Fragile X syndrome (FXS) is the leading cause of inherited intellectual disability and autism spectrum disorder. Individuals with FXS often present with a wide range of cognitive deficits and problem behaviors. Educational, behavioral and pharmacological interventions are used to manage these and other complex issues affecting individuals with FXS. Despite the success of preclinical models and early-phase drug clinical studies in FXS, large-scale randomized-controlled trials have failed to meet primary endpoints. Currently, no targeted or disease-modifying treatments for FXS have received regulatory approval. Here, we examined the placebo response in FXS clinical trials conducted between 2006 and 2018. Specifically, we performed a meta-analysis of placebo-treated groups in eight double-blind, randomized controlled trials. Placebo groups demonstrated significant improvements on caregiver-rated efficacy endpoints, which were greater in adolescents and adults than in children. Among the latter measures, the Visual Analog Scale scores displayed the greatest improvements, whereas the positive effects on the Vineland-II Adaptive Behavior Composite and the Aberrant Behavior Checklist-Community/fragile X version were statistically significant in both children and adolescents/adults. Although the Clinical Global Impression scale Improvement appears to have exhibited a substantial placebo effect in multiple clinical trials in FXS, limited data availability for meta-analysis, prevented us from drawing conclusions. No placebo-related improvements were observed in performance-rated measures. These findings raise substantial concerns about placebo effects in outcome measures commonly used in the randomized-controlled trials in FXS and suggest several potential improvements in the study design and implementation of such trials. Considering the small number of trials available for this study, larger and more detailed follow up meta-analyses are needed. Meanwhile, efforts to improve the measurement properties of endpoints and rater training in drug trials in FXS should be prioritized. FMR1) gene . The FM triggers epigenetic silencing (hyper-methylation) of the gene, which results in marked reduction of the FMR1 product . FMRP is an RNA-binding protein that regulates the translation of ~4% of the brain’s mRNA, including many transcripts from genes implicated in ASD [FMR1 genotype and FMRP and the neurobehavioral phenotype, including ASD, continue to grow [Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability (ID) and single-gene disorder associated with autism spectrum disorder (ASD) ,2,3,4. Fd in ASD ,6, and s to grow ,8.Individuals with FXS present with cognitive impairment and a wide range of problem behaviors, such as ASD, general and social anxiety, attentional network deficits, repetitive and perseverative behaviors, and sensory over-reactivity ,2,3,4. AAnimal and in vitro FXS models have led to great progress in drug identification and clinical trials in humans with FXS. Nevertheless, translating preclinical successes targeting core phenotypes associated with FXS into clinical trials has been a challenge ,8. At prFMR1 expansion, gene methylation, FMRP deficit and overall severity of neurobehavioral phenotypes [Placebo response has steadily increased over time in neurologic and, in particular, psychiatric randomized controlled trials (RCTs), potentially obscuring any improvements due to drug treatment ,14,15,16enotypes ,22. Despenotypes .Here, we present the first meta-analysis of RCTs in FXS with a focus on the placebo effect response. Rather than evaluating the impact of the placebo effect on the overall outcome of these trials, we have focused on examining whether the placebo groups showed significant improvements in eight RCTs of individuals with FXS conducted between 2006 and 2018. We aimed at answering the following questions: Did placebo groups show significant and clinically meaningful responses?Were clinician- and caregiver-rated outcome measures equally affected by placebo response?Did age influence response in placebo groups?https://clinicaltrials.gov) and personal communications with other investigators. Most of these FXS trials evaluated compounds that modulate either glutamatergic or GABAergic transmission, which were analyzed in this study and described in Eight double-blind RCTs, conducted between 2002–2015 and published in English between 2006 and 2018 ,28,29,30Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) Memory 10 subtests; and the Test of Attentional Performance for Children  alertness, distractibility, flexibility and go/no-go subtests. Clinician-rated outcome measures include ratings of clinician observations and standardized performance-rated instruments. The first type included the Clinician Global Impression scale-Improvement (CGI-I), which is predominantly informed by caregiver reporting but administered and rated by the clinician in order to best determine the participant’s overall condition. Performance-rated measures are direct assessments of a participant’s skills captured by a trained examiner. These measures are thought to capture a more valid and direct evaluation of the participant’s functionality as the scoring is based on the participant’s performance of specific assessment tasks. Participants’ active effort in the assessment tasks directly impacts their outcome ,32. The Caregiver-rated outcome measures are informed by caregiver report and observation of the participant. For an individual to qualify as an acceptable caregiver rater, they typically need to meet certain requirements specific to the length of time knowing the participant or spending with the participant. The most common caregiver-rated outcome measure used in the FXS studies was the Aberrant Behavior Checklist-Community (ABC-C), scored using both the original scoring and fragile X (ABC-CFX)-specific factor structure scoring . The triIt is important to note that the aforementioned outcome measures were classified using the previously stated criteria for the purposes of the meta-analysis. Some of these outcome measures have been categorized differently across trials given the overlap between caregiver- and clinician-rated input, specifically the CGI-I and Vineland-II . The VinStudy information such as specific treatment, sample size, participant age range, trial phase, duration, primary and secondary outcome measures and details about design were recorded in an Excel spreadsheet. In a separate spreadsheet, baseline and End-of-Treatment (EOT) data for primary and secondary efficacy endpoints in placebo groups were extracted independently by H.P., and S.L. Differences (delta) between EOT and baseline for outcome measures were extracted directly from the original publication, when available, or calculated from baseline and EOT values. Raw data were requested from authors if published data were reported in a format other than descriptive values . Outcome measures used in a single study did not qualify for the meta-analyses; however, data were displayed in 2 and its corresponding p-value were calculated as a measure (percentage) of variance that is attributable to heterogeneity between studies [2p > 0.05), were analyzed by fixed effects meta-analyses. For those efficacy endpoints showing significant heterogeneity (I2p ≤ 0.05), random effects meta-analyses were carried out. Other aspects of the meta-analyses’ procedure have been previously reported [Analyses included descriptive statistics, such as calculations of proportions, for dataset characterization and meta-analyses. Data from groups receiving active drug were not included in the meta-analyses since the focus of the analyses was whether placebo per se led to improvement in outcome measures not its overall contribution to efficacy. Thus, meta-analyses included score differences (deltas) between EOT and baseline time points in the placebo groups as primary variable efficacy endpoint. If these differences were not provided, baseline and EOT scores were extracted and the difference calculated. In these instances, a previously applied pre-post correlation of 0.7 was assumed to estimate the standard error of the difference . As sens studies . Efficacreported ,35. To determine the functional or clinical significance of the improvements in placebo groups, the effect size of delta scores was calculated as a function of SDs at baseline. In addition, for performance-rated outcome measures, we compared differences between baseline and EOT scores with expected developmental changes  for the endpoint as estimated by natural history studies . For meMeta-analyses were performed using the R “meta” package ,37, whilThe table includes mean score differences between baseline and EOT in placebo-treated groups, 95% confidence intervals (CIs), heterogeneity indices among studies in the meta-analysis, and the type of employed meta-analysis. Score differences and CIs were calculated using an estimated pre-post correlation of 0.7 . p = 0.56) or adolescents/adults . In contrast, The clinician-rated outcome measures used in these trials were the same across the two age groups . Mean changes in CGI-I scores after placebo treatment were non-significant in a total seven studies of either children  of the studies had available CGI data ,26,27,28All performance-rated outcome measures used in the eight FXS drug trials were applied to a single study, either of children or adolescent/adults. No placebo improvements were noted in the analysis of these measures. While not included in the meta-analyses, it is important to note that only the PLS-5 Comp showed borderline significant changes in scores.p = 0.012) and adolescent/adults . The VAS changed in two studies involving children (p < 0.001). Changes in total and subscales scores of the ABC-C and the ABC-CFX were variable, and most studies used the latter FXS-revised version [p < 0.001), and on the ABC-CFX Stereo  and ABC-CFX Hyper  subscales improved significantly only in trials of older participants. In contrast, comparisons of delta scores on the ABC-CFX SA showed age-dependent changes; while there were marked improvements in adolescent/adult trials , there were no significant changes in trials with children . The two original ABC-C subscales revealed no improvements in seven studies: the Irr  and the SU/L . Changes in caregiver-rated efficacy endpoints differed between trials depending on the participants’ age. The Vineland-II Comp score significantly improved in six studies of both children  also improved significantly. To determine the influence of participants’ age on improvements in response to treatment with placebo, we examined the portion of studies in each age group showing positive effects on caregiver-rated measures. There were no sufficient clinician- or performance-rated endpoints for similar analyses. Since multiple ABC-CFX and ABC-C scores were used in the trials, only subscales were included in the analysis to avoid redundancy. Regardless of the applied pre-post correlation, two of the five subscales (40%) improved in children while five of the seven (71%) improved in adolescents/adults. These observations suggest that caregivers of older individuals with FXS tend to report positive responses to placebo more frequently.d of 1.0 [To determine whether the statistically significant improvements in placebo groups represented functionally meaningful changes, we evaluated several parameters. The first was the effect size of these improvements; considering that one unit of standard deviation of a cognitive or behavioral measure corresponds approximately to a Cohen’s d of 1.0 ,40, we mFX, effect sizes were complemented by gender- and age group-based mean scores and SDs. These parameters lead to the calculation of “corrected” effect sizes, based on comparable populations rather than the cohorts under study.If available, effect size was complemented by comparisons with reference values for endpoints. In the case of the Vineland-II Comp, natural history data for FXS have been published for both genders and a wide age range. The latter allowed for calculations of estimated changes in standard scores per year. For the ABC-CFX and the VAS corresponded to small to medium and large to very large score change ranges, respectively. Improvements in the Vineland-II Comp were smaller, at the negligible effect size level. Nonetheless, the reported increases in its standard scores are in contrast with the natural evolution of a decrease in adaptive behavior scores in FXS [FX, cannot be analyzed over time like the Vineland-II Comp [FX Total or subscale scores show that in males older than 12 years, SDs tend to be large with respect to mean scores (80–110% mean range). The mean decrease of 8.6 total score reported here is equivalent to 31% of the baseline SD. Since in the present study at baseline the SD of the ABC-CFX represented only 63% of the mean score, a more accurate estimate of the effect size could be based on the mean score. In this case, the ABC-CFX score change would correspond to a “corrected” effect size of 0.19 .In terms of effect size, score changes in the ABC-Cs in FXS . Males ws in FXS . In cont-II Comp . Nonethe-II Comp  for ABC-The present study is, to our knowledge, the first meta-analysis demonstrating positive responses in participants with FXS receiving placebo in multiple drug trials. We identified significant improvements in several caregiver-rated outcome measures, but not in the single clinician-rated endpoint under evaluation. No performance-based measure was used in more than one study, making this type of endpoint not suitable for meta-analyses. Improvements in caregiver-rated outcome measures were detected in studies involving either children or adolescents/adults, with a larger proportion of the latter showing a positive outcome. In terms of the magnitude of improvements, as measured by effect size or reference values, two out of three outcome measures showed functionally significant changes. These data are in line with reports of increasing placebo responses in RCTs of neurologic and psychiatric disorders, and raise concerns about study design and conduct of drug trials in FXS and other neurodevelopmental disorders ,14,16,422) and were strong amidst assumptions regarding pre-post correlations . As the studies used predominantly caregiver-rated endpoints, we could not evaluate the effect of the type of measure on outcomes. The fact that the CGI-I did not reach statistical significance, contrary to a study reporting on placebo response in pediatric ASD [The main goals of this meta-analysis were to determine whether placebo-treated patients with FXS show improvements and, if so, whether positive responses were influenced by age of the participant and type of outcome measure. Multiple endpoints demonstrated improvements in placebo groups even in the face of effect heterogeneity in some trials , but also the magnitude of the change in the reported values of these outcome measures. Our analyses of effect size showed that in children with FXS, the VAS Comp score improved at a very large level . As a reference, a minimal clinically important difference (MCID) is a metrics equivalent to ≥0.5 SD ,40, whicThe present meta-analysis shows that patients with FXS receiving placebo in drug trials can demonstrate statistically significant and clinically relevant improvements. The reporter must believe there are actual improvements in behavioral symptoms or cognition of the participant for these improvements to be captured in the outcome measures. This is important, as positive changes subjectively reported by caregivers could be due to caregiver expectations of treatment efficacy. As such, even measures like the CGI, which is clinician-rated but by and large caregiver informed in behavioral studies, appears to have captured a substantial placebo response in the recently failed clinical trials in FXS ,30,38,43The present study shows that patients with FXS receiving placebo demonstrate significant improvements on caregiver-rated efficacy endpoints, which were greater in adolescents and adults than in children. Among the latter measures, the VAS scores displayed the greatest improvements, while the positive effects on the Vineland-II Comp and the ABC-CFX were statistically significant in both children and adolescents/adults. Although the CGI-I appears to have exhibited a substantial placebo effect in multiple clinical trials in FXS, limited data availability for meta-analysis prevented us from drawing conclusions about this widely used clinician measure. No placebo-related improvements were observed in performance-rated measures. These findings raise substantial concerns about placebo effects in outcome measures commonly used in randomized-controlled trials in FXS.FMR1 expansion, gene methylation, and FMRP deficit, and overall severity of neurobehavioral phenotype [The present study of a heterogeneous group of clinical trials in FXS focused on improvements in groups assigned to placebo, rather than a direct assessment of placebo (versus drug) responses in these trials. Moreover, it could only arrive to conclusions regarding caregiver-rated measures because of the small number of studies using other types of endpoints. Future analyses should also attempt to differentiate objective from subjective improvements in individuals with FXS. It is also important to appreciate differences between actual placebo-related improvements in behavior/cognition due to increased attention during the trial from positive changes subjectively reported by caregivers . Considering these and other limitations of this study, larger follow up meta-analyses are needed. Developmental placebo response in longer (≥6 month) studies where the child grows and improves need to be considered as well . Moreovehenotype ,8. Newerhenotype ,20,51. Ahenotype . Overallhenotype , which whenotype ,53 paralhenotype .


Disturbance of networks was recently proposed to be associated with the occurrence of depression in Parkinson’s disease (PD). However, the neurobiological mechanism of depression underlying PD remains unclear.This study was conducted to investigate whether intra-network and inter-network brain connectivity is differently changed in PD patients with and without depression .Forty-one PDD patients, 64 PDND patients, and 55 healthy controls (HCs) underwent resting-state functional magnetic resonance imaging (fMRI). The default mode network (DMN), executive control network (ECN), salience network (SN), precuneus network (PCUN), and sensorimotor network (SMN) were extracted using independent component analysis (ICA), and then the functional connectivity (FC) values within and between these networks were measured.PDD patients exhibited abnormal FC values within the DMN, ECN, SN, PCUN, and SMN. In addition, PDD patients demonstrated decreased connectivity between anterior SN (aSN) and bilateral ECN, between posterior SN (pSN) and dorsal DMN (dDMN), and between PCUN and dDMN/SMN/bilateral ECN. Connectivity within the left hippocampus of dDMN and the right medial superior frontal gyrus of aSN was a significant predictor of depression level in PD patients.Aberrant intra- and inter-network FC is involved in several important hubs in the large-scale networks, which can be a biomarker for distinguishing PDD from PDND. Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease ; it is cThe recently proposed “triple-network” model emphasized the cooperation among networks. The triple-network model includes the default mode network (DMN), executive control network (ECN), and salience network (SN) . They arResting-state functional magnetic resonance imaging (RS-fMRI) is a novel, non-invasive approach without performing any specific task. Previous RS-fMRI studies in depressed PD patients using the amplitude of low-frequency fluctuation (ALFF) and regional homogeneity (ReHo) analysis have demonstrated alterations in regional brain activity in the prefrontal–limbic system, the basal ganglia (BGN), and the DMN . StudiesAlternatively, the ICA is a seed-free approach for detecting connectivity of brain networks. ICA offers an effective means for identification of functional systems within the brain during rest, typically referred to as “resting-state networks” (RSNs) or “intrinsic connectivity networks” (ICNs) . The ICAz-maps , and then we computed the FC correlation values between each network pair representing the intrinsic FC between networks (inter-FC). Second, we would compare the intra-FC and inter-FC data among PDD patients, PDND patients, and healthy controls (HCs).This study was proposed to use the RS-fMRI and ICA to systematically investigate not only the connectivity within but also the connectivity between DMN, ECN, SN, PCUN, and SMN in PDD patients and PD without depression (PDND) patients. Specifically, we first used the ICA to identify and decompose the networks into independent Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V) criteria by an experienced psychiatrist, and patients had a 17-item Hamilton Depression Rating Scale (HDRS-17) score higher than seven . The criteria for recruited patients were as follows: (1) they met the United Kingdom Parkinson’s Disease Society Brain Bank Criteria ; (2) thean seven . Afterwaan seven . An MMSEan seven . The samThe procedure and parameters to obtain resting-state fMRI images were previously described . During 1). The procedures based on this toolbox included three steps: (1) data reduction, (2) applying ICA algorithm, and (3) back-reconstruction for individual-level components. In this study, 38 ICs were auto-estimated through the minimum description length (MDL) criteria, and Group ICA was performed 20 times. Nine meaningful components were identified as anatomically and functionally classical RSNs via visual inspection. They were extracted from all participants, including the ventral DMN (vDMN), dorsal DMN (dDMN), PCUN, two SMNs, right ECN (RECN), left ECN (LECN), anterior SN (aSN), and two posterior SNs (pSN). Seven were related to the “triple-network” model, with three components reflecting the SN (one for the aSN and two for the pSN), two reflecting the DMN (one each for the vDMN and dDMN), and two reflecting the ECN (one each for the LECN and RECN) subnetworks.The ICs were analyzed by the Group ICA  reflected the measures of intra-FC. Linear detrending, despiking, and temporal filtering were conducted for all TCs of the networks of interest before evaluating the inter-FC . The differential brain regions were identified among the three groups using one-way analyses of variance (ANOVAs) and t-test SPM12 . Comparisons of connectivity within each ICN among PDD, PDND, and HC groups were performed using a design model of one-way ANOVA in SPM12, followed by post hoc two-sample t-test . The between-groups differences of connectivity among selected ICNs were assessed using ANOVA, and post hoc two-sample t-test was used to determine connectivity changes between each pair of the three groups .To statistically analyze the FC within each selected ICN, it was calculated voxel-wise with participants’ reconstructed spatial maps using one-sample p < 0.05 was considered significant.The correlations between the detected connectivity abnormalities and the HDRS as well as the UPDRS-III scores were assessed using Pearson correlation coefficient for the PDD and PDND patients. A To test whether the intra-connectivity can predict depression level in the PD patients, hierarchical regression analyses were performed on the depression level as indicated by HDRS scores. Five regression models were run in total. Age, gender, educational years, MMSE scores, UPDRS-III scores, and illness duration were entered as predictors in step 1. To evaluate which connectivity could significantly predict depression level and beyond the predictors included in step 1, regression model 1 included connectivity of all four brain regions, which were different between PDD and PDND as predictors in step 2, and a stepwise method was used to determine which predictors to retain in the final model. To evaluate the predictive value of connectivity of each of the four regions, the connectivity of one of the four regions was entered as the only predictor in step 2 for each of the other four regression models.p < 0.001).No significant differences in H-Y staging, UPDRS-III, and PD duration were observed between the PDD and PDND patients, while no significant differences in gender, age, education level, relative gray matter volume (REL-GMV), MMSE scores, relative white matter volume (REL-WMV), and mean FD were observed between the three groups . In contt-test (p < 0.05), including the LECN (IC20), RECN (IC06), dDMN (IC11), pSN (IC09/12), vDMN (IC04), aSN (IC05), SMN (IC15/IC25), and PCUN (IC26).The respective spatial patterns of 10 networks of interests for the three groups  were revealed by the one-sample p < 0.05, p < 0.05, aSN, dDMN, and SMN exhibited significant changes in FC among the PDD, PDND, and HC groups  in the patients with PD.p < 0.05/4). Connectivity within the right Cerebelum_Crus2 of the SMN reached a level of p < 0.05, while it could not survive the correction  and motor or non-motor scale scores (UPDRS-III/HDRS-17/BDI-21). This may be biased due to the small sample size in PDD group. Further studies in a larger sample size will be necessary to explore the relationship between connectivity abnormalities and the severity of depression in PDD. Third, it should be noted that none of our results could survive at the strict AlphaSim correction level of  studies . Our powIn conclusion, abnormal FC between RSNs may reflect the neuroimaging characteristics of the prefrontal–limbic–striatal–cortex circuitry-related networks. The RSNs in PDD or PDND patients are not restricted to the triple-network model but rather spread to other neural networks such as PCUN and SMN. Specifically, we found that both the aberrant intra- and inter-network FC is involved in several important hubs in the large-scale networks. This study highlighted that aberrant intra-network FC and inter-network FC can be biomarkers for distinguishing PDD from PDND, and our findings can help understand the neural foundation of the depression in PD.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Medical Research Ethical Committee of the Second Xiangya Hospital. The patients/participants provided their written informed consent to participate in this study.CT and XZ contributed to the conception and design of the study. HL, SC, QS, TW, YZ, ZM, TZ, and WS contributed to the data collection. HL and JF contributed to the data analysis. HL contributed to writing the manuscript. JL and JY contributed to the English-language revision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycobacterium avium subspecies paratuberculosis (MAP). Other studies associated nicotine with upregulation of miR-124 expression in macrophages, which led to anti-inflammatory response. This review discusses published work on the role of nicotine in modulation of the innate immune response and subsequent signaling in macrophages in IBD subsets.Cigarette smoke (CS) has adverse effects in patients with Crohn’s disease (CD), an inflammatory bowel disease (IBD) that has been associated with microbial infection, immuno-dysregulation, and mucosal dysfunction. However, CS seems to provide relief and protection to patients with another IBD known as ulcerative colitis (UC). These two subsets are featured as M1- and M2-mediated responses, respectively. Nicotine is the most active, addictive, and studied ingredient in CS. The mechanism of how nicotine and/or other CS ingredients induce pro-inflammatory or anti-inflammatory phenotypes in IBD patients remains under investigation. Our most recent in vitro nicotine study provided significant insights toward understanding the contradictory effects of nicotine on IBD patients, and it elucidated the mechanistic role of α7nAChR in modulation of macrophages in tobacco smokers. Shifting the beneficial effect of nicotine to a harmful outcome in CD patients was linked to a nicotine-microbe interaction that supports a microbial etiology in CD pathogenesis. Among the most debated pathogens in CD etiology is  Mycobacterium avium subspecies paratuberculosis (MAP) [Inflammatory bowel disease (IBD) is a chronic condition that involves severe inflammation in the lining of the digestive tract . IBD, whis (MAP) ,8. This is (MAP) . On the is (MAP) . It alleis (MAP) ,11. Suchis (MAP) . This obis (MAP) ,11. Smokis (MAP) ,11.M. tuberculosis, or Klebsiella pneumoniae was contrary to that observed in UC-like macrophages [To understand this dogma in IBD subsets, our research team has been investigating the effects of cigarette smoke and nicotine on the cellular and molecular changes in CD- and UC-like macrophages. AlQasrawi et al. reported recently that they were able to mimic macrophages similar to those in CD and UC patients with history of prior or active cigarette smoking and studied cellular response in the presence and absence of active bacterial infection . Among trophages . These fMany studies strongly support smoking as an independent risk factor of CD, and it may even affect the severity of the disease . CD patiGenetics and/or ethnicity factors have been reported in the literature to influence the degree of how smoking impacts CD severity. Aldhous and Satsangi reported that there was no association between smoking and CD susceptibility among Jewish populations . AnotherParadoxically, smoking has been a favorable and protective environmental association in patients with UC . This prInterestingly, different nicotine replacement forms have been recently recommended by clinicians to help UC patients. This includes transdermal patches, chewing gum, and nicotine-based enemas . In a stNicotiana tabacum. Although nicotine itself is not toxic, it is considered a highly addictive component of tobacco [Tobacco leaf extracts contain roughly 4500 components, and around 150 of those have been labeled as harmful to human health, such as dioxins, which are known to be both carcinogenic and an immunomodulator . Heavy m tobacco . This maDespite the extensive studies about the involvement of nicotine in IBD pathogenesis, the mechanisms by which nicotine modulates immune response in CD and UC require more elucidation. We propose that these mechanisms require a multi-factorial interaction between nicotine and the immune system, dysbiosis leading to infection, or are epigenetic, as illustrated in Nicotine is known to have a suppressive effect on both innate and humoral immune systems . Many stIt is well-known that nicotine activates cells through α7nAChR, which is responsible for the activation of cholinergic anti-inflammatory pathway . The preClostridium difficile in CD [Clostridium coccoidos, Eubacterium rectale, and Clostridium leptum), Actinobacteria (high guanine and cytosine content bacteria (Propionibacteriaceae and Bifidobacteria)), and a decrease in Bacteroidates (Prevotella spp. and Bacteriods spp.) [The gut microbial diversity and load have been studied in IBD. Dysbiosis occurs in IBD as defined by decrease in gut microbial diversity with shifting the balance between beneficial and pathogenic bacteria, which may lead to aberrant immune response in genetically susceptible individuals . In CD ple in CD . Howeverle in CD . Many rele in CD ,42,43. SThe term “epigenetic” refers to heritable changes of gene expression events, which is caused independently of genetic information from the primary DNA sequence . The maiNoncoding microRNAs (miRNAs) pathways such as miRNA-18b (miR-18b), miRNA-140 (miR-140), and miRNA-124 (miR124) explain one of the epigenetic mechanisms by which nicotine regulates gene expression of pro-inflammatory cytokines . miR-124Nicotine has been shown to be responsible for miR-124 elevation in samples in both UC and CD, while the latest has contradictory effect in the two diseases . RecentlOne of the most debated explanations for the contradictory effect of nicotine is the association of CD with microbial infection, which lacks in UC. Among the most discussed pathogens associated with CD is MAP ,56,57,58MAP is an intracellular pathogen capable of modulating the innate immune response in CD leading to significant production of pro-inflammatory cytokines . It was The effect of nicotine on IBD patients is clearly demonstrated through changes to gastrointestinal tissue. However, the effect of nicotine and smoking on the respiratory tract is well established. The similarity between intestinal inflammatory response in CD smokers and others with pulmonary inflammation is well studied and reported in the literature. Specifically, the effect of smoking on tuberculosis (TB) and exacerbating of infection has been well elucidated ,40. For Several mechanisms may explain the protective effect of nicotine on innate immunity in UC. First, nicotine’s effect is mediated by activation of the cholinergic anti-inflammatory pathway via α7nAChR, on macrophages, monocytes, and dendritic cells . This caActivation of α7nAChRs downregulates the production of pro-inflammatory cytokines, including TNFα, IL-6, and IL-1β . The dowIn conclusion, CS plays a contradictory role in IBD subsets; it is a risk factor in CD development and provides protection in UC. Our most recent in vitro study was the first to provide significant insights toward understanding the cellular events involved in the mysterious divergent effects of CS ingredients on IBD patients . Nicotin


Antiretroviral therapy (ART), while essential in combatting tuberculosis (TB) and HIV coinfection, is often complicated by the TB-associated immune reconstitution inflammatory syndrome (TB-IRIS). Depending on the TB disease site and treatment status at ART initiation, this immune-mediated worsening of TB pathology can take the form of paradoxical TB-IRIS, unmasking TB-IRIS, or CNS TB-IRIS. Each form of TB-IRIS has unique implications for diagnosis and treatment. Recently published studies have emphasized the importance of neutrophils and T cell subtypes in TB-IRIS pathogenesis, alongside the recognized role of CD4 T cells and macrophages. Research has also refined our prognostic understanding, revealing how the disease can impact lung function. While corticosteroids remain the only trial-supported therapy for prevention and management of TB-IRIS, increasing interest has been given to biologic therapies directly targeting the immune pathology. TB-IRIS, especially its unmasking form, remains incompletely described and more data is needed to validate biomarkers for diagnosis. Management strategies remain suboptimal, especially in the highly morbid central nervous system (CNS) form of the disease, and further trials are necessary to refine treatment. In this review we will summarize the current understanding of the immunopathogenesis, the presentation of TB-IRIS and the evidence for management recommendations. Mycobacterium tuberculosis infection (TB) remains the world’s most deadly infectious disease with 10 million cases and nearly 1.5 million deaths in 2018 alone . Sym. Sym4]. Head and spine imaging are also important for diagnosis as radiologic worsening is itself sufficient for diagnosis of IRIS in some situations , and sigWhile neurologic worsening after ART-initiation in a patient being treated for TBM is strongly indicative of CNS-TB-IRIS, several other conditions should be considered. When CSF culture or molecular diagnostics are positive, it is important to rule out drug-resistant TB, which can occur in more than 10% of TBM cases and is associated with poor outcome . CryptocAlthough the immunopathogensis of TB-IRIS in the CNS is likely to mirror that of IRIS outside of the CNS, there are important differences. Many TBM-IRIS associated cytokines, chemokines, and mediators involved in neutrophil response or inflammasome activation are found in the CSF of TBM-IRIS patients in high concentrations, but blood concentrations do not differ significantly between TBM patients with and without IRIS, suggesting a highly compartmentalized inflammatory response . DespiteThis understanding of the immunopathology of TBM-IRIS has encouraged efforts to better predict which TBM patients will go on to develop IRIS . As in en = 98), and therefore was not able to definitively conclude whether mortality was improved in this subpopulation , or to assess incidence of IRIS [Protocols for prevention of IRIS are where TBM-IRIS differs most from other forms of TB-IRIS. Unlike in extra-meningeal TB, the high morbidity and mortality of CNS-TB-IRIS makes the risk of IRIS related to early ART initiation higher than the risk of developing additional infections related to delaying ART. The American Thoracic Society and Centers for Disease Control recommend delaying ART initiation until 8 weeks after ATT initiation , and oth of IRIS . There i of IRIS . One tri of IRIS .While many immunomodulating therapies have been tried with some success in treating CNS-TB-IRIS once it has developed, high-quality evidence is lacking . In factUnmasking TB-IRIS is an increasingly recognized complication of initiating ART in a patient with subclinical or occult TB infection . Many epThe pathogenesis of unmasking TB-IRIS is not fully elucidated, but its final immunologic response is thought to involve similar mechanisms to the uncontrolled immune response that occurs after rapid immune restoration in paradoxical IRIS . SubclinThe presentation of unmasking TB-IRIS is similar to the paradoxical form, though disseminated TB is more common ,155,157.Management strategies are not well defined or evidence-based for unmasking IRIS. While earlier studies describe withdrawal of ART in severe cases , more reCareful history taking, physical examination, and review of chest radiography at the time of ART initiation is important to prevent unmasking IRIS ,165. RisPoint-of-care screening assays have been developed to identify those patients with extrapulmonary TB that might be otherwise missed. The urine lateral flow assay for LAM has become an attractive option to diagnose disseminated TB in people with HIV due to their ease of incorporation into HIV clinics for rapid screening . While sCryptococcus meningitis IRIS in particular is a notable factor [Prophylactic TB treatment for patients initiating ART has become more widespread given WHO recommendations for combined prophylaxis with trimethoprim-sulfamethoxazole and isoniazid/pyridoxine . While te factor . Severale factor ,180,181.TB-IRIS in all forms causes significant morbidity and mortality in persons with HIV/TB coinfection and requires special focus given its partially iatrogenic nature. While significant strides have been made recently, both in the realms of immunology to better understand pathogenesis, and in clinical trials to better optimize prevention and treatment strategies, we are still lacking the knowledge we need to avoid poor outcomes of TB-IRIS—particularly in IRIS related to TBM. As the arsenal of targeted immunosuppressive medications expands, there is newfound opportunity to target our treatments of TB-IRIS to its known immunologic mediators, such as IL-6 or the inflammasome. This will require continued research to better characterize the immune response, as well as rigorous trials to demonstrate improved efficacy and decreased side effects compared to corticosteroids.Unmasking TB-IRIS remains incompletely described and its prevention will require improved TB detection at ART initiation. As availability of ART and implementation of HIV test and treat continues to increase, ability to rapidly rule out TB coinfection is important. Continued research to identify IRIS biomarkers is of particular importance in unmasking IRIS given its diagnostic challenges.TB-IRIS in the CNS must be a priority for further research given its high mortality. CSF laboratory tests have been shown to predict TBM-IRIS development, which opens an avenue for targeting advanced immunosuppressants or intensified TB treatment to those at highest risk, but such approaches require clinical trials. Given the localized disease characteristic of CNS-TB-IRIS, it is possible that unique immune-targeted therapies might be beneficial for CNS-TB-IRIS compared to disease outside of the nervous system.


Bioinformatic methods for the efficient analysis of these large-scale datasets are instrumental for advancing our understanding of adaptive immune responses. However, while well-established solutions are accessible for the processing of single-cell transcriptomes, no streamlined pipelines are available for the comprehensive characterization of T-cell receptors. Here, we propose Scirpy source code and documentation are available at https://github.com/icbi-lab/scirpy.Bioinformatics online. B and T lymphocytes are equipped with a vast repertoire of immune cell receptors that can recognize a wealth of different antigens. High-throughput sequencing technologies have enabled the study of these immune repertoires at unprecedented resolution  in single cells. However, while the study of single-cell transcriptomes is facilitated by tools like Seurat , a Python-based Scanpy extension that provides simplified access to various computational modules for the analysis and visualization of immune repertoires from single cells. Due to its tight integration with Scanpy, Scirpy allows the combination with scRNA-seq transcriptomic data to comprehensively characterize the phenotype and TCR of single T cells.Here, we present Scirpy integrates different bioinformatic methods for importing, analyzing and visualizing single-cell TCR-sequencing data from human and mouse , but can further identify clonotype clusters based on CDR3 amino acid sequence identity or similarity. The latter approach, inspired by TCRdist  [project number T 974-B30 to F.F. and I3978 to Z.T.] and by the European Research Council (ERC) [advanced grant agreement number 786295 to Z.T.]. Z.T. is a member of the German Research Foundation (DFG) [project number TRR 241(INF)].Conflict of Interest: none declared.btaa611_supplementary_dataClick here for additional data file.


In the field of developmental psychology, the Stress-and-Coping model  posits that individual differences in biological, psychological, and social risk and protective factors serve to increase or buffer the impact of stressful experiences on psychological well-being later in life. Importantly, research suggests that residents of public housing generally experience more risk factors than elders at large . The present study examines the impact of a programmatic intervention, The Happy Campaign, on individuals living in public housing in a small Midwestern city . Goals of the Happy Campaign were to improve residents’ coping skills and increase perceived support. Results demonstrated significant improvement in key aspects of well-being post-intervention; these included significant increases in exercise (p = .04), self-reported health (p = .01), as well as decreases in negative affect (p= .008). Data also show a moderate increase in residents’ hope post-intervention (p = .06). Although future research is needed to account for confounding variables that arose in conducting research in this community setting, these data provide preliminary evidence that a broad-based, environmental intervention may offset the myriad risks faced by particularly vulnerable elders, and even augment well-being.


Reactive oxygen species (ROS) are attractive weapons in both antibiotic-mediated killing and host-mediated killing. However, the involvement of ROS in antibiotic-mediated killing and complexities in host environments challenge the paradigm. In the case of bacterial pathogens, the examples of some certain pathogens thriving under ROS conditions prompt us to focus on the adaption mechanism that pathogens evolve to cope with ROS. Based on these, we here summarized the mechanisms of ROS-mediated killing of either antibiotics or the host, the examples of bacterial adaption that successful pathogens evolved to defend or thrive under ROS conditions, and the potential side effects of ROS in pathogen clearance. A brief section for new antibacterial strategies centered around ROS was also addressed. ROS have been called “double-edged swords of life” (Reactive oxygen species (ROS), including hydrogen peroxide  cycle enzymes, leading to dysfunction of TCA cycle and the hyperactivation of respiration  in response to microbe recognition . Howeverathogens .S. aureus via NOX2-dependent ROS accumulation in mouse model , promoting antibacterial autophagy . In addise model .Salmonella typhimurium and Listeria monocytogenes (S. aureus (MRSA) infection, mitoROS were generated and delivered directly to the phagosome by mitochondrial-derived vesicles (MDVs) in a Toll-like receptor signaling-dependent manner , PerR , OhrR , and SoxRS  can be activated by direct oxidation of their sensor proteins and then adjusting the bacterial response appropriately , the iron-sensing transcriptional repressors, such as Fur and DtxR (diphtheria toxin repressor), can also be utilized to sustain redox homeostasis by controlling the expression of genes encoding iron acquisition systems and iron-dependent enzymes , GS enzyme isocitrate lyase deficient mutants were significantly more susceptible than wild-type strain toward isoniazid, rifampicin, and streptomycin  pathway is an important target to mitigate ROS damage, since NADPH is the cofactor for antioxidant systems. In olerance .E. coli and Pseudomonas fluorescens tend to pool ketoacids both inside the cell and in the extracellular matrix under ROS conditions. For example, the increased generation of KG can be achieved by the modulation of TCA cycle enzymes like isocitrate dehydrogenase when using citrate, glucose, and malate as carbon sources  and pyruvate can undergo non-enzymatic decarboxylation in the presence of ROS, generate non-toxic byproducts, and thus alleviate the ROS damage. Bacteria like  sources . It can  sources , 2017. Wn source .E. coli and Pseudomonas aeruginosa -2 mediated virulence in S. enterica , an important virulence factor of S. aureus . During the exit from phagosome, B. pseudomallei senses host GSH via sensor protein VirA, which then activates the expression of the T6SS  to sustain intracellular redox homeostasis. Certain intracellular pathogens have even evolved to exploit the host GSH for adaptions. In the T6SS . In L. mctivator .S. typhimurium evidently makes use of host-derived ROS during intestinal inflammation. ROS generated by phagocytes convert thiosulfate to tetrathionate, which in turn can be used as a respiratory electron sink by S. typhimurium, allowing it to outcompete the native microbiota  regulated by OxyR allows E. coli to employ H2O2 as a terminal oxidant for respiration  could act as a chemoattractant by reversibly oxidizing TlpD that inactivates the chemotransduction signaling complex  is entirely dependent on H2O2. Also, numerous H2O2-non-producing species like Streptococcus mutans and Streptococcus pyogenes release eDNA in a H2O2-dependent manner   .S. aureus into a metabolic state with reduced respiration, which is incompatible with the killing mechanism of most bactericidal antibiotics (E. coli. The activation of redox regulators SoxRS results in increasing expression of the AcrAB-TolC multidrug-resistant (MDR) pump, which in turn lowers the fluoroquinolones concentration and promotes antibiotic tolerance  were more susceptible to rifampicin, when compared with wild-type leucocytes  and cold atmospheric plasma (CAP), have been suggested as efficient alternative approaches. Although their antibacterial efficacy also centered around ROS, the modes of action for these new techniques are distinct from conventional antibiotics, as ROS-mediated damage in such therapies is the primary stress-induced damage . For exaROS are attractive weapons to kill pathogenic microbial cells. However, ROS, as a double-edged sword, should be regulated with care since under non-lethal ROS, certain pathogens have evolved delicate mechanisms to utilize ROS for their adaption to thrive. Also, numerous pathogens can use ROS as a stepstone for the evolution of antibiotic tolerance and resistance. Efforts to target bacterial adaptive pathways, as well as the use of novel ROS-inducing antibacterial strategies, will be promising approaches for antibacterial therapy.HL, XZ, YH, BL, LC, and BR: conception/design of the work, agreement to be accountable for all aspects of the work. HL, LC, and BR: drafting the work. XZ, LC, and BR: final approval of the manuscript to be published. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pigmented purpuric lichenoid dermatitis of Gougerot and Blum is an uncommon variant of pigmented purpuric dermatosis (PPD) characterized by confluent purpuric papules and plaques on the legs . The ove3 and viral load was 22 copies/mL. Serologies for syphilis and hepatitis B and C virus were negative.A 32-year-old black male, born in a West African country, reported the development of multiple asymptomatic brown-to-violaceous plaques on the legs in the previous 2 months. He was diagnosed with human immunodeficiency virus (HIV) type 1 infection 9 years before but had bad compliance with highly active antiretroviral treatment (HAART). One month before noticing the development of skin lesions, HAART was reinitiated with emtricitabine, tenofovir and dolutegravir. At the time of presentation, CD4+ lymphocyte count was 870 cells/mmOn skin examination, the patient had multiple red-brown nummular plaques with an oval-to-irregular outline on the legs . The lesDermoscopy revealed a diffuse coppery-red structureless background permeated by a brownish-to-gray network with ill-defined borders and a polymorphic pearly white central area with polygonal-to-oval red and gray dots, globules, and patches .Histopathologic examination revealed a dense band-like lymphocytic infiltrate in the upper dermis, endothelial cell swelling and extravasation of red blood cells with hemosiderin inside of macrophages. There was no visible vessel wall damage .The clinical diagnosis of pigmented purpuric lichenoid dermatitis of Gougerot and Blum is challenging, as this dermatosis can closely resemble Kaposi sarcoma—an important differential diagnosis in HIV patients like ours—cutaneous vasculitis, and other PPDs, particularly lichen aureus. Dermoscopy is a noninvasive technique that may increase accuracy in the differential diagnosis. One previous report described its dermoscopic findings . In compPPD has been associated with many conditions, such as hypertension, diabetes mellitus, and autoimmune diseases . Althoug


Introduction: Survival of ALK-rearranged NSCLC patients has dramatically improved by the use of multiple ALK-tyrosine kinase inhibitors (ALK-TKI). However, still little is known about the impact of drug sequencing and clinical features on survival in a real-world setting.Methods: Patients with stage IV ALK-rearranged NSCLC treated at six centers in Switzerland and Italy were identified and standard clinical variables collected. OS curves were constructed using the Kaplan–Meier method and compared with the log-rank test. Multivariate Cox proportional hazard analysis was applied to determine the correlations between clinical features and OS. In four patients, biopsies were subjected to NGS.Results: One-hundred and twenty-one patients with stage IV ALK-rearranged NSCLC diagnosed between 2011 and 2016 were included. With a median follow-up time of 39.5 months, the median OS from diagnosis of stage IV disease was 48.0 months. First-line treatment consisted of an ALK-TKI in 24% of patients, with crizotinib in 83% of them. Chemotherapy as first-line treatment did not influence OS (p = 0.955). The use of more than one ALK-TKI line positively correlated with OS (p = 0.016), as well as the use of alectinib or lorlatinib in any treatment line, as compared to the use of crizotinib ± ceritinib (p = 0.022). A never smoking history was an independent prognostic factor for OS (p = 0.032). Moreover, treatment with alectinib significantly improved OS.Conclusions: Targeted treatment for ALK-positive NSCLC patients lead to prolonged OS. Smoking status was a negative independent prognostic factor in a multi-variate analysis. The use of alectinib or lorlatinib in any treatment line improved overall outcome. ALK (anaplastic lymphoma kinase) gene have been a particularly successful target in terms of the development of new targeted therapies, achieving important improvements in survival (ALK gene rearrangements are present in about 5% of NSCLC patients (EML4) with ALK and produce EML4-ALK-fusion proteins are the most commonly observed rearrangement, but at least 27 fusion variants have been identified so far  have been made. In particular, the identification of genetic alterations responsible for initiation and maintenance of the malignant phenotype in several cancers including NSCLC has led to the development of targeted therapies. These new molecules specifically target the so-called oncogenic driver mutation and have improved patient outcomes, in terms of overall survival (OS), but also in terms of quality of life (QoL). Among different oncogenic driver mutations known in NSCLC, rearrangements of the survival –6. ALK gpatients . They wepatients , 9. Inved so far .ALK rearrangement is of therapeutic relevance at the time of initiation of first-line treatment, as it confers sensitivity to ALK tyrosine kinase inhibitors (ALK-TKIs). Importantly, the rare occurrence of KRAS co-mutations leads to primary resistance to ALK-TKIs  by the use of ALK-TKIs and their sequencing.All the clinical ALK-rearranged NSCLC patients' data were retrospectively collected and analyzed. If available, biopsies both before starting ALK-TKI and at progression were subjected to next-generation sequencing (NGS) to further explore resistance mechanisms. All this study was approved by the local ethics committee (EK-ZH-2018-01919) and in accordance with the local laws and regulations.ALK-rearranged NSCLC on treatment with an ALK-TKI were collected by each participating center and assembled in a central database.This is a retrospective study study performed in collaboration among Swiss and Italian cancer centers. Data on patients with metastatic Several demographic and background clinical characteristics were documented for each patient, such as age, gender, smoking status, histology, patterns of metastases, co-mutations, and treatments received in any line. Since the ALK-TKIs were utilized in different lines of systemic treatment, the OS was calculated from the date of diagnosis to the date of death or last time the patient was seen. Patients still alive at the time of data collection were censored at the date of the last available medical record. Several other clinical endpoints were also assessed: objective response rate (ORR) was defined as the proportion of patients achieving either a complete or a partial remission as best clinical response to an ALK-TKI according to the local radiologists' interpretation based on the response Evaluation Criteria for Solid Tumors (RECIST) 1.1 or clinically, when indicated. The ORR rate, as well as the progression-free survival, were not reported for the overall population due to the different treatment lines, in which ALK-TKI were administered.ALK gene rearrangement by standard diagnostic procedures used in the respective institution, as recorded in the patient's medical record, 18 years of age or older at diagnosis of ALK-rearranged NSCLC. If a biopsy at diagnosis and at progression was available, those samples were subjected to NGS.The inclusion criteria were as follows: diagnosed with metastatic NSCLC, confirmation of an p <0.05. All statistical analyses were performed using two-sided tests. All data were analyzed for the pooled study sample comprising patients from Italy and Switzerland combined.Descriptive statistics were used to summarize patients' demographic and treatment characteristics at diagnosis and subsequent recurrence or progression. All analyses were conducted using SigmaPlot statistical software . OS was analyzed using the Kaplan–Meier method and was reported as median with confidence limits , with statistical significance of survival differences assessed using a non-parametric log-rank test. In order to study any possible influence of main prognostic factors on the OS, multivariate analysis was performed according to the Cox proportional hazards survival model considering statistically significant In four patients, pre- and post-treatment biopsies were subjected to NGS using the Ion AmpliSeq Comprehensive Cancer Panel (CCP), or the Thermo Fisher Oncomine Focus Assay Panel (OFA). NGS was performed centrally at one of the sites . The variant calling was done using Ion Reporter (Thermo Fisher Scientific). Only variants predicted to be damaging by SIFT and PolyPhen were included. Additionally, the variant frequency cut-off was adjusted to the estimated tumor cell content in each specimen.in situ hybridization was used in almost all cases to detect ALK rearrangement (n = 119). In the remaining two cases, ALK immunohistochemistry was performed. Baseline patients' characteristics are summarized in n = 86, 71%), or ALK-TKIs . Seventy-five patients (62%) received ALK-TKIs after first-line treatment, 58 (48%) as second-line treatment. Forty-six patients (45%) received more than one ALK-TKI treatment line, including the use of alectinib or lorlatinib in 26 of them (25%). Seventeen patients (14%) have not received a treatment with ALK-TKI at the time of analysis .Between January 2011 and June 2016, a total of 121 patients at six centers were identified. Break-apart fluorescence p = 0.323), with no significant difference in OS between those treated or not with brain radiotherapy . No significant difference in OS was observed between patients treated with ALK-TKIs or ChT as first-line treatment (p = 0.955). A significant difference in OS was observed in favor of patients treated with more than one treatment line of ALK-TKIs as compared to those treated only with one line of ALK-TKI  and whose ALK-TKIs included alectinib or lorlatinib . A non-significant trend toward a better prognosis was observed for adenocarcinoma histology , median OS was 48.0 months (95% CI: 12.9–83.0). A non-significant trend in OS was observed between patients with or without brain metastases at diagnosis  as best response, which lasted for 7 months. He was first oligo-progressing and received a pulmonary lobectomy of the left lower lobe with continuation of crizotinib, but was switched to docetaxel three months later upon systemic progression. After two months he was progressing and switched to ceritinib for 5 months, but was then progressing in the brain. After that, alectinib was initiated, which achieved a PR for almost 1.5 years. He then progressed in the liver and a hepatic lesion was biopsied. This biopsy and the lobectomy specimen were subjected to NGS using the CCP and an ALK p.Ile1171Ser point mutation was detected in the liver biopsy, which was absent before, most likely explaining the resistance to alectinib .Case #2 was a female patient who was diagnosed with stage IIIA adenocarcinoma of the lung. She was operated upfront and received an adjuvant chemotherapy with four cycles of cisplatin/pemetrexed, but relapsed 7 months later with metastases in retroclavicular lymph nodes. At that point, an ALK-translocation was diagnosed and she was started on crizotinib. Upon progression, she received cisplatin/pemetrexed again, later a re-challenge, followed by docetaxel and then ceritinib. Upon progression in the brain and liver she was started on lorlatinib. After an initial response she was progressing in the liver and in the bones. An NGS analysis performed on a biopsy of a progressing hepatic lesion using the OFA panel at that stage showed expression of the EGFR variant III with deletion of exons 2–7  adenocarcinoma of the lung. After an initial course of palliative cisplatin/pemetrexed he was started on crizotinib and was responding for 14 months. He then presented with oligo-progressive disease (oligo-PD) in two locations (bone), on which he was irradiated while continuing crizotinib. However, 2 months later he was progressing in the primary tumor, which was biopsied. The initial diagnostic specimen and this re-biopsy were subjected to NGS using the CCP, which showed emergence of a BCL9 p.Arg1401_Pro1402delinsThrAla mutation . The priCase #4 was a female patient with stage IV  adenocarcinoma of the lung. She received one cycle of palliative cisplatin/pemetrexed, but due to side effects and her reduced general condition was switched to crizotinib shortly thereafter. She was progressing 7 months later, at which timepoint a broncho-alveolar lavage (BAL) was performed. Cells from this BAL and cells from the initial malignant pleural effusion were subjected to NGS using the CCP, which showed a newly emergent LPP p.Arg415Cys mutation. The patient was then started on ceritinib with a partial remission as best response, which was maintained for 6 months. She then deteriorated rapidly and eventually died due to a pulmonary infection and pulmonary PD.ALK-fusion positive stage IV NSCLC represent a subgroup with favorable OS compared to patients with NSCLC and no actionable molecular alterations. Real-life data allow for evaluating the impact of treatment sequences, as well as giving detailed information about clinical features, which can be associated with patients outcome. Here we present the analysis of 121 ALK-fusion-positive patients treated in several institutions with comprehensive data and access to ALK-TKI through early access programs and/or clinical trials with ALK-TKI. In this cohort of ALK positive patients, the median OS was 48 months. This reflects the impressive efforts made in the development and access to ALK-TKIs when compared to NSCLC patients without actionable molecular alterations. Since the very first results with crizotinib in late and first-line, the treatment of these patients has dramatically changed . In order to identify possible clinical features with impact on patients survival, we first evaluated the localization pattern and number of metastases at time of diagnosis. Clinical characteristics of this patient cohort did not differ from the ones reported in clinical trials, e.g., brain metastases were present in 30% of patients at time of diagnosis. It has been previously reported that liver and multiple-organ metastases might have a negative prognostic impact in patients with advanced adenocarcinoma of the lung . This is the first report of such an alteration as a potential mechanism of resistance to lorlatinib. The EGFRvIII splice variant has been reported in solid tumors and has been reported to generate a highly immunogenic peptide that is currently being studied as target for immunotherapeutic approaches  and all participants provided informed consent to participate in this study. Written informed consent was obtained from every patient for the publication of any potentially identifiable images or data included in this manuscript.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.CB reports personal fees from AstraZeneca, Pfizer, Takeda, Roche, Janssen-Cilag, Novartis and Boehringer-Ingelheim; non-financial support from AstraZeneca and Takeda; all outside the submitted work. AA reports personal fees from AstraZeneca, Pfizer, Takeda, Roche, Takeda, MSD, and BMS; grants and personal fees from Boehringer-Ingelheim; all outside the submitted work. RD reports personal fees from Janssen-Cilag, Boehringer-Ingelheim, and IPSEN pharma, outside the submitted work. MF reports grants and other from BMS; other from BMS, MSD, Astra Zeneca, Boehringer-Ingelheim, Roche, and Takeda; grants from Astra Zeneca; all outside the submitted work. GM reports personal fees from Boehringer-Ingelheim and non-financial support from AstraZeneca, outside the submitted work. SR reports grants, non-financial support and other from AstraZeneca, Roche, and Boehringer-Ingelheim; personal fees, non-financial support and other from Takeda; non-financial support, and other from MSD Oncology and Bristol-Myers-Squibb; other from Pfizer, Novartis, Eli Lilly, Eisai; grants and other from Meck Serono; grants from AbbVie; all outside the submitted work. PW reports personal fees from AstraZeneca, Pfizer, Takeda, Roche Janssen-Cilag, Novartis, and Boehringer-Ingelheim, outside the submitted work. GB reports personal fees from Janssen-Cilag, Boehringer Ingelheim, and Roche; non-financial support from Bristol-Myers Squibb, AstraZeneca/MedImmune, Pierre Fabre, and Ipsen; all outside the submitted work. AC-F reports personal fees from Astra Zeneca, Roche, Pfizer, Takeda, BMS, Boehringer Ingelheim, and MSD, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Extracellular Vesicles (EVs) are emerging as pivotal elements in cancer. Many studies have focused on the role of Small- (S)-EVs but in recent years Large-(L)-EVs have progressively gained increasing interest due to their peculiar content and functions. Tumor-derived L-EVs carry a lot of oncogenic proteins, nucleic acids and lipids to recipient cells and are involved in the reshaping of the tumor microenvironment as well as in the metabolic rewiring and the promotion of the pro-metastatic attitude of cancer cells. Several techniques have been developed for the isolation of L-EVs and commercial kits are also available for efficient and easy recovery of these vesicles. Also, the improvement in DNA sequencing and “omics sciences” profoundly changed the way to analyze and explore the molecular content of L-EVs, thus providing novel and potentially useful cancer biomarkers. Herein, we review the most recent findings concerning the role of L-EVs in cancer and discuss their possible use in oncology as “liquid biopsy” tools as compared to the other classes of EVs. Extracellular Vesicles (EVs) are emerging as important intercellular messengers regulating tumor progression and cell metabolism. Based on diameter, different classes of EVs are known, including either Small- (S) or Large- (L)-EVs. Among the S-EVs, exosomes are nano-sized vesicles deriving from the late endocytic pathway, carrying a large variety of molecules, such as proteins, DNA fragments and RNAs . In the Although their role is not completely understood, L-EVs have progressively gained increasing interest in the last years. Recently, L-EVs have been proposed to be involved in cancer metabolic rewiring , as wellHere, we summarize the main characteristics of EVs with a particular focus on L-EVs and discuss their involvement in cancer progression, as well as their possible applications in oncology. +/−, ANXV+/− or HSP70+/− vesicles). Another possible nomenclature is based on the EV origin, such as podocyte- or platelet-derived EVs, as well as oncosomes or apoptosomes, for L-EVs released from cancer or apoptotic cells, respectively.In 2018 the International Society for Extracellular Vesicles revised the definition of EVs as “particles naturally released from the cells that are delimited by a lipid bilayer and cannot replicate” . HoweverThis nomenclature was structured to organize the chaotic literature on EVs. However, the EV name was misused or improperly used in many papers, making it hard to correctly define EV classes due to the lack of proof of their origin.Despite the biology of S-EVs has been widely explored , L-EVs’ They originate from the outward budding of the plasma membrane, which is guided by specific molecular machinery . One of It has been debated whether L-EVs are waste cellular products or if they exert specific functions in relation to their content. The second hypothesis is more likely to be correct, since a number of proteins involved in this active loading have been identified. The most widely studied is the ADP-ribosylation factor 6 (ARF6), namely a protein expressed on the surface of endosomes . AlthougThe biogenesis of L-EVs in cancer cells follows the same mechanisms described for normal cells, although some differences were reported for large oncosomes. Notably, the formation of oncosomes is prompted by the disruption of both cytoskeletal and membrane plasticity regulators as recently demonstrated by Minciacchi et al. . DiaphanOne of the most studied topics of L-EVs is the exploration of their nucleic acid content, which is pretty unique concerning all the other EVs. With respect to S-EVs, oncosomes contain up to 7-fold more DNA and structural analyses showed that it is condensed in a chromatinized form, with large DNA fragments ranging from 100 Kbps to 2 Mbps . InteresSince tumor-derived RNAs packaged within the phospholipidic bilayer of L-EVs are protected from degradation by ribonucleases , their aDifferences in protein content have been reported between tumor-derived S-EVs and L-EVs with some classes of proteins found up to 60-fold increase in L-EVs. In particular, enzymes implied in glucose (GAPDH), glutamine (GLS) and aspartate (GOT1) metabolism were found constantly up-regulated in L-EVs, suggesting their involvement in the metabolic rewiring of recipient cancer cells . AdditioTo a minor extent, L-EVs also carry proteins implied in DNA packaging (Histone H2B type 2F), angiogenesis (Glycoprotein I), apoptosis control (Voltage-dependent anion channels 2) and cell migration  . CytokerSeveral fatty acids are enriched in L-EVs, either with a structural or functional role, including the ceramide and phosphatidylserine implied in the biogenesis of all kinds of vesicles ,11,12,21It was recently demonstrated that cells can modulate the lipid expression on L-EVs, depending on the function they are committed to. For example, platelet-derived L-EVs at the inflammatory sites can convert their membrane lipids into the signal molecule 12-HETE (12-hydroxy eicosatetraenoic acid) through the extracellular phospholipase A2 and the vesicular 12-lipoxygenase . The 12-EVs can be isolated from either culture media and biological fluids, including blood, urine, saliva and ascites. The EV isolation techniques can be grouped into five distinct categories—ultracentrifugation, density-gradient separation, polymer-based precipitation, immune selection and microfluidic isolation  Table 1Table 1. g) and type of rotor [g), L-EVs isolation is performed at lower acceleration. Many authors reported that consistent isolation can be performed at 10,000× g and this makes the isolation of L-EVs simpler [The classical method for isolating EVs is based on the separation of particles by centrifugation. Initially, the high buoyant density particles  are pelleted at low-speed centrifugation. The non-EV proteins are then removed by resuspension of the pellet, followed by repeated high-speed centrifugation . The effof rotor . While t simpler .Although centrifugation is time-consuming, it needs a relatively small set of reagents and consumables, as well as it can be easily applied to large starting volumes. Although it produces consistent yields of L-EVs, they are generally tainted by a lot of contaminating proteins, such as albumin for plasma isolated EVs or uromodulin/Tamm–Horsfall protein (THP) for vesicles of urinary derivation. Thus, adequate controls are needed to verify the purity of harvested vesicles, since inadequate samples may affect downstream analyses of functional experiments .It is based on the use of commercial membrane filters with pores of various sizes. The EV preparation goes through the membrane, losing all the components larger than the pores. Large particles are removed first by filters with broad pore diameters and the particles with a size smaller than the target EVs are separated from the filtrate at the next stage. Further ultracentrifugation stages can be added and the yields are inversely proportional to the number of filtration stages, while directly proportional to the purity of EV preparations ,31. A grGel filtration is based on the separation of molecules according to their hydrodynamic radius and it is largely used for separating biopolymers . This method is also applicable to the separation of EVs from both plasma and urine protein complexes as well as lipoproteins . Brieflyg). Different polymers may be used such as polyethylene glycol, sodium acetate , protamine (a positively charged molecule) or, as recently proposed, the PRotein Organic Solvent Precipitation (PROSPR) [The second most popular method for EV isolation is based on precipitation technique through hydrophilic polymers. EVs are dispersed in a solution of super hydrophilic polymers that restrains their solubility. Thus, the vesicles can be sedimented by low-speed centrifugation (1500× (PROSPR) . Althoug(PROSPR) . It is gThe use of specific mAb, directed against typical EV surface molecules, such as tetraspanins, can be used in conjunction with magnetic beads, to selectively bind EVs . Other pFollowing isolation of L-EVs from either cell culture supernatants or biological fluids, quality controls are mandatory in order to schedule downstream analysis of EVs. For this purpose, typical assays are based on flow cytometry and Western blotting for protein content characterization, Tunable Resistive Pulse Sensing (TRPS) for dimensional assessment and transmission electron microscopy (TEM) for morphological study.The MISEV2018 defined the criteria that must be fulfilled to demonstrate the EV nature and purity . SpecifiFinally, proteomics of the recovered EVs should be addressed to verify the parental origin of these vesicles or document their functional activity . The preTumor cells shed a high number of L-EVs as compared to normal cells . Thus, iAnother described role of L-EVs includes angiogenesis and the reorganization of the tumor microenvironment. To this regard, other molecules carried by L-EVs include the vascular endothelial growth factor (VEGF), the tumor growth factor (TGF)-β and the microRNA (miRNA or miR)-1246, which are factors with known angiogenic potential. Noteworthy, the miR-1246 regulates the TGF-β/SMAD signaling pathway, promoting endothelial cell proliferation and vessel sprouting ,44. BertThe role of L-EVs in the interplay between tumor cells and the surrounding microenvironment is also another topic of interest, although at present is still poorly investigated. The bidirectionality of the EVs transfer between tumor cells and tumor stroma, in fact, has been recently demonstrated in gastrointestinal tumors since vesicles shed by cancer-associated fibroblasts (CAF) were found able to induce chemoresistance in either pancreatic  or colorFinally, the L-EVs also play a role in cancer immune escape. As recently reported, breast cancer (BC)-derived L-EVs can contain large amounts of indoleamine-2,3-dioxygenase (IDO) , an enzyThe release of L-EVs is thought to function as a mechanism of acquired drug resistance exploited by cancer cells. In this regard, L-EVs can transfer the P-glycoprotein, also known as multidrug resistance protein (MDR)-1 and the multidrug resistance-associated protein (MRP)-1 from drug-resistant to drug-sensitive cancer cells . The conOther putative mechanisms implicated in the drug resistance are the expression of surface receptors on the L-EVs functioning as decoys for monoclonal antibodies (moAbs). In this context, Simon et al. showed that L-EVs released by GBM cells in the presence of bevacizumab can reduce the amount of drug available by its trapping on the vesicle surface . Such a Liquid biopsy is currently under investigation as a diagnostic tool alternative to tissue biopsy in cancer. It consists of isolating tumor-derived components, such as circulating tumor cells (CTCs), circulating-free tumor DNA (ctDNA) and EVs, from blood or potentially any other biological fluid . The dowThe use of EVs as liquid biopsy, particularly, is an fast growing field of research. Although L-EVs have been less investigated as compared to S-EVs, their peculiar molecular features and easiness of isolation render them a very attractive tool. In fact, while the ctDNA analysis refers only to small DNA sequences that are hard to discriminate from non-tumoral circulating-free DNA (cfDNA), the L-EV-DNA is more similar to the original cell DNA; thus, it is suitable for more complex analyses, such as epigenome-wide analysis, as well as methylation studies .Moreover, circulating L-EVs are found in at least one order of magnitude higher as compared to the number CTCs detectable from single blood drawn  and therA number of studies evaluated the possible use of L-EVs as cancer diagnostic biomarkers. Minciacchi et al., for example, proposed the measurement of CK-18 in L-EVs as a surrogate of the presence of PCa and found higher expression in plasma-derived vesicles from PCa patients when compared with healthy subjects . HoweverSince the mutational status of circulating EVs is considered an ideal surrogate of naive tumor cells, the molecular characterization of L-EVs isolated from body fluids may be an useful biomarker for precision therapy. In this context, limited evidence derives from a study with EVs purified from lymphatic drainage or exudative seroma, obtained after lymphadenectomy from 17 patients with stage III melanoma . The AutBoth the protein and mRNA content of L-EVs may be also investigated for predictive aims. For example, the expression of the Breast Cancer Resistance Protein (BCRP) in L-EVs from patients undergoing neoadjuvant treatment for locally advanced BC proved to be correlated with primary resistance to anthracycline-based chemotherapy, indicating its possible use as a negative predictive biomarker .Since the circulating L-EV count reflects disease status, as well as relative tumor burden, its longitudinal evaluation during treatment could be used for monitoring response. In this context, Julich-Haertel et al. identified a particular subset of AnnexinV+/EpCAM+/ASGPR1+ L-EVs which were highly specific for the identification of hepatic malignancies, whose circulating levels rapidly decreased after curative tumor resection, thus suggesting their possible use as biomarkers of minimal residual disease . In the Finally, analyzing the cargo of the L-EVs is another potential tool for monitoring tumor cell changes during anti-tumor therapy, to early detect the onset of acquired drug resistance. The identification of specific L-EVs, in fact, can be the distinctive of chemoresistant tumor cells as occur for TrpC5+ L-EVs that were demonstrated in metastatic BC patients following adriamycin-based chemotherapy, while being undetectable in chemotherapy naïve patients .L-EVs are interesting vesicles, with potential clinical applications as they play an important role in the processes of cancerogenesis, metastasis and drug resistance. Their content in protein, DNA and regulatory RNAs, is several-fold higher than any other vesicle class, mirroring the composition of the parental cell. All these characteristics make L-EVs an ideal tool for liquid biopsy in oncology.At present, promising results for the use of circulating L-EVs, as either a prognostic or predictive tool, have been produced with GBM and melanoma. Due to its intracranial localization, in fact, GBM is hard accessible for traditional biopsy and, thus, L-EVs liquid biopsy was proposed for prognostic stratification of patients, as well as to predict response to targeted therapy. On the other hand, the L-EV-DNA analysis has been proposed for the mutational analysis of melanoma patients amenable to anti-BRAF therapies. Moreover, mutational tracking along the therapy is thought to early detect the onset of acquired resistance with potential implications in the treatment decision.Despite the original optimism for the translatability of L-EVs in the clinical arena, several limitations need to be first overcome. In this context, the major challenge concerns the lack of standardized protocols for L-EVs isolation and downstream analyses, while the current absence of universally accepted markers for quality control restricts their use in the pre-clinical setting. Additionally, another unsolved issue depends on the contamination of tumor-derived L-EVs with the EVs that have not a tumoral origin, meaning that tumoral products result greatly diluted in the multitude of “physiological” L-EVs and hardly accessible to further molecular testing. A possible solution could be the use of highly specific analytical techniques able to differentiate the tumor-derived material from the physiological material. In this direction, instruments for single EVs analysis are under development . MoreoveIn conclusion, although the pre-clinical data of L-EVs appear very promising, further methodological improvements and validation from large clinical trials are needed to support the applicability of these vesicles as tumor biomarkers for tailoring treatment decisions or monitoring tumor progression.


A high prevalence of cardiovascular risk factors including age, male sex, hypertension, diabetes, and tobacco use, has been reported in patients with Coronavirus disease 2019 (COVID-19) who experienced adverse outcome. The aim of this study was to investigate the relationship between cardiovascular risk factors and in-hospital mortality in patients with COVID-19.MEDLINE, Cochrane, Web of Sciences, and SCOPUS were searched for retrospective or prospective observational studies reporting data on cardiovascular risk factors and in-hospital mortality in patients with COVID-19.Univariable and multivariable age-adjusted analyses were conducted to evaluate the association between cardiovascular risk factors and the occurrence of in-hospital death.The analysis included 45 studies enrolling 18,300 patients. The pooled estimate of in-hospital mortality was 12% (95% CI 9–15%). The univariable meta-regression analysis showed a significant association between age , diabetes  and hypertension  with in-hospital death. Male sex and smoking did not significantly affect mortality. At multivariable age-adjusted meta-regression analysis, diabetes was significantly associated with in-hospital mortality ; conversely, hypertension was no longer significant after adjustment for age . A significant association between age and in-hospital mortality was confirmed in all multivariable models.This meta-analysis suggests that older age and diabetes are associated with higher risk of in-hospital mortality in patients infected by SARS-CoV-2. Conversely, male sex, hypertension, and smoking did not independently correlate with fatal outcome. Coronavirus disease 2019 (COVID-19) is a recently recognized infective disease caused by a new betacoronavirus, which has sparked in Hubei province, China, and has spread rapidly worldwide taking on pandemic proportions.  OR “COVID 19”[All Fields] OR “severe acute respiratory syndrome coronavirus 2”[All Fields] OR “2019-nCoV”[All Fields] OR “SARS-CoV-2”[All Fields]) AND ; Cochrane Library, Web of Science and SCOPUS—“COVID-19” OR “COVID 19” OR “SARS-CoV-2” OR “severe acute respiratory syndrome coronavirus 2” OR “2019-nCoV”. Search was conducted by using the Thomson Reuters EndNote X7 software.Citations were screened on the title and abstract level by three independent reviewers , and potentially eligible reports were retrieved and scrutinized in full text. Divergences were resolved by discussion and consultation with a fourth investigator (G.G.). The full-size articles published in peer-reviewed journals were considered for this meta-analysis.Prospective and retrospective observational studies were included if they met the following pre-specified criteria: (I) inclusion of patients hospitalized for COVID-19; (II) data on in-hospital mortality; (III) data on baseline cardiovascular risk factors. Studies reporting only surrogate outcome measures or with largely incomplete data on in-hospital outcome were excluded. Studies reporting data on special populations  were excluded. For papers collecting overlapping data, only studies with the largest number of patients were selected. In some doubtful cases, corresponding authors were contacted for requests for clarifications.Two investigators (A.S. and L.E.) independently extracted data by using an agreed predefined spreadsheets reporting first author, journal, year of publication, study design, region and country, period of enrollment, sample size, in-hospital mortality, demographic and clinical cardiovascular risk factors . Data on the severity of the disease expressed by the percentage of acute respiratory distress syndrome (ARDS) and of invasive mechanical ventilation (IMV) use, as well as data on clinical setting expressed by the percentage of admission in intensive care unit (ICU) were collected. Disagreements were resolved by consensus among all the investigators.Two unmasked reviewers  evaluated the quality of the studies on pre-specified electronic forms according to the Newcastle–Ottawa scale items. The reviewers independently appraised study selection, comparability, and outcome of each report, and divergences were resolved after consensus.The study outcome measure was the occurrence of death during hospitalization.The meta-analysis was performed by estimating the mean in-hospital mortality over all studies using random effects models with restricted maximum-likelihood estimator. Random-effects model was preferred for estimating the average effect and its precision, which would give a more conservative estimate of the 95% confidence interval (CI), due to the heterogeneity within and between studies. Studies with larger sample size and therefore a smaller standard error received more weight when calculating the mean survival proportions. The individual study proportions of outcomes were converted using the Freeman–Tukey double arcsine transformation method before the pooled analysis. The summarized proportions in the original scales were calculated as the back-transformation of the arcsine transformed estimates. Raw data on the prevalence of cardiovascular risk factors were collected for each study included in this meta-analysis.Multiple univariable meta-regression analyses were performed to appraise the possible association between the proportion of single cardiovascular risk factors and the risk of death, as well as the degree of heterogeneity among studies. In order to assess whether the associations resulted at univariable analyses were age-independent, data on each cardiovascular risk factor were entered in age-adjusted multivariable meta-regression models.These analyses were performed using the logarithmic transformation of the proportions, then back transformed through the exponential function.To appraise for the influence of clinical setting on the study outcome, we conducted a subgroup analysis for studies including only ICU patients versus studies enrolling mixed population (both ward and ICU) and tested any interaction between subgroups.The hypothesis of statistical heterogeneity was tested by means of Cochran Q statistic and the null hypothesis of statistical homogeneity was refused if p values were less than 0.10. I  values <Of 12,038 reports initially identified, we retrieved 8,514 studies through merging of data from independent searches and removing duplicates. Sixteen studies have been excluded because of overlapping populations. During screening and eligibility assessment, we identified 45 full-size articles enrolling 18,300 patients. , 15–54 T2: 96.5%; p < 0.001; Fig. The pooled estimate of in-hospital mortality was 12% (95% CI 9–15%) although affected by a high heterogeneity degree among studies (Iinteraction = 0.004).Data on clinical setting were available in 19 studies Fig. . Seven sThe univariable meta-regression analyses between cardiovascular risk factors and in-hospital mortality are displayed in Fig. At multivariable age-adjusted meta-regression analysis, diabetes was significantly associated with in-hospital mortality ; conversely, hypertension was no longer significant after adjustment for age , suggesting that in-hospital mortality did not depend on the size of the studies  studies, and selective reports from special populations. .By including 45 studies and 18,300 patients, this is the largest meta-analysis investigating the effect of cardiovascular risk factors on in-hospital mortality in COVID-19, by using crude and multivariable meta-regression models, available so far. The main findings of this study can be summarized as follows: (I) COVID-19 was associated to a high risk of in-hospital death, which occurred in about one patient in 8; (II) studies with high percentage of ARDS and IMV use, two indexes of disease severity, as well as of ICU admission, reported the highest risk of death during hospitalization; (III) age and diabetes were independent predictors of mortality; (IV) after adjustment for age, hypertension did not show any significant association with the risk of in-hospital death.The mortality estimate of COVID-19 in the general population depends on the number of deaths relative to the number of confirmed cases of infection, which is not representative of the actual death rate, and may be substantially overestimated. , 57 RealThe pooled mortality estimate in our analysis was affected by a wide inconsistency between patient cohorts. There are many reasons for the effect variability detected across observational studies including clinical setting , the prevalence of in-hospital complications (e.g. ARDS) and the need of invasive mechanical supportive measures, which may play as effect modifiers. We tried to account for this variability by performing exploratory analyses of the relationships between the rate of mortality and the proportion of ARDS and IMV use, which are indexes of disease severity. As expected, the higher the percentage of ARDS/IMV among patients enrolled, themm higher was the rate of death during in-hospital course. In addition, the subgroup including only studies conducted in ICU, compared to the mixed group, showed a significantly higher risk of death. These analyses were still affected by high residual heterogeneity suggesting that these variables, although able to stratify prognosis, could not account for the differences among the studies included in this meta-analysis. Our analysis could not account for multiple variables as well as for concealed confounders such as the availability of health-care resources, in-hospital paths, healthcare systems and strategies adopted in the context of pandemic between centers or countries.  ActuallyThe association of age with mortality is an expected finding; noteworthy, this significant correlation persisted in multiple adjusted meta-regression models including other cardiovascular risk factors. This result underlies the importance of individual preventive and protective measures during COVID-19 public health crisis and of dedicated healthcare strategies for elderly patients during hospitalization.Diabetes is the second most common comorbidity (after hypertension) in hospitalized patients with COVID-19, and its prevalence increase with age. , 26, 60 SARS-CoV-2 binds to the zinc peptidase angiotensin-converting enzyme 2 (ACE2), a surface molecule expressed by endothelial cells of arteries and veins, arterial smooth muscle, epithelial cells, and immune cells.  Some autOur study demonstrates that the association between hypertension and mortality in hospitalized patients with COVID-19 reflect largely the older age of these patients. In fact, the significant correlation observed at the univariable meta-regression model was not confirmed after adjustment for age. Preliminary studies suggested that hypertension was a risk factor for in-hospital outcome in patients with COVID-19. , 26, 60 The association between smoking and mortality in COVID-19, though plausible, is still debated , 66. A rThe recent onset and the variable dissemination pattern of the outbreak make difficult to draw firm conclusions on the clinical characteristics and epidemiology of COVID-19. Our data indicate that elderly and diabetic patients are higher-risk populations, and suggest taking utmost care in clinical management of these patients during hospitalization.Continuous surveillance, with reporting of patient characteristics worldwide, are required to confirm our findings and more deeply understand the relationship between cardiovascular risk factors and outcome of patients infected by SARS-CoV-2.Some limitations of our study should be acknowledged. Although we included 45 studies enrolling 18,300 patients, results of meta-analyses are hypothesis-generating and should be interpreted accordingly.The observational nature of the studies included might have contributed to the heterogeneity observed in this analysis. We tried to manage this issue by performing univariable meta-regressions for ARDS and IMV as well as a subgroup analysis for ICU vs ICU/ward. Although these analyses showed significant and clinically valuable results, they were still affected by a substantial residual heterogeneity.Owing to the intrinsic limitations of study-level analyses, we could not account for multiple potential confounders and we adjusted only for age, considered the most important confounding factor in this clinical setting. Moreover, we could not adjust for hidden confounders as well as for patient characteristics non-systematically or rarely reported.Many authors reported incomplete clinical and instrumental information  due to factors such as critically ill patients, operator’s fear of contagion, and in-hospital paths. We may hypothesize that difficulties in data collection/storage and limited feasibility/repeatability of instrumental exams may have affected the completion and granularity of data.To meet the urgent need of clinical reports on COVID-19, many studies encompassed a relatively short follow-up time as compared to the course of the disease, and some of them ended before the discharge or death of each patients enrolled. Although we tried to manage this issue by excluding the studies with higher number of open cases at the end, a certain underestimation of mortality is likely. We do not claim to provide the exact estimate of in-hospital mortality in COVID-19 pandemic, but to explore the potential association between cardiovascular risk factors and outcome. Since the number of pandemic-focused studies is increasing, and clinical observation of these patients is ongoing, the current mortality pattern might change to some extent in the next weeks or months.The majority of studies included in this analysis reported data from Chinese populations, and only ten reports were from other countries (3 from Europe and 7 from US). This issue might have affected the generalizability of our findings, and requires confirmation by large worldwide studies.Among the cardiovascular risk factors, we did not analyze unfrequently reported conditions such as obesity, dyslipidemia, and family history of cardiovascular disease. Indeed, these data were seldom reported in the items selected for this meta-analysis, and were not sufficient for inference analysis.This study suggests that older age and diabetes are associated to higher risk of in-hospital mortality in patients infected by SARS-CoV-2. Conversely, hypertension, male sex and smoking did not independently correlate with fatal outcome at multivariable meta-regression analysis.Although difficult to realize in a pandemic scenario, large multicenter cross-national studies are warranted to improve our understanding on the association between cardiovascular risk factors and mortality in COVID-19.Additional file 1: Main characteristics of the studies included in the analysis.Additional file 2: Quality assessment of the studies based on the Newcastle–Ottawa scale.