can mobilize mesenchymal stem cells and may help maintain cardiac function33 after cell therapy in this subset of patients. In one study, overexpression of SCF by cardiomyocytes was shown to promote stem cell migration and improve outcome after MI34, and thus patients with elevated levels of SCF might be more receptive to cell therapy.The PLS-DA analysis identified candidate biomarkers that were consistently up-regulated in patients who responded via multiple outcome metrics. These highly cross-related markers included SCF, MCSF, and IL12p70, which could have roles in either priming the stem cell niche or supporting the implanted stem cells. MCSF and IL12p70 are implicated in the activation of monocytes and T-cells, respectively, and thus may have a role in immune clearance of necrotic tissue; SCF supports stem cell recruitment and activation. SCF, the ligand for c-Kit, was reported to activate multiple signaling pathways including RAS/ERK, PI3-Kinase, Src kinase, and Janus kinase/signal transducer, the activator of JAK/STAT transcription pathways36. Rodent studies showed an anti-apoptotic role for IL-15 with anti-tumor activity37. Elevated IL-15 might protect stem cells and encourage proliferation. A recent study of showed that IL-15 and MCP-1 indicate myocardial response to ischemic insult in the early phase after MI via an inflammatory response38. Other cytokines like IL-8 and ENA78 correlated with improved outcomes  cell proliferation and hematopoiesismes Figs . These cOne limitation of this study is its retrospective design. It was impossible to do biomarker profiling before therapy due to the retrospective study design, however we compared all treated subjects and placebo subjects (ignoring responders versus responders) to see if there were any differences in biomarkers levels. The following biomarkers had a significant  difference: TNFA, IFNG, PAI1, IL18, IFNA, IL6, NGF, MCP3, RANTES, EOTAXIN, IL17A, IL4, IL10, IL22, GMCSF, BDNF, IL12P40, IL1RA, GCSF, and MCSF. However, none of these biomarkers were implicated as predictive in our PLS-DA analysis. Importantly, there was no statistically significant difference in the placebo and treatment cohort for IL-15, SCF, or PDGFBB, which were the most predictive biomarkers.41, exosomes, connexin 4342, and novel small molecules including miRNA.Future prospective studies are needed to confirm the panel of biomarkers identified in our study as predictors of cell therapy response in the early phase following STEMI. However, the use of both placebo and cell-treated subjects was an important step to validate the findings. The leads generated in the cell-treated cohort were not implicated in the placebo-treated group. Other targets for future panels include osteopontinWe observed that the patients who improved after placebo also had biomarkers that correlated with outcome. Some of these biomarkers were also seen in the BM-MNC-treated cohort including Eotaxin and MIP1a, which suggests that these improvements might occur regardless of cell therapy. However, IL-15 and SCF were the two lead candidates from the BM-MNC-treated group that had no correlation in the placebo subjects. Thus, elevated levels of these markers may have a specific role in increasing the efficacy of autologous BM-MNC therapy.Baseline biomarker measurement is a relatively quick and affordable approach to personalized management of patients, which could increase the efficacy of cell therapy via more rational and precise inclusion criteria. This study demonstrated specific cytokines in both BM-MNC and control groups, which led to improved cardiac function measure. The limitations of this study include the relatively low number of subjects leading to small sample bias. Clearly, our findings require confirmation in replication cohorts with larger patient populations. Another limitation is that the TIME and Late-TIME studies did not show aggregate improvements in the cell-treated patients relative to the placebo-treated patients.In this study, we identified a set of circulating plasma proteins associated with a favorable response to BM-MNC therapy after STEMI. The identified biomarkers might improve the selection criteria to conduct precision medicine, which personalizes the therapeutic decision and enhances the clinical outcome of STEMI patients undergoing stem cell treatment.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Information


Pain management following dental procedures, particularly pulpotomies and extraction, is of great importance in pediatric dentistry. The aim of this study was to investigate the efficacy of pre-treatment with ibuprofen on post-operative pain following pulpotomy of primary molars.  In a split mouth double-blinded randomized clinical trial, 49 children aging between 6-10 years old were given either ibuprofen or a placebo 45 min prior to the treatment. After pulpotomy and placement of a stainless steel crown (SSC), the pain level was evaluated using the Wong-Baker face visual analogue scale for up to 7 days post-treatment. McNemar and Wilcoxon tests were used for data analysis. P=0.032). However, there was no significant difference in the pain levels between placebo and ibuprofen groups at 48 and 72 h post-treatment . The number of times patients needed analgesics in ibuprofen group was significantly lower compared to that in the placebo group (P=0.008). Forty-five patients were eligible to participate in this study. Pre-medication with ibuprofen significantly reduced pain during the first 24 h post-treatment (Pre-medication with ibuprofen resulted in less pain following pulpotomy and SSC placement in primary teeth. One of the main reasons that children find dental procedures unpleasant is pain and discomfort during and after this treatment. The memory of the pain during primary experiences may prevent children from going to the dentist to receive regular check-ups and treatment even years after the first unpleasant experience .It has been reported that 37% to 95% of children may feel pain following various dental procedures, such as tooth extraction or pulpotomy and stainless steel crowns (SSC) -7. PulpoSeveral studies have reported that pre-treatment with analgesic medications significantly reduces post-operative pain after root canal treatment in adults -10. IbupParacetamol and ibuprofen are widely prescribed in children and are the most frequently used over-the-counter analgesics and antipyretics. It has been reported that single doses of ibuprofen (4-10 mg/kg) and paracetamol (7-15 mg/kg) have similar efficacy for relieving moderate to severe pain, and similar safety as analgesics or antipyretics in children. While post treatment prescription of ibuprofen (5-10 mg/kg) is more effective antipyretic than that of paracetamol (10-15 mg/kg) . Most stThis split mouth randomized clinical trial was approved by the Ethics Committee of Kerman University of Medical Sciences in Kerman, Iran (Ref No. KA/93/210) and was also registered online .The mean pain score during the first 24 h following treatment was considered the primary outcome. The sample size calculation was based on figures derived from a relevant study in Turkey [16] considering α=5% and power of 80%, to detect a significant 20% difference in pain score (SD=0.9) a sample size of 44 patients was calculated. A total number of 49 patients aged between 6 to 10 years old  who had been referred to the postgraduate clinic of the Pediatric Department at Kerman Dental School in Kerman, from October 2013 to June 2014, participated in this study.The patients’ parents were fully informed about the nature, aim and method of the study and they provided their informed consent on behalf of their children to participate in the study. The inclusion criteria were: patients aging 6-10 years old who had bilateral primary molar teeth with carious lesions, the teeth were suitable for restoration, no tenderness to percussion and no pain except after eating, teeth without symptoms, no clinical and/or radiographic signs of pulp degeneration, no sinus tract, no or minimal physiological resorption (less than two-thirds of the root length affected), and there was bleeding of the pulp at the time of cavity preparation and pulp exposure.Exclusion criteria were: any systemic diseases that contra-indicated conservative pulp therapy, or radiographic findings such as internal resorption, widening of periodontal ligament space, furcation or periapical radiolucency, and root canal calcifications.In order to carry out a double blinded study, ibuprofen and placebo elixirs were placed in similar single-dose glass bottles. The placebo had the same color, odor and taste as the ibuprofen elixir. The medication dose was calculated based on the patient’s weight. Therefore, each bottle of medication contained 4-10 mg/kg of ibuprofen, while the placebo bottle contained a strawberry-flavored starch suspension. The bottles were encoded and only the pharmacist who prepared the medications was aware of their content. Two bottles containing either ibuprofen or placebo were prepared for each patient and 45 min prior to each appointment, one of the bottles was given to the patient. The operator watched the patient to ensure that the patient drank the entire content of each bottle. After administering local anesthesia using 2% lidocaine with 1:80000 epinephrine  and placing rubber dam, a pulpotomy was performed using a sterile round bur  in a high-speed handpiece with copious water irrigation. Any remaining coronal pulp tissue was removed with a sharp spoon excavator. After all the pulp had been removed, the cavity was rinsed with 0.9% normal saline and a wet cotton pellet was applied to obtain hemostasis. If hemostasis was not achieved in both teeth, the patient was excluded from the study and a pulpectomy was performed for the tooth. Calcium-enriched mixture (CEM) cement  was used as the pulpotomy agent in the teeth with successful hemostasis. Once the material had set, a restorative glass ionomer cement  was placed over the CEM followed by final restoration using a SSC .All dental procedures including anesthesia, pulpotomy and SSC placement were performed by a second year postgraduate pediatric dentistry student. The patients’ parents were asked to complete a Wong-Baker faces pain rating scale (Wong-Baker FPRS) by asking their children how much pain they had. The pain intensity was rated for up to 7 days post-treatment. The Wong-Baker FPRS consists of a row of 6 numbered faces ranging from “no hurt” (score=0) to “hurts worst” (score=5). The pain severity scoring was outlined as follows: 0; no pain, 1-2; mild pain, 3; moderate pain, and 4-5; severe pain. The parents were also given an elixir of ibuprofen  after the treatment to be used as an analgesic in case the child had pain requiring relief. They were also asked to record the number of times the analgesic was used. The maximum use of the analgesic was one dose every 6 h and the parents were given an emergency phone number if there was any need for consultation in case of the patient had severe pain.Data were analyzed with McNemar and Wilcoxon tests and the level of significance was set at 0.05.Forty-nine patients were eligible to participate in this study ; howeverP=0.032)  (P=0.008). Furthermore, the number of the patients who used analgesics in the ibuprofen group (37.8%) was also significantly lower (62.2%) compared to the placebo group (P=0.013)  . Howeverctively) . The patP=0.013) . None ofP=0.013) .P<0.05).The results of the present study have shown that pre-treatment with ibuprofen significantly decreased post-operative pain and the need for analgesics during the first 24 h following pulpotomy and SSC placement in primary molar teeth  and CEM cement, have been used in dentistry for pulp capping, perforation repair, apical plug and root-end filling -23. BothAll previous investigations of pain control in children have focused on strategies to manage distress and discomfort following dental extractions, particularly after general anesthesia , 27, 28.Previous investigations that compared post-operative pain following extraction of primary molar teeth reported that administration of paracetamol, ibuprofen and diclofenac reduced the pain scores following primary teeth extraction in children -16, 28. However, a more recent meta-analysis has shown that ibuprofen is a more effective pain reliever compared to paracetamol which is in accordance with other study that has previously reported [30]. Paracetamol may be considered by some clinicians to be a safer analgesic than ibuprofen which is a non-steroidal anti-inflammatory drug. However, it has been reported that both medications have similar safety spectra .Therefore, in the present study ibuprofen was used as the pre-medication analgesic. Ibuprofen and paracetamol are among the most widely used analgesic compounds for treating mild to moderate dental pain. However, the results of a multicenter and double blind study in patients with postoperative dental pain has been showed that 400 mg ibuprofen provided superior analgesia over 1000 mg paracetamol that was both clinically and statistically significant. Similar efficacy for relieving moderate to severe pain, and similar safety as analgesics or antipyretics in children has been reported with single doses of ibuprofen (4-10 mg/kg) and paracetamol (7-15 mg/kg) . Therefore, in the present study ibuprofen was used as the pre-medication analgesic. Ibuprofen and paracetamol are among the most widely used analgesic compounds for treating mild to moderate dental pain. However, the results of a multicenter and double blind study in patients with postoperative dental pain has been showed that 400 mg ibuprofen provided superior analgesia over 1000 mg paracetamol that was both clinically and statistically significant. Similar efficacy for relieving moderate to severe pain, and similar safety as analgesics or antipyretics in children has been reported with single doses of ibuprofen (4-10 mg/kg) and paracetamol (7-15 mg/kg) .In a preliminary investigation, it has reported that the children pre-treated with an analgesic experienced less pain following various dental procedures . HoweverIn the present study, all children reported pain following the procedure. However, most of them had only mild pain. The reason for the more severe pain in this study compared to previous reports -7 can beIn conclusion, the present study has shown that pre-medication with ibuprofen significantly reduced the amount of post-operative pain during the first 24 h following pulpotomy and SSC placement. It has also reduced the need for using analgesics post-treatment for children undergoing these procedures.


In this study, we developed a high-throughput in vivo model system for identifying muco-active reagents using Xenopus laevis embryos. We tested mucus secretion under various conditions and developed a screening strategy to identify potential muco-regulators. Using this novel screening technique, we identified narasin as a potential muco-regulator. Narasin treatment of developing Xenopus embryos significantly reduced mucus secretion. Furthermore, the human lung epithelial cell line, Calu-3, responded similarly to narasin treatment, validating our technique for discovering muco-active reagents.The airway epithelium in human plays a central role as the first line of defense against environmental contaminants. Most respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and respiratory infections, disturb normal muco-ciliary functions by stimulating the hypersecretion of mucus. Several muco-active agents have been used to treat hypersecretion symptoms in patients. Current muco-active reagents control mucus secretion by modulating either airway inflammation, cholinergic parasympathetic nerve activities or by reducing the viscosity by cleaving crosslinking in mucin and digesting DNAs in mucus. However, none of the current medication regulates mucus secretion by directly targeting airway goblet cells. The major hurdle for screening potential muco-active agents that directly affect the goblet cells, is the unavailability of  The muco-ciliary epithelium of the airway tract is one of the most vulnerable tissues which are constantly challenged by external pathogens or contaminants. The airway epithelium consists of several cell types, such as the mucus secreting goblet cells, multi-ciliated cells, and basal cells . The gobXenopus embryonic epidermis is an emerging in vivo model system with several advantages for studying the muco-ciliary epithelium. The embryonic epidermis of Xenopus is composed of multiciliated cells, goblet cells, ionocytes, and small secretory cells [Xenopus embryonic epidermis to those of mammalian multiciliated cells facilitated research on muticilia formation [Xenopus embryonic epidermis are tractable, and constitute an accessible model for investigating mucus secretion [The ry cells . The simormation , 7. Althormation , recent ecretion , 9, 10.Xenopus laevis embryos. Using this unbiased screening technique, we showed that several known muco-regulators function in similar ways in the Xenopus muco-ciliary epithelium. Furthermore, we identified a new muco-active reagent, narasin. Narasin is a potent inhibitor of mucus secretion in both Xenopus epidermis and the human lung epithelial cell line, Calu-3. We believe that the screening technique developed in this study would benefit researchers studying the pathophysiology of mucus-related disorders.In this study, we developed a unique high-throughput screening strategy for identifying muco-active reagents using Xenopus laevis were housed under 12-h light/dark cycles at 18°C in containers according to the guidance of Animal Care and Use Committee from the Institutional Review Board of UNIST.The Animal Care and Use Committee from the Institutional Review Board of Ulsan National Institute of Science and Technology (UNIST) approved this work . This study was performed in accordance with documented standards of the Animal Care and Use Committee from the Institutional Review Board of UNIST. All members of the research group attended educational and training courses for care and use of experimental animals. Adult Xenopus laevis were obtained from Nasco (U.S.A). The average age of the Xenopus laevis is less than 5 years old and frogs were fed three times in a week with trout pellets from a local supplier. Xenopus were housed in the XR4 husbandry system (aquatic habitats) with a 12-hour light and 12-hour dark cycle. 12 frogs were housed in a 40 liter tank. The water quality for the system was set to the manufacturer’s recommendation . Human chorionic gonadotropin (HCG) was used to trigger ovulation in an adult female one day before the experiments and the animal was squeezed to release the oocytes. A dissected testis from an adult male was used to fertilize the ovulated eggs. Cysteine solution  was used to remove the jelly layer encasing the eggs and the fertilized eggs were grown in 1/3× MMR until stage 32. For the ELLA (Enzyme-Linked Lectin Assay) analysis, four of the St. 32 embryos were nourished in 48 multi-well plates and the media from the wells were collected for further ELLA analysis. At least 5 independent experiments were performed for each condition.Adult 2, 1.6mM HEPES) to clear the secreted mucus particles. After washing, the embryos were incubated for 3 hours in 1/3× MMR containing each library compound in a 48-well plate with a mesh-attached chamber. We used the SCREEN-WELL natural product library (Enzo BML-2865) and 10 mM Diversity Set provided by the National Cancer Institute’s (NCI) Developmental Therapeutic Program's Open Compound Repository. Then, the treated embryos were moved into fresh media without the compounds for an hour, following which the media was collected into a 96-well plate with a U-shaped bottom. The collected media was incubated at 4°C overnight. After incubation, the 96-well plate was washed twice with PTW , followed by incubation in 1% bovine serum albumin (BSA) for an hour. After incubation, HRP-conjugated wheat germ agglutinin (WGA)  was added and incubated for an hour. After binding, the samples were washed twice with PTW. Then, one tablet of o-phenylenediamine dihydrochloride (Sigma P8287) in phosphate citrate buffer  was used for chromogenic detection. The reaction was stopped using 2.5 N sulfuric acid. The intensity was measured using a microplate reader at 492 nm. Z score was used to calculate the differences in detected mucus level with respect to the average, which was set at zero.Stage 32 sibling embryos were washed vigorously several times in 1/3× MMR  / σx; Raw measurement, μ; Mean, σ; Standard deviationFor immunofluorescence microscopy, the embryos were fixed using MEMFA  salt, 4% formaldehyde) solution. The fixed embryos were washed with TBST  and then incubated in a blocking solution , 2% dimethyl sulfoxide in TBST) for 30 minutes at room temperature, followed by incubation with primary antibodies at 4°C overnight. Next, the embryos were washed with TBST and incubated with secondary antibodies for an hour. Immunofluorescence analysis was performed with the following antibodies: anti-GFP (Abcam ab13970), anti-α-tubulin (DSHB 6G7), anti-actin (DSHB JLA20), WGA-Alexa 488 , and anti-Muc5ac (Abcam ab77576) as primary antibodies. The stained embryos were imaged using a confocal microscope (Zeiss LSM880). Images were analyzed using Zen (Zeiss).Human lung epithelial cells, Calu-3 (ATCC HRB-55), were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Lonza 12-604F). For RT-PCR, the cells were split into six-well plates and harvested for RNA extraction. For immunofluorescence analysis, the cells were split on a coverslip in six-well plates. Thapsigargin (1 μM) and narasin (2 μM and 4 μM) were used. After 3 hours of incubation, the cells were harvested for RT- PCR or fixed using 4% formaldehyde.GAPDH expression  .Total RNA was extracted using a PureLink RNA mini kit (Invitrogen 12183018A), and the cDNA was synthesized using a random primer (NEB S1330S) and GoScript Reverse Transcriptase (Promega A50047). Quantitative RT-PCR was performed using the LightCycler 480 instrument (Roche LC480). The expression of ER stress marker genes was used to assess ER stress levels after normalization to in vivo mouse models or primary air-liquid interface (ALI) culture systems require cautious handling and a considerable amount of resources. Cancer cell lines provide alternative and compensatory models for studying the muco-ciliary epithelium; however, cancer cell lines are not physiologically relevant in certain contexts. Therefore, the muco-ciliary epithelium of Xenopus embryonic tissue is an excellent alternative model system that provides an accessible source of tractable tissues for investigating the pathophysiology of human respiratory disorders [Xenopus embryonic epidermal cells -based methods.The mouse tracheal epithelium and human bronchial cells are frequently used to study the muco-ciliary epithelium. The mouse model is well-established as a means of exploring the pathophysiology of the muco-ciliary epithelium; however, isorders , 11. Theal cells . The genet cells , 12. Xenaminants . The secXenopus embryos may provide a suitable alternative for high-throughput screening-based strategies. For identification of muco-active reagents from unbiased chemical libraries using a high throughput approach, we first examined if the secreted mucus level in Xenopus embryo culture media can be efficiently detected by ELLA  and a 10 mM Diversity Set provided by the National Cancer Institute’s (NCI) Developmental Therapeutic Program's Open Compound Repository, to identify compounds that regulate mucus secretion. The final concentration of the chemicals used was 15 μM. Embryos were incubated in the media with individual chemicals for 3 hours, after which they were moved to new media which were without the chemicals. After 1 hour of further incubation, the media was collected for ELLA based-mucus detection. The graph represents the ratio of optical density measurements of drug-treated media and the averaged control media in ELLA. Data were analyzed by calculating the Z-score representing the differences of individual values from the mean value. Using this new strategy, we tested approximately 600 natural compounds and 200 synthetic chemicals. While none of the chemical compounds from the Diversity Set provided by the National Cancer Institute made any significant changes in ELLA signal intensity, the natural compound library yielded several compounds that potentially affected mucus levels  stress in cancer cells . We specXenopus . In addiXenopus , althougXenopus , which wXenopus embryonic epithelium to develop an unbiased drug discovery strategy for novel muco-active reagents. Indeed, our novel technique efficiently and effectively detected changes in mucus levels in the nourishing media by a simple two-step analysis procedure involving WGA-HRP detection and chromogenic measurement. Furthermore, we tested the muco-regulatory activities of a natural compound library to validate our high throughput screening procedure, which is currently challenging in other popular model systems such as the HBEC ALI culture or mouse models. We have identified several muco-active reagents from this pioneering natural compound library screening. Among several candidates, two chemicals, phorbol 12,13-dibutyrate and bicuculline, are known to control mucus secretion as a PKC activator and GABA receptor antagonist respectively. These data validated the effectiveness of our technique. In addition, we identified a novel muco-active compound called narasin, which can possibly be used as a therapeutic for respiratory diseases. Genomic analysis in Xenopus tropicalis, revealed a huge expansion of Muc genes in its genome [Xenopus genome contains 26 gel forming mucin genes including 12 Muc2 genes (Muc2A-L), and 11 Muc5 genes (Muc5A-K). Although, the Muc genes in Xenopus are highly evolved and differ structurally from those of humans, our data suggest the secretory mechanism may be conserved in Xenopus and can be used to discover the muco-regulator affecting mucus secretion from the goblet cells. Further research on the physiology and molecular mechanism of Muc gene regulation in Xenopus would strengthen the utility of embryonic epidermis as an alternative model system to study mucociliary epithelium.In this study, we exploited the advantages of the s genome . ApparenXenopus embryonic epidermis.Abnormalities in muco-regulation are one of the most frequently observed symptoms in respiratory disorders. Therefore, novel reagents that effectively regulate mucus secretion from the airway epithelium are being actively researched. However, current muco-regulation research models do not provide high throughput screening protocols for identification of muco-regulators in an unbiased manner. In this study, we developed a cost-efficient and unbiased strategy targeting muco-active reagents using Xenopus has been extensively used to study multicilia as a tractable and alternative model. In addition, several types of secretory cells such as the goblet cells, ionocytes, and small secretory cells are also in the limelight for research on muco-regulation. The Xenopus embryonic epidermis possesses several advantages compared to other research models such as human bronchial epithelial cells (HBEC) and the mouse model. One Xenopus female can produce thousands of embryos per day, which generates an unlimited source of research material. Unlike HEBCs, which take several weeks to differentiate into the muco-ciliary epithelium in ALI culture, the Xenopus embryonic epidermis differentiates to a completely functional muco-ciliary epithelium within two days. The mouse airway epithelium is an excellent in vivo model; however, internal organs are not suitable for massive drug screening purposes, as they require a substantial amount of labor and resources. In contrast, the Xenopus embryonic epidermis is located on the external skin of embryos, and is favorable for experimentally analyzing muco-regulation. Moreover, Xenopus embryos grow in minimally salted water and the secreted mucus can be accurately and simply measured using ELLA.The embryonic epidermis of Narasin is an ionophoric antibiotic used for treating coccidial infections in poultry and livestock. Although narasin is widely used in farm animal feed, its cytotoxic effects on several organs such as the cardiovascular, muscular, and gastrointestinal systems have been reported. However, the underlying mechanism of its cytotoxicity is not yet understood. In this study, we observed that narasin severely alters mucus secretion and induces accumulation of mucus granules in the cytosol. Our data suggest that the inhibitory effect of narasin on mucus secretion is not due to secondary effects stemming from ER stress responses, which was previously shown to be induced by narasin in a human cell line. Extensive research is required to understand the underlying mechanisms of the muco-regulatory activity of narasin.The muco-ciliary epithelium of the airway tract is one of the most vulnerable tissues of the human body and mucus hypersecretion is one of the major symptoms of respiratory disorders. We believe that the high throughput screening strategy developed in this study will accelerate the identification of effective muco-regulators with novel functions and mechanisms.