Chronic obstructive pulmonary disease (COPD) is projected to become the third cause of mortality worldwide. COPD shares several pathophysiological mechanisms with cardiovascular disease, especially atherosclerosis. However, no definite answers are available on the prognostic role of COPD in the setting of ST elevation myocardial infarction (STEMI), especially during COVID-19 pandemic, among patients undergoing primary angioplasty, that is therefore the aim of the current study.In the ISACS-STEMI COVID-19 registry we included retrospectively patients with STEMI treated with primary percutaneous coronary intervention (PCI) between March and June of 2019 and 2020 from 109 high-volume primary PCI centers in 4 continents.A total of 15,686 patients were included in this analysis. Of them, 810 (5.2%) subjects had a COPD diagnosis. They were more often elderly and with a more pronounced cardiovascular risk profile. No preminent procedural dissimilarities were noticed except for a lower proportion of dual antiplatelet therapy at discharge among COPD patients . With regards to short-term fatal outcomes, both in-hospital and 30-days mortality occurred more frequently among COPD patients, similarly in pre-COVID-19 and COVID-19 era. However, after adjustment for main baseline differences, COPD did not result as independent predictor for in-hospital death  nor for 30-days mortality . No significant differences were detected in terms of SARS-CoV-2 positivity between the two groups.This is one of the largest studies investigating characteristics and outcome of COPD patients with STEMI undergoing primary angioplasty, especially during COVID pandemic. COPD was associated with significantly higher rates of in-hospital and 30-days mortality. However, this association disappeared after adjustment for baseline characteristics. Furthermore, COPD did not significantly affect SARS-CoV-2 positivity.Trial registration number: NCT 04412655 (2nd June 2020). Chronic obstructive pulmonary disease (COPD) has been estimated affecting more than 200 million people worldwide, representing the fourth highest cause of death in the world, with a projection to be the third leading cause of death in next years . It reprThis is reflected on the clinical side, where COPD patients are recognized at higher risk of cardiovascular diseases and mortality , 4. MoreTherefore, we aimed to inquire from a large, contemporary, multicenter registry of STEMI patients if COPD diagnosis at hospital admission could be deemed a risk factor of adverse outcome, especially during COVID-19 pandemic.st until June 30th of 2019 with those admitted within the same period of 2020. We included patients treated within 48 h from symptoms onset. We did not exclude those patients undergoing mechanical revascularization after failed thrombolysis (rescue angioplasty).Study population is constituted by patients enrolled in the ISACS-STEMI COVID-19 (NCT 04412655), a retrospective multicenter registry including STEMI patients enrolled by 109 high-volume primary PCI centers from Europe, Latin America, South-East Asia and North-Africa. This study was conducted to compare STEMI patients treated from March 1Collected baseline characteristics included demographic, clinical, procedural data, data on total ischemia time, door-to-balloon time, referral to primary PCI facility and PCI procedural data. COPD diagnosis was determined at admission according to international guidelines society . The outThe study was approved by the Ethical Committee of AOU Maggiore della Carità, Novara. Detailed data have previously been provided , 14.All analyses were performed by using SPSS Statistics Software 23.0 . Patients were grouped according to COPD diagnosis. Absolute frequencies and percentages were used for categorical variables. Conversely, continuous variables were reported using median and interquartile range. Normal distribution of continuous variables was tested by the Kolmogorov–Smirnov test. Chi-square test was adopted for categorical variable, while ANOVA or Mann–Whitney test, as appropriate, were used for continuous variables.P‐value < 0.1) were entered in block into the model. A P < 0.05 was considered statistically significant. The data coordinating center was established at the Eastern Piedmont University, Novara, Italy.Multivariable logistic regression analyses were performed to identify the association of COPD with in-hospital and 30-day mortality and SARS-CoV-2 infection after adjustment for baseline confounding factors. All significant variable  patients were affected by COPD, while 14,876 (94.2%) patients were not. Table Angiographic and procedural characteristics are summarized in Table A similar percentage of SARS-CoV-2 positive patients between the two groups was recognized .A significant higher rate of mortality was experienced by COPD patients than ones without, both considering in-hospital  and 30-day  mortality , neither for in-hospital  nor for 30-day mortality .Main findings from our large, multicenter, contemporary registry of STEMI patients are consistent with a low but not negligible percentage of patients with COPD. They displayed a significant higher baseline cardiovascular risk profile that portended an increase rate of in-hospital and short-term mortality. However, after adjustment for baseline clinical confounders, COPD did not result an independent predictor of fatal outcomes.The strict relationship between lung and heart is well known, even if the mechanisms of this interplay are less intuitive and not fully delineated, especially on the pathological side: of course, sharing two key risk factors, age and cigarette smoking, enforces the link between COPD and ischemic heart disease. However, during last years the paradigm of COPD as localized inflammatory disease has shifted to systemic involvement, driven by the evidences of higher level of C-reactive protein, fibrinogen and pro-inflammatory cytokines , 16. TogResults from clinical studies have shown a prevalence of coronary artery disease among COPD patients ranging from 10 to 38% , 20 withSimilarly, individuals with acute myocardial infarction are more prone to adverse outcomes if a concomitant COPD diagnosis was present . Data frFurther distinctions have been enlightened by prior studies with regards of STEMI or Non-STEMI presentation. Enriquez et colleagues have shown no significant independent role of COPD into prediction of in-hospital mortality among STEMI patients, whilst it was exclusive of among those with Non-STEMI . MoreoveIn our large multicenter cohort of patients admitted for STEMI a relatively limited proportion of subjects diagnosed with COPD. Their enhanced cardiovascular risk of COPD patients was made-up by more frequent co-presence of active smoking, diabetes mellitus, hypertension, hypercholesterolemia, prior acute cardiac ischemic event, in line with previous reports on the topic . MoreoveCOPD patients were more prone to fatal outcomes displaying both for in-hospital and 30-day mortality a higher percentage that patients without COPD. However, these significant differences were not confirmed at multivariate logistic regression analyses that did not show an independent role of COPD diagnosis to predict short-term mortality. Our findings are inserted in the controversy still persistent on whatever COPD might represent a risk factor unassociated with others or if it reflects the burden of comorbidities linked to this condition , 33. AddAdditional value of this investigation is the crucial information regarding the ongoing COVID-19 pandemic impact, especially considering our inspection of a lung disease. COPD is known providing higher risk of infection: lung inflammation, airflow obstruction, and impaired clearance of respiratory virus are elements supporting a greater risk for COVID-19 and its severity among COPD patients , 36–38. Findings from our large multicenter registry provide an updated snapshot of the impact of COPD on short term outcomes in the acute setting of STEMI. Based on our data, COPD does not represent itself an independent negative prognostic determinant. Therefore, COPD should not be used for risk stratification and COPD patients should not be object of more intensive and aggressive therapies as compared to the standard care for STEMI patients. Dedicated, prospective studies may offer definite and comprehensive answers about the COPD contribution on mortality after an acute MI.Our findings should be viewed in light of some limitations. The retrospective non-randomized design of our study precludes to definite answer on the impact of COPD on morality in patients experiencing a STEMI. Not all variable of interest could have been collected, reducing the allowed investigations.The diagnosis of COPD was based on medical records of every patients, and systematic confirmation through pulmonary function tests in case of suspect of doubt diagnosis was not performed. As well, we were not able to classify COPD patients according to their inflammatory endotype , that haThe ISACS-STEMI registry was not conceived to examine the role of COPD, therefore data are missing on complete in-hospital and post-discharge drug treatment regimens, specifically regarding the COPD, preventing analyses on essential aspect of the treated topic , even ifThis is one of the largest studies investigating characteristics and outcome of COPD patients with STEMI undergoing primary angioplasty, especially during COVID-19 pandemic. COPD was associated with significantly higher rates of in-hospital and 30-days mortality. However, this association disappeared after adjustment for baseline characteristics. Furthermore, COPD did not significantly affect SARS-CoV-2 positivity.


The invasive nature of brain tumours, particularly glioblastoma, severely limits its therapy. Matrix-metalloproteinases (MMPs), enzymes involved in the degradation of the extracellular matrix, are associated with the invasiveness of brain tumours; hence, the determination of MMPs is critical for the monitoring of cancer patients. The aim of our comparative study was to evaluate the possible additional utility of the MMP-9 level of serum-derived small extracellular vesicles (sEVs) for characterising brain tumours. We established a relationship between low MMP-9 content in sEVs and improved survival, and discovered that MMP-9 levels considerably differed between tumour types and stages, showing a positive correlation with aggressiveness. We demonstrated on a large number of samples that the high MMP-9 level of serum-sEVs may serve as a negative prognostic marker for brain tumours.t-test, Welch’s test, ANOVA, ROC). We found that the MMP-9 content of sEVs is independent of gender and age, but is affected by surgical intervention, treatment, and recurrence. We found a relation between low MMP-9 level in sEVs (<28 ppm) and improved survival (8-month advantage) of glioblastoma patients, and MMP-9 levels showed a positive correlation with aggressiveness. These findings suggest that vesicular MMP-9 level might be a useful prognostic marker for brain tumours.Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell invasion and metastasis, and its elevated level in brain tumour tissues indicates poor prognosis. High-risk tissue biopsy can be replaced by liquid biopsy; however, the blood–brain barrier (BBB) prevents tumour-associated components from entering the peripheral blood, making the development of blood-based biomarkers challenging. Therefore, we examined the MMP-9 content of small extracellular vesicles (sEVs)—which can cross the BBB and are stable in body fluids—to characterise tumours with different invasion capacity. From four patient groups , 222 serum-derived sEV samples were evaluated. After isolating and characterising sEVs, their MMP-9 content was measured by ELISA and assessed statistically (correlation, paired  In tumour diagnostics, computed tomography (CT) and magnetic resonance imaging (MRI) scans are used to determine disease status, as well as to evaluate the response to treatments ; howeverFurther standard methods for profiling tumours require obtaining tumour samples through invasive surgical procedures. In addition to carrying a high risk of complications, the limitations of such invasive procedures include difficulty in acquiring tumour samples with high heterogeneity or in inaccessible positions and taking multiple biopsies in the occurrence of metastases, as well as monitoring tumour response or relapse. Thus, neuro-oncological research aims to discover novel methods suitable for monitoring CNS tumours in clinical practice .Considering the challenges associated with traditional biopsies, recent oncology research has turned its focus from analysing whole tissues to analysing various biological fluids for tumour-derived components ,8,9; thiExtracellular vesicles (EVs) are small, lipid bilayer-enclosed vesicles released by both normal and neoplastic cells into the extracellular space  which enFinding blood–based biomarkers for CNS tumour monitoring is more challenging, as the blood–brain barrier (BBB) may prevent the release of tumour-related biomarkers into peripheral blood. The available evidence supports that tumour-derived EVs can cross the BBB ,30. HoweIn a previous study, we analysed the protein content of whole serum as well as serum-derived small extracellular vesicles (sEVs)—EVs with a diameter of 30–200 nm size , theoretMatrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play an essential role in the physiology of cells by degrading the extracellular matrix (ECM) . It has Matrix metalloproteinase-9  is one of the most complex forms of MMPs . The rolIn recent decades, several studies reported the upregulation of MMP-9 in the source of tumour tissue, which provides the opportunity to identify MMP-9 in the serum as well. In the case of some peripheral tumours, elevated MMP-9 levels in plasma were detected and associated with cancer development, invasion, and poorer survival ; howeverBased on the findings of our previous study , we hypoFor this purpose, in this study, 222 serum samples were collected from four patient groups according to the criteria of the National Ethical Committee, and MMP-9 analysis was performed on serum-derived sEV samples using enzyme-linked immunosorbent assay (ELISA). According to our knowledge, this was the first study that investigated the MMP-9 level of serum sEVs in human brain tumours. The large sample size allowed us to demonstrate the factors influencing the MMP-9 level , and was suitable for determining a measurable, significant difference in the sEV MMP-9 level associated with different diseases and patient survival outcomes. Calculating the MMP-9 concentrations of serum-derived sEVs enabled us to establish the cut-off values and the specificity and sensitivity of the analysis required in clinical practice.Blood samples of 222 patients treated at the Department of Neurosurgery, of the University of Debrecen were analysed. Samples were obtained from patients with glioblastoma multiforme (GBM), meningioma (M), and brain metastasis originating from non-small cell lung cancer (BM). Control samples (CTRL) were collected from patients with lumbar disc herniation without evidence of cancer. In addition to the four main groups, subgroups were also distinguished based on histopathology, sampling time (prior or after to surgery), grading, recurrence, and treatment . DetaileBlood samples were collected, processed to serum, and stored by the Neurosurgical Brain Tumour and Tissue Bank of Debrecen according to the criteria of the National Research Ethics Committee. An informed consent form was signed by each patient; the study was conducted in accordance with the Declaration of Helsinki. This study was carried out according to two ethical approvals, namely 51450-2/2015/EKU (0411/15), Medical Research Council, Scientific and Research Ethics Committee, Budapest, 30 October, 2015 and 121/2019-SZTE, University of Szeged, Human Investigation Review Board, Albert Szent-Györgyi Clinical Centre, Szeged, 19 July 2019.g, 10 °C. The serum samples were stored at −80 °C until further processing.Blood samples were collected into BD Vacutainer SST II Advance Tubes, allowed to clot for at least 1 h at room temperature, and centrifuged for 20 min at 3000× g, 4 °C, then for 70 min at 110,000 g, 4 °C, using a fixed-angle rotor . The pellet was resuspended in particle-free Dulbecco’s phosphate-buffered saline  and stored at −80 °C until further processing. This sEV isolation protocol served to reach intermediate recovery and intermediate specificity according to the guideline ‘Minimal Information for Studies of Extracellular Vesicles 2018’ (MISEV2018) [sEVs were isolated from the sera via differential centrifugation as described previously . BrieflySEV2018) .Following the main suggestions and requirements included in MISEV2018, sEVs were characterised by transmission electron microscopy (TEM), Western blot analysis (WB), and nanoparticle tracking analysis (NTA). The TEM and WB measurements were performed on a representative sample of each main group.In order to examine sEV morphology, TEM analysis was performed using a Tecnai G2 20 X-Twin type instrument , operating at an acceleration voltage of 200 kV. The samples were dropped on a grid ) and dried without staining or other fixation procedure.Confirming the presence of sEVs, CD81, CD5L, and calnexin markers were presented by Western blot analyses using NuPAGE reagents and an XCell SureLock Mini-Cell System  according to the manufacturer’s protocols. The protein content of sEV samples was determined using a Pierce BCA Protein assay kit  and a benchtop microplate reader  according to the manufacturer’s instructions. For detection of the sEV markers in the four main groups, rabbit anti-human Alix (1:1000), rabbit anti-human CD5L (1:2000), and rabbit anti-human Calnexin  primary antibodies , and HRP-conjugated anti-rabbit IgG  secondary antibody were used. THP-1 cell line  lysate was used for the positive control for Calnexin.Determining the nanoparticle size distribution and concentration of the tested samples, sEVs were diluted in particle free DPBS and analysed using a NanoSight NS300 instrument with 532 nm laser . Six videos of 60 s were recorded for each sample under constant settings .The LC-MS analysis was performed in a previous study  based onThe detailed methodology of LC-MS measurements was described in the named article . BrieflyTo accurately measure MMP-9 content of sEVs, LEGEND MAX Human MMP-9 ELISA Kit  was used according to the manufacturer’s protocol. sEV isolates of 222 serum samples were measured individually, and the vesicles were disrupted in a detergent-free manner by five repeated freeze–thaw cycles to expose the entire protein content. As we examined sEV isolates, potential interferences—observed in clinical immunoassays of sera —are avoiA standard curve was applied for each assay, and all samples were run in duplicate on separate plates. In the first step, 50μL assay buffer and 50 uL standard dilutions or 12× diluted samples were added to the appropriate wells, and the plates were sealed and incubated at room temperature for 2 h while shaking at 200 rpm. After the incubation, plates were washed four times with 1× wash buffer. The first step was followed by three more incubation steps, namely 100 μL of human MMP-9 detection antibody solution, 100 μL of Avidin-HRP solution, and 100 μL of substrate solution D were added to each well and incubated at room temperature for 1 h, 30 min, and 15 min, respectively. The plates were washed four times with 1× wash buffer between each incubation procedure. The last step was performed in the dark, and then the reaction was stopped by adding 100 μL of stop solution; the absorbance was immediately read at 450 nm and 570 nm on a benchtop microplate reader .Before conducting any statistical tests, the MMP-9 level was normalised to the protein content of serum sEVs, meaning that MMP-9 concentration (ng/mL) was divided by protein concentration of the EV-enriched isolates (ng/mL) for every sample. The results of all the analyses are notated in parts-per-million (ppm), describing the individual values of MMP-9 in patients.Outliers from the analysed groups were always excluded using the ROUT (robust regression followed by outlier identification) method with the Q parameter set to 1%. Then we examined the assumptions of normal distribution utilising the Shapiro–Wilk test and the homogeneity of variances by using the F test (comparing two groups) and the Brown–Forsythe test (comparing more than two groups).t-test, respectively. One-way ANOVA was used to examine differences between more than two groups.The MMP-9 ppm concentrations of independent groups and matched samples were compared with Welch’s test and the paired To determine the relationship between continuous variables, linear regression analyses were conducted. To improve linearity in regression, skewed data were logarithmised.The diagnostic potential of the MMP-9 ppm was evaluated using receiver operating characteristic (ROC) analyses. Kaplan–Meier analyses with log-rank tests were used to compare the overall survival rates of different groups.The collected data about the MMP-9 content of sEVs were analysed statistically using GraphPad Prism 8.3.4.All datasets generated during the current study are available from the corresponding author upon reasonable request. All relevant data of our experiments had been submitted to the EV-TRACK  knowledgOur main goal was to compare the MMP-9 content of serum-derived small extracellular vesicle (sEV) samples from 222 patients with different CNS diseases. The four main investigated groups were glioblastoma (GBM), brain metastasis originated from non-small cell lung cancer (BM), meningioma (M), and lumbar disc hernia patients as controls (CTRL).p = 0.0065, multi ROC AUC = 0.86).In a previous study, following the isolation and characterisation of sEV samples, LC-MS analysis was performed on 96 sEV samples. Each of the four groups  contained 24 individuals, and six sample pools were created from the individuals allowing four parallel samples to be tested per group. As a result of the analysis, a 17 membered protein panel was constructed from the identified proteins which were able to separate the four groups with 100% efficacy . In thisr = 0.7264; p = 0.0022)  were compared with the averaged MMP-9 concentrations (from ELISA) of the six-sample-pools to ascertain that ELISA is also capable of distinguishing the groups. The correlation analysis confirmed that the MMP-9 concentrations measured by ELISA were highly comparable to LC-MS measurements ( 0.0022) .Therefore, we aimed to perform ELISA measurements on the larger cohort; the MMP-9 concentrations and sEV characteristics can be found in Performing comparative studies between different tumours requires examining what factors  may influence MMP-9 levels of serum-derived sEVs .The MMP-9 level was normalised to the total protein content of serum sEVs, meaning that MMP-9 concentration (ng/mL) was divided by protein concentration of the EV-enriched isolates (ng/mL) for every sample. The results of all the analyses are notated in parts-per-million (ppm) describing the individual values of MMP-9 in patients .Age and gender analyses were carried out in the control group, eliminating the possible MMP-9-modifying effect of any tumour disease. Correlation analysis revealed that there is no distinct relationship between age and MMP-9 levels a. The MMt-tests were completed in the GBM and BM groups  before and after the surgical resection in the case of primary GBM patients, while BM samples showed marked increase  after resection.Examining the effects of surgical resection on the MMP-9 level of sEVs, paired p < 0.0001).Further examination on preoperative GBM samples found a distinct difference between the original tumour and the recurrence based on MMP-9 levels of serum sEVs e, and thp < 0.0001) indicates that therapy might decrease the MMP-9 levels of the circulating sEVs  were performed on all primary and secondary GBM samples, as well as on all BM samples to determine the effect of surgical resection on a broader cohort  using a cut-off point of 16 MMP-9 ppm with 74% sensitivity, 61% specificity, and an AUC of 0.70  using 0–2, 3–8, 10–23 months as the cut-offs a. Long-tp = 0.0063), which represents an eight-month increase in the median OS , so we performed an analysis where we observed that lower survival time was associated with higher MMP-9 level e. To detedian OS f.p = 0.0340) in median survival  survival found to be associated with low MMP-9 level (<28 ppm). Our results support that the high level of MMP-9 in serum-derived sEVs might be a negative prognostic marker of the probability of survival in GBM patients.In recent years, oncology research has turned its focus from analysing whole tumour tissues to analysing various biological fluids for tumour-derived components ,9. AlthoUsing EV samples instead of whole sera, it is possible to amplify the signals released by brain tumours into the circulation . ExtraceDue to the priority of living-cell secretion, large amounts and stable circulation compared to circulating tumour cells and ctDNA, exosome-based liquid biopsy has been tested in clinical trials and several of them have been approved and reached the market. In 2016, Exosome Diagnostics proposed the first exosome-based liquid biopsy in the world , ExoDx™ According to Lihares et al., screening for gliomas has no clinical relevance at this time . This isBased on these considerations, we sought to determine whether the MMP-9  level of serum-derived sEVs can provide information about patient survival. ELISA was used to measure the MMP-9 content of 222 sEV samples isolated from whole serum for this purpose. Serum samples were collected from patients with the most common malignant, benign, and metastatic brain tumours, namely glioblastoma multiforme, meningioma , and braFollowing the measurements, comparative analyses on the MMP-9 level of sEVs were conducted. We determined that surgical resection, recurrence, and therapy can modify the MMP-9 level, but neither age nor gender can. We also detected statistically significant differences in the sEVs’ MMP-9 levels associated with various diseases, as well as a correlation between low MMP-9 level and longer survival time, which highlighted the prognostic value of vesicular MMP-9 level. However, functional studies, including MMP-9 activity measurements, would be needed to explore the causal relationship between tumour progression and the MMP-9 level of serum sEVs.We hypothesise that the higher MMP-9 level of sEVs observed in patients with a poor prognosis is due to the tumour itself. However, there is a debate surrounding the source of the tumour-specific EVs in the blood. Osti et al., as a confirmation of the tumour origin of extracellular vesicles, employed an orthotopic transplantation of tumour-initiating cells (TICs) isolated from human GBM. GBM TICs expressing the exosomal marker CD9 coupled with the GFP protein were produced using lentiviral transduction. Because GBM-derived extracellular vesicles express CD9 on their surface, they were distinguished from natural GFP-negative mouse extracellular vesicles by following the GFP signal. According to their data, nearly half of the extracellular vesicles in the peripheral circulation of transplanted mice were tumour-derived . Howeverp < 0.001) [To the best of our knowledge, this study was the first one that analyses the MMP-9 content of serum sEVs; nevertheless, a few serum MMP-9 investigations have been published with contradictory findings. Hormingo et al. found that YKL-40 and MMP-9 could be monitored in patients’ serum and help confirm the absence of active disease in GBM . However< 0.001) . This laSeveral technical issues may have contributed to the poor and contradictory serum/plasma results. It is important to note that the measurement of MMPs in body fluids can be influenced by the type of fluid and method of collection and storage. EDTA or heparin, for instance, can affect the baseline serum/plasma concentrations of MMP-9 . To solvAnother difficulty is that plasma MMP-9 may be unstable and degrades rapidly even when stored at −80 °C ,62. ThisRecently, several research groups have studied vesicular mRNA that can be isolated from body fluids ,66,67,68It is also important to note that there is no gold standard method for sEV isolation, which may impede the reproducibility of experimental findings in EV research. The applied technique impacts the isolation efficacy in terms of yield, purity, and EV composition, which may interfere with downstream analyses, including the molecular cargo profiling ,79,80,81Prior studies have suggested that MMP-9 measurements in serum do not reliably reflect circulating MMP-9 levels and may be artificially high compared to results obtained from plasma samples ,91,92,93In order to find the putative source of the MMP-9 as biomarker, Ricci et al. correlated the expression of serum MMP-9 with the expression of the same protein in the tumour tissue. To address this aim, glioma tissues were subjected to immunohistochemistry with MMP-9 antibody. They discovered that the delocalisation of the MMP-9 signal from glioma cells to endothelial cells of neoplastic blood arteries is directly associated with tumour malignancy. In line with these findings, previous immunohistochemical and in situ hybridization studies demonstrated that, in high-grade glioma, MMP-9 expression is primarily restricted to perivascular regions at the infiltrating borders of the tumour and, in the majority of cases, to endothelial cells, with a close association to tumour malignant behaviour ,95. To dThe successiveness of a biomarker candidate can be determined by comparing its efficacy to that of existing clinical procedures. We were unable to correlate the sEVs’ MMP-9 level of the patients with their MRI status; nevertheless, contradictory findings exist in the literature. Hormigo et al. observed that levels of MMP-9 were higher in the serum samples of patients with high-grade glioma after surgery, while the MMP-9 concentrations were significantly lower in glioblastoma patients with no radiographic evidence of disease in comparison to the subjects with active tumour . In contIn addition to disease status, other patient characteristics may influence MMP-9 levels in the patient sera. Otero-Estévez et al. found that MMP-9 was significantly correlated with gender and age when examining the serum of colorectal cancer patients . In contp < 0.001) in plasma extracellular vesicle concentration after surgery [p = 0.1843; n = 14), while BM samples showed marked increase  after resection. This intriguing phenomenon relies on the basis of a few BM samples (n = 6), and therefore should be supported by further measurements.The surgical resection also can affect the extracellular vesicle concentration and the MMP-9 content of serum/plasma EVs. Osti et al. measured a significant drop ( surgery . Hormigo surgery . In our p < 0.0001) indicated that therapy might decrease the MMP-9 level of circulating sEVs (p < 0.0001), the majority of these GBM patients received treatment, therefore the decrease in MMP-9 cannot be attributed solely to relapse. Tabouret et al. reported that urine protein levels of MMP-2 also decreased and were considered to be related to treatment response, although this was not confirmed in an additional patient cohort [Determining the influence of the administered therapy, MMP-9 levels were also compared based on whether or not GBM patients had received treatment at the time of sampling. Our result  survival was found to be associated with a low MMP-9 level (<28 ppm). Our results support that the high level of MMP-9 in serum-derived sEVs might be a negative prognostic marker of the probability of survival in GBM patients. Osti et al. tested for possible associations between plasma extracellular vesicle concentration and patient outcomes. Patients with GBM were ranked according to their extracellular vesicle content and divided into high-content or low content groups; no significant differences in PFS or OS were found . Jiguet-By measuring the level of MMP-9, we may determine the efficacy of the treatment. For instance, the majority of patients with GBM will experience relapse despite surgery and standard first-line treatment consisting of radiotherapy with concurrent and adjuvant temozolomide. In certain cases, therapeutic options at the time of recurrence include surgery or reirradiation, whereas in other cases, bevacizumab is the preferred option worldwide. Jiguet-Jiglaire et al. demonstrated that low baseline plasma levels of MMP-9 were associated with a high response rate and a prolonged PFS and OS in patients with recurrent GBM treated with bevacizumab but not cytotoxic chemotherapy. In addition, they observed that MMP-9 plasma levels decreased during treatment with bevacizumab and tended to rise with disease progression. In a retrospective analysis performed in the Avaglio trial , a low plasma level of MMP-9 at baseline consistently predicted PFS and OS gain associated to bevacizumab .Unmet medical need exists for biomarkers able to predict response to antiangiogenic agents. According to the ClinicalTrials.gov database, only a few studies are currently being conducted to determine the predictive value of MMP-9 level on cancer treatment response. One study aims to investigate the predictive impact of circulating MMP-2 and MMP-9 on the progression-free survival of patients with metastatic kidney cancer treated with anti-angiogenic agents (Sunitinib or Pazopanib) in comparison with two untreated cohorts (NCT03185039). Another study evidenced the role for MMP-9 in the primary or acquired resistance to bevacizumab; therefore, in their trial they use monoclonal antibody GS5745 that may overcome resistance to bevacizumab through a specific inhibition of MMP-9. The phase I study is the first step to analyse the tolerance, determine the recommended dose of the combination, and explore the impact of GS5745 on MMP-9 plasma levels and multimodal imaging in patients with recurrent glioblastoma (NCT03631836).On the other hand, Farina and Mackay draw attention to the Janus-faced nature of MMP-9. This important molecule plays an essential role in tumour biology, from initiation/promotion to angiogenesis, dissemination, immune surveillance, and metastatic growth. However, MMP-9 also possesses antitumour activity and serves essential physiological functions . In ordeIn summary, we have demonstrated that sEVs’ MMP-9 content is suitable for estimating the probability of patient survival and it allows us to obtain information about CNS tumour aggressiveness. This biomarker’s benefits include its accessibility (a simple blood test), affordability, fast detectability, ease of implementation, and reproducibility. Collectively, these advantageous characteristics would permit the application of serum sEVs’ MMP-9 for non-invasive patient monitoring.Glioblastoma is the most prevalent primary brain tumour in adults, comprising 45.2% of all malignant primary CNS tumours, with a median survival time of 15 months. On average, 5.5% of patients live five years after their diagnosis .The link between the survival of glioblastoma patients and their MMP-9 level of serum-derived sEVs is revealed in our article; hence, the identification of sEVs’ MMP-9 is of great importance. Our presented fast-to-perform and non-invasive method can support clinical decision making.


The COVID-19 pandemic has disproportionately impacted economically-disadvantaged populations in the United States (US). Precarious employment conditions may contribute to these disparities by impeding workers in such conditions from adopting COVID-19 mitigation measures to reduce infection risk. This study investigated the relationship between employment and economic conditions and the adoption of COVID-19 protective behaviors among US workers during the initial phase of the COVID-19 pandemic.Employing a social media advertisement campaign, an online, self-administered survey was used to collect data from 2,845 working adults in April 2020. Hierarchical generalized linear models were performed to assess the differences in engagement with recommended protective behaviors based on employment and economic conditions, while controlling for knowledge and perceived threat of COVID-19, as would be predicted by the Health Belief Model (HBM).Essential workers had more precarious employment and economic conditions than non-essential workers: 67% had variable income; 30% did not have paid sick leave; 42% had lost income due to COVID-19, and 15% were food insecure. The adoption of protective behaviors was high in the sample: 77% of participants avoided leaving home, and 93% increased hand hygiene. Consistent with the HBM, COVID-19 knowledge scores and perceived threat were positively associated with engaging in all protective behaviors. However, after controlling for these, essential workers were 60% and 70% less likely than non-essential workers, who by the nature of their jobs cannot stay at home, to stay at home and increase hand hygiene, respectively. Similarly, participants who could not afford to quarantine were 50% less likely to avoid leaving home  than those who could, whereas there were no significant differences concerning hand hygiene.Our findings are consistent with the accumulating evidence that the employment conditions of essential workers and other low-income earners are precarious, that they have experienced disproportionately higher rates of income loss during the initial phase of the COVID-19 pandemic and face significant barriers to adopting protective measures. Our findings underscore the importance and need of policy responses focusing on expanding social protection and benefits to prevent the further deepening of existing health disparities in the US.The online version contains supplementary material available at 10.1186/s12889-022-13259-w. Socio-economic status (SES), defined by constructs such as education, occupation, and income , has lonSome sectors of the population bore the double brunt of economic strain and COVID-19 risk. During the early months of the COVID-19 pandemic, low-income workers disproportionately experienced job losses . ConcurrUS studies have documented growing SES and health disparities during the COVID-19 pandemic –9. TheseThis study investigated the potential mechanisms through which disparities in SES, with a specific focus on employment conditions and income, influence people’s ability to engage in COVID-19 protective behaviors. To do so, we analyzed data collected from a sample of currently working adults in the US and used the Health Belief Model (HBM), a widely used model informing behavior change interventions, as a framework to guide our analytic model . The HBMHypothesis 1: Knowledge of recommended COVID-19 protective behaviors and COVID-19 threat perception are positively associated with engagement in such protective behaviors, such as frequent handwashing.Hypothesis 2: Adoption of protective behaviors related to social distancing, such as staying at home, is determined by employment conditions. Specifically, from the perspective of the HBM, employment conditions impede self-efficacy to engaging in certain COVID-19 protective behaviors but not others.Our study investigates the following hypotheses:N = 5,062). Respondents received no incentives for participation. The current analytic sample comprised 2,845 respondents who reported being currently employed and identified themselves as working full-time (n = 2067), part-time (n = 402), self-employed (n = 371), or in the military (n = 5). The [redacted] Institutional Review Board approved the study as exempt and waived the need for explicit written or oral consent.The study methodology is described in detail elsewhere . In brieQuestionnaire development was guided by the HBM , a modelKnowledge of COVID-19 protective behaviors was assessed by two binary  items, asking respondents whether they could protect themselves from being infected with COVID-19 by “Washing your hands frequently with soap and water” and “Stopping going to school/work.” Whereas as the pandemic evolved, resuming day-to-day activity with a mask became the norm, this was not the case in the early stages of the pandemic when many locations instituted stay-at-home orders. Thus, these questions were intended to capture handwashing and social distancing, two of the primary COVID-19 prevention measures promoted at the time of survey implementation (April 2020).Perceived COVID-19 risk was assessed by the question “On a scale from 0-10, what do you think is your risk of getting infected with Coronavirus?” Perceived COVID-19 severity was assessed by a single item, “On a scale from 0-10, if you were infected with Coronavirus, how severe do you think it would be?” On this scale, 0 signifies very low perceived risk or severity, and 10 signifies very high perceived risk or severity.The HBM deconstructs disease threat as perceived susceptibility or a person’s subjective perception of the risk of acquiring an illness or disease and perceived severity of the seriousness of contracting an illness or disease . As recoThe socio-demographic information of respondents included: sex, age , marital status , race (coded as non-Hispanic white and non-white because of low numbers among minority groups), educational attainment , type of residence , US Census region  (coded beconomic precarity index was developed based on the sum of the following binary items: 1) annual household income ; 2) variable income ; 3) no paid sick leave (coded ‘yes’ if employers did not offer paid sick leave); 4) no health insurance ; 5) lost income due to COVID-19 (yes/no); and 6) food insecurity in the last 3 months  “Are you considered an essential worker ?” or 2) “If you were required to remain at home because of a quarantine or work closure, would you be able to do at least part of your job from home?” In addition, a separate binary measure was used to assess whether people thought that they could afford to quarantine if mandated to do so. Within the framework of the HBM, we used these two measures as a proxy for self-efficacy to engage in protective behaviors related to physical distancing , but not in behaviors related to hygiene .The primary outcome measures were four binary (Yes/No) measures assessing whether respondents endorsed the following four COVID-19 protective behaviors in response to the question “To protect myself from getting infected with Coronavirus, I ...”: 1) “Avoided leaving home except for food or medical supplies”; 2) “Started using hand-sanitizer and/or washing my hands more often”; 3) “Started cleaning and/or disinfecting things that I might touch ”; and 4) “Avoided seeking medical or dental care for other [non COVID-19] health concerns.” The first three behaviors were recommended by the CDC to protect against COVID-19 infection at the time of survey administration. The fourth behavior was recommended by some policymakers at the beginning of the pandemic for the double purpose of reducing the risk of COVID-19 exposure at medical centers and prioritizing strained healthcare resources for the treatment of COVID-19 cases.n = 252, 8.9%) and annual household income  had the most missing data. Because of missing data in different cells, the GLMs included 2,800 observations. All analyses were performed in 2021 on Stata version 15.1 .Descriptive statistics were calculated for the total sample and by essential worker status. Polychoric correlations were estimated to assess the relationships between the variables that make up the economic precarity index. Hierarchical generalized linear models (GLM)  with robTable p<0.0001 (Table Svs. 33.7%), and more likely to not have health insurance (5.7% vs. 3.4%). Close to one-third of essential workers did not have paid sick leave (30.4%), 41.9% had lost income due to COVID-19, and 15% were food insecure. Overall, 81.0% of essential workers had at least one economic precarity factor  and did not have a fixed income (54.9%), while the majority had paid sick leave (75.4%), had health insurance 95.1%), did not lose income due to COVID-19 (60.8%), and were not food insecure (87.7%). All of these economic precarity indicators were correlated at %, did notor Fig. .Fig. 2DiKnowledge of COVID-19 protective behaviors was high. Overall, 82.8% of respondents identified not going to work or school as a protective behavior, and almost all (99.7%) endorsed that frequent handwashing and handsanitizing were protective against COVID-19 infection. While there were no significant differences concerning these personal hygiene behaviors, a smaller proportion of essential workers (78.2%) endorsed not going to work or school as a protective behavior, compared to those who could work from home (91.0%).In terms of perceived threat of COVID-19, respondents reported an average risk  and severity . While there was no difference in perceived risk between the two groups, essential workers perceived COVID-19 as less severe than non-essential workers.Engagement in COVID-19 protective behaviors was high in the sample. Increase in hand hygiene was almost universally endorsed (92.8%), and a large proportion of respondents avoided leaving home (76.6%) and seeking medical or dental care for non-COVID-19 health concerns (64.2%). A small proportion of respondents engaged in cleaning and disinfecting more (34.3%). As can be seen in Table Tables Each additional point in the economic precarity index was associated with a 10 percent increase in the odds of avoiding seeking medical or dental care for other health concerns  but was not associated with the odds of engaging in other protective behaviors. Even after controlling for knowledge, perceived threat, and socio-demographic factors, essential workers were significantly less likely to engage in COVID-19 protective behaviors than non-essential workers, except for cleaning and disinfecting more; AORs ranged from 0.3 (95% CI: 0.2 - 0.5) for increased hand hygiene to 0.7 (95% CI: 0.6 - 0.8) for avoiding seeking medical or dental care for other health concerns. Not being able to afford to quarantine was negatively associated with avoiding leaving home  and increased hand hygiene , but not with the other two behaviors.In sensitivity analysis, essential workers were more likely to have higher economic precarity scores and report not being able to afford quarantine. When analyzing each of the economic precarity items’ individuality, essential workers were more likely than non-essential workers to endorse each of the individual economic precarity items, except for not having health insurance (See Table SConsistent with the HBM, findings confirm our first hypothesis that COVID-19 knowledge and threat perception would be positively associated with engagement in protective behaviors among US workers. These findings echo past COVID-19 research showing a positive association between accurate knowledge, perceived threat and likelihood of engaging in protective behaviors, such as handwashing, forgoing of large gatherings, and mask-wearing , 32. Howfait accompli and related feelings of futility in engaging in prevention. On the other hand, essential workers may face structural problems to handwashing. Essential work that involves travel to a workplace and regular interaction with coworkers and the public may present barriers to hand hygiene protocols either because of unavailability of running water or soap and/or because jobs are overly demanding, and afford minimal breaks during work hours [We found partial support to our second hypothesis. Whereas we observed that not being able to stay at home negatively impacted respondents’ self-efficacy to follow social distancing directives, we also observed that these factors were negatively associated with hand hygiene. The study design precludes us from determining the causal mechanisms of this association. Whereas we propose that essential workers faced economic barriers to staying at home, we are less clear on the reasons behind lower handwashing despite high knowledge that this behavior was protective. Some have proposed that strong illness-related beliefs may mediate the association of knowledge and adherence to health promoting behaviors. Several studies documented lower adoption of protective behaviors among those with strong beliefs that they would get infected by COVID-19 , 33, 34.rk hours , 36. Fedrk hours . While trk hours . Furtherrk hours . In addirk hours . The facThe prevalence of economic precarity indicators was high even in this sample of employed, relatively high-income earners. More than half of respondents in this category did not have fixed income; more than a quarter did not have paid sick leave; and nearly 40% reported having lost income due to COVID-19. Socio-economic vulnerabilities and employment precarities disproportionately burdened essential workers in our sample; for example, 15% of essential workers were classified as food insecure, compared to 10.5% nationwide . Our finFurther, not being able to stay at home was negatively associated with most COVID-19 protective behaviors. This points to broader structural barriers for protection against COVID-19 infection and supports that social class is a significant health determinant in the US . It is pOur study is not without limitations. First, the data is cross-sectional; therefore, causality cannot be inferred. Importantly, we cannot assess temporality; therefore, we tacitly posit that COVID-19 threat perception precedes behavior, but the data structure precludes us from confirming this. Further, this manuscript provides a snapshot of the situation at the initial stages of an evolving pandemic with rapidly changing dynamics. Second, as a convenience sample of English-speaking social media users, the sample is not generalizable to the US population in important ways. At the same time, our sample of respondents had representation from every US state and had a balanced distribution by age group and type of residence. The recruitment strategy potentially under-sampled people from lower SES. For example, this survey was not accessible to respondents without access to the Internet. In the US, it is estimated that 70% of people have Facebook accounts, and three-quarters use them daily . It is iOur study provides quantitative evidence to expand our understanding of health disparities among the working population with different SES in the US. First, findings from this study reiterate the importance of safety net policies for the working population, such as universal health coverage, paid sick leave, and fixed income, as necessary to engage in health-related protective behaviors. In our study, these emerged as significant factors associated with health disparities. Moreover, they underscore the importance of “equitable distribution” of the impact of health policies, that is the need to consider the underlying difference in disease incidence and/or exposure to risks stemming from SES disparities. US Census data show that the expansion of benefits, such as unemployment insurance benefits and direct cash benefits, may have helped to stave off the worse economic effects of the pandemic on households, despite declines in earnings in 2020 compared to the prior year . FurtherThe complex and intertwined relationships between different factors including SES and COVID-19-related protective behaviors, warrant further qualitative analyses to understand the underlying factors and pathways of how various SES components influence the working population’s engagement with protective behaviors and health. Ideally, a combination of multiple policies that address employment conditions, socio-economic vulnerabilities and working conditions should work hand in hand to reduce disparities shown in our study .Findings from this study can contribute to the evidence base supporting national policies that afford a safety net for workers, such as universal health coverage and mandatory paid sick leave, and encourage the private sector to promote secure, quality employment conditions as critical policies to revert the widened socio-economic gap and health disparities in the US since the 1970s, aggravated by the COVID-19 pandemic.Additional file 1.


Psychological consequences of COVID-19 contact, symptoms, or diagnosis are being increasingly reported. Few studies have examined the psychological effects tied to these events, using an unaffected comparison group. Most did not consider confounding factors like fear and stigma. This study aims to (1) identify individual characteristics associated with COVID-19 contact/symptoms or diagnosis and (2) examine the independent association between COVID-19 contact/symptoms or diagnosis and psychological symptoms.From September 2020 to February 2021, 20,327 adults participated in community-based surveys in Quebec. Using repeated cross-sectional online questionnaire, data were collected on probable generalized anxiety disorder (GAD) and major depression episode (MDE), using the GAD-7 and the PHQ-9 scales, respectively. Self-reported events of (1) contact with a case or symptoms of COVID-19, and (2) diagnosis of COVID-19 were examined, along with several sociodemographic and pandemic-related factors.COVID-19 contact, symptoms, or diagnosis was more frequent in young adults, healthcare or social services workers, adults living with children, and those reporting a greater sense of threat, stigma, financial losses, or daily stress. COVID-19 contact or symptoms and diagnosis were associated with probable MDE relative to the unaffected group . Suicidal thoughts and psychomotor retardation were the symptoms most closely associated with a COVID-19 diagnosis.Results from this study stress the need for better understanding, recognition, and support for people suffering from psychological symptoms following a COVID-19 diagnosis. As we now have a more accurate understanding of the acute symptoms of SARS-CoV-2 infection, a vast array of persistent symptoms are being increasingly reported, including various neuropsychiatric symptoms such as difficulty thinking or concentrating (referred to as “brain fog”) or anxiety and depression. Some question whether these persistent symptoms may be related to long COVID, a syndrome corresponding to the persistence of symptoms for several weeks to months in people recovered from COVID-19 , with symptoms remaining after 3 months . The community-based surveys fall into the first axis of the larger international study, where repeated cross-sectional online questionnaires were completed by adults in eight countries , only the most recent participation was included in the final sample.Recruitment and data collection were undertaken by a professional polling firm. The sample was drawn randomly from Web panels including adults 18 years and older residing in the province of Quebec, Canada. Members of the panels were recruited voluntarily using a variety of strategies  ; however, adaptive questioning could have shortened the questionnaire. Each question appeared once the previous question was completed. A nonresponse option was provided for questions concerning sensitive topics. Details about the length, purpose, and consent to the study were provided before the start of the survey.First, probable generalized anxiety disorder (GAD) and major depression episode (MDE) were measured using the GAD-7 and the Patient Health Questionnaire-9 (PHQ-9) scales, respectively Swinson, . These qThe main exposure variable was based on self-reported events of COVID-19 contact, symptoms, or diagnosis. Participants were asked which type of COVID-19 experience they had faced since the start of the pandemic. These experiences were classified as (1) no direct COVID-19 experience, (2) having experienced self-isolation or quarantine due to a contact with a case or symptoms of COVID-19 (without diagnosis), or (3) having been diagnosed by a doctor with COVID-19 or having experienced self-isolation or quarantine due to a diagnosis of COVID-19. The first group was considered as a comparison (unaffected) group.Several factors previously identified as positively or negatively influencing the psychological response to the pandemic were examined following the results of the larger international study into which this study falls . Multivariate logistic regression was also performed. Sensitivity analyses using cases that were isolated due to a diagnosis of COVID-19 during wave 1 (March–July 2020) only allowed exploring whether the psychological effects persisted over time .The data from completed questionnaires were analyzed using the SPSS statistics version 26. ChiThe final sample totalled 20,327 adults . Among the adults surveyed, 599 adults (3%) had reported being diagnosed with COVID-19 and 3008 (15%) had reported requiring quarantine or self-isolation due to a contact with a case or symptoms of COVID-19 (without diagnosis), for a total of 18% of the sample having had a self-reported event of COVID-19 contact, symptoms, or diagnosis. Of the 599 self-reported cases of COVID-19, 244 were acquired during the first wave.p≤0.05) was also observed between reporting either a COVID-19 contact, symptoms, or diagnosis and reporting a greater sense of threat, stigma, financial losses, or daily stress.Table A higher prevalence of recent psychological symptoms was observed in participants who had reported COVID-19 contact, symptoms, or diagnosis compared to those reporting no COVID-19–related events Table . COVID-1Findings from bivariate and multivariate analyses shown in Table In contrast, individuals who had been in contact with a case or who had symptoms of COVID-19 (without diagnosis) presented with levels of probable GAD that were comparable to those of individuals who had reported a diagnosis of the disease  and those having received a diagnosis displayed similar anxiety levels. In contrast, the report of recent major depression symptoms was far more frequent among people having reported a COVID-19 diagnosis in the past year, with as many as 4 out of 10 reporting moderate or severe depressive symptoms. Perhaps even more worrisome is the fact that suicidal thoughts were among the symptoms most closely associated with the experience of a previous infection, with prevalence four times higher in COVID-19 patients relative to the general population.Our findings are consistent with those established in a previous study. Personal experiences related to the COVID-19 pandemic have been associated with clinically significant higher levels of stress, anxiety, depression, and functional impairment. An online survey among 565 American adult respondents evaluated participants’ experiences during the coronavirus pandemic. Participants who reported having received a diagnosis of COVID-19 had the highest odds of a probable anxiety/depression diagnosis  were found to be just as frequent in people infected during the first wave of the pandemic as in those infected more recently (both 38%), suggesting that the psychological effects of a COVID-19 diagnosis may last several months following the event. This prevalence may also be higher than originally expected as it has been estimated that 10–20% of people infected with SARS-CoV-2 exhibit symptoms associated with long COVID, which can include psychological symptoms, within weeks or months of recovery  and the general population. In addition, this study assessed these impacts while taking many potential confounding factors into consideration such as isolation, financial insecurities, and sociodemographic variables.What are the key implications for public health interventions, practice or policy?The results obtained in this study may encourage the need for recognition and support for people suffering from persistent psychological effects following an experience of COVID-19 diagnosis.The results of the study may also urge further research to be conducted in order to increase the comprehension of these effects, especially through the use of longitudinal studies, which can allow a more thorough understanding of the evolution of the persistent psychological symptoms noted in this study.


Eating behavior regulation emerges during early development and involves general self-regulation , appetite regulation (homeostatic metabolic need) and appetite self-regulation (including both Bottom-Up Food Approach and Bottom-Up Food Avoidance and top-down purposeful self-control of eating). Limited research has investigated developmental trajectories of the regulation of eating behavior before the preschool years. The current study used a novel food delay task to assess infant distress as an early emerging marker of eating behavior regulation constructs across early infancy and examine associations with amount of milk consumed.n = 179) completed the Ability to Delay Gratification for Food in Infants Task (ATDG-FIT) at 2 weeks, 8 weeks, and 16 weeks of age. The ATDG-FIT required infants to wait before being fed while their bottle was present, but not accessible (3-min Pre-Feeding Delay). After this, the infant was fed for 1 min, then the feeding was paused for 30 s (Mid-Feeding Delay). Infant distress was coded during each feeding delay period and the amount of milk consumed was measured.Mother-infant dyads  = 15.02, p < 0.001), whereas the mean proportion of distress during the Mid-Feeding Delay increased from 2 to 8 weeks of age  = 27.04, p < 0.001). There was a positive interaction between distress during Mid-Feeding Delay and infant age predicting the amount consumed in the protocol , suggesting that the association between distress during this part of the task and amount consumed strengthens as infants get older.The mean proportion of distress during the Pre-Feeding Delay period decreased from 8 to 16 weeks of age (The ATDG-FIT may be an effective method to assess emerging eating behavior regulation constructs during early infancy. Obesity established during early childhood is often sustained , and difThe regulation of eating behavior involves a nuanced interplay between homeostatic, hedonic, and cognitive control factors, and is not well-characterized prior to age 12 months , 12. YetIn their recent overviews of the literature, Russell and Russell , 11 provThe developmental capabilities of infants younger than 6 months require that responses to ATDG tasks are captured through infant negative affect or frustration, as such tasks involve tolerating the typically unpleasant state of waiting for a desired outcome , 17. RegConsidering responses to an ATDG-for-food task in infants younger than age 6 months also requires consideration of the fact that while infant distress is a very early indicator and precursor of later GSR, it is also an essential indicator of hunger, an aspect of AR  and a fuFinally, individual differences in Bottom-Up Food Approach and Bottom-Up Food Avoidance, or appetitive traits such as food reward and avoidance have been studied in older children and identified as promoting or reducing risk for obesity, respectively –33. RussThe goal of the current study was therefore to examine infant distress in a novel ATDG-for-food task – the ATDG-FIT – as a marker of early eating behavior regulation, specifically GSR, AR, and Bottom-Up ASR. We developed the ATDG-FIT to parallel ATDG-for-food tasks in older children so we could determine how infant responses in the ATDG-FIT would evolve across the first year of life and lay the groundwork to test whether they would relate to later ATDG performance. As noted in n = 106) did not significantly differ from those included in the analyses on any of the study variables of interest . Infants who were bottle-fed for at least one of the three timepoints were retained as the analytic sample for the current study (n = 179).Mother-infant dyads were recruited from a community in the Midwest United States through flyers, postcards, and social media. Mothers provided written informed consent for themselves and their infants. The study was approved by the University of Michigan Medicine Institution Review Board (IRB MED). Inclusion criteria for the study consisted of the following: (1) Child was born at 37.0 – 42.0 weeks gestation with weight appropriate for gestational age, and no significant perinatal or neonatal complications. Participants were excluded from the study if they met any of the following criteria: (1) Mother is not fluent in English; (2) infant is not the biological child of the mother; (3) mother < 18 years old; (4) medical problems or diagnosis affecting current or future eating, growth, or development; (5) child protective services involvement in the neonatal period; and (6) infant does not consume at least two ounces in one feeding from an artificial nipple and bottle at least once per week. Dyads were recruited to begin the study when infants were 2 weeks of age. To facilitate recruitment, families could also enter the study at infant age 8 weeks (2 months) or 16 weeks (4 months). Data were collected for each family at the infant’s first assessment point (termed “baseline”) and as described at each timepoint thereafter. Infants who were breastfed at all three timepoints were dropped from study analyses because there were no available objective measurements of amount consumed. Infants excluded from analyses for this reason , which was video recorded for observational coding. The ATDG-FIT was conducted at the point during the home visit when mothers indicated that they thought the infant was hungry based on the infant fussing or crying, which have been identified as relatively reliable indicators of infant hunger in prior literature . The timThe goal of the ATDG-FIT was to assess infant behaviors when the infant is not allowed to eat immediately , mild (1), or moderate/intense (2) based on facial, vocal, and body movement indicators. Mild distress included instances of whimpering, mild fussing, and facial expressions or body movements indicating distress or frustration . Moderate/intense displays of negative affect included stronger displays of distress such as hard crying, active squirming or arching back while crying, or screaming. In the current study we considered any distress as an indicator so we created a composite code to indicate distress present  or not present . Undergraduate research assistants, who did not administer the protocol, were trained to achieve interrater reliability (κ > 0.70) on coding at each of the three timepoints prior to coding the videos. Approximately 20% of videos were double-coded throughout the coding process to assess interrater reliability at the 2-week (κ = 0.73), 8-week (κ = 0.75), and 16-week (κ = 0.78) timepoints. For data analysis purposes, we calculated the proportion of distress in each segment . We analyzed infant distress during the 3-min Pre-Feeding Delay  and 30-s Mid-Feeding Delay  periods, as these two segments required infants to wait without milk or a pacifier being offered. Hereafter, we refer to these variables as Pre-Feeding Delay Distress and Mid-Feeding Delay Distress.Amount of breastmilk or formula consumed by the infant in grams was calculated by subtracting weight of the bottle and contents remaining from the initial weight of the bottle and contents. Bottles were weighted using a Taylor TE32FT digital scale . It was only possible to directly calculate the amount consumed variable in this manner for infants who were bottle-fed. Values for amount consumed were imputed for any timepoints when infants were breastfed, but this was true only for those infants who were bottle-fed for at least one of the three study timepoints.Infant weight-for-length z-score (WLZ) was included as a covariate, given associations with infant consumption amount . Infant Mothers reported whether they were Hispanic or not, as well as their race, choosing from United States Census categories White, Black, American Indian or Alaska Native, Asian, Native Hawaiian or Pacific Islander, Multiracial , and Other; categories were collapsed to indicate race/ethnicity  was calculated by dividing income by the poverty income threshold for a household of that size in the given year; an ITNR of 1.0 indicates that a household is living at the poverty level, with higher values indicating greater income-to-needs  .Descriptive statistics were used to characterize the sample and bivariate statistics were used to assess associations among key variables. To examine developmental change, analyses of variance (ANOVAs) using repeated measures were used to assess differences in Pre-Feeding Delay Distress and Mid-Feeding Delay Distress) across the 3 ages. Repeated measure ANOVAs were also used to assess differences in mean amount consumed across the 3 ages.To examine whether Pre-Feeding Delay Distress or Mid-Feeding Delay Distress related to amount consumed and whether the association changed with age, multilevel modeling (MLM) in Stata 17  was usedMulti-level model was used to examine predictors of the amount consumed across development  for Pre-Feeding Delay Distress or Mid-Feeding Delay Distress. This MLM included an infant age by distress interaction term in order to test whether the association between Pre-Feeding Delay Distress or Mid-Feeding Delay Distress and amount consumed changed as infants grew older. A positive interaction between infant age and either distress variable would indicate that the strength of the association between distress and amount consumed increased as infants grew older. The model to predict amount consumed was estimated including time variant independent variables using the following equation:yit = β0 + β1(Infant age) + β2(Pre-Feeding Delay Distress) + β3(Mid-Feeding Delay Distress) + β4(Pre-Feeding Delay Distress*Infant age) + β5(Mid-Feeding Delay Distress*Infant age) + β6(Feeding mode) + β7(Milk type) + β8(WLZ) + β9(Time elapsed since last feeding) + ui0 + eitMissing values for amount consumed at the timepoints when infants were breastfed were handled alongside all other missing data using multiple imputation with covariates –46. Impun = 99), infant age 8 weeks , and/or infant age 16 weeks  and were included in the analytic sample for the current study (see SD = 2.23) for this sample, where values of 1.0 indicate that a household is living at the poverty level and higher values indicate greater income-to-needs .The total sample recruited consisted of 285 mother-infant dyads. Of these, 179 had data from at least one ATDG-FIT assessment at infant age 2 weeks . Specifically, infant distress during Pre-Feeding Delay was positively associated with amount consumed at 16-weeks of age , and positively but not significantly associated with amount consumed at 2-weeks  or 8-weeks . Infant distress during Mid-Feeding Delay was positively and significantly associated with amount consumed at all timepoints .Bivariate analyses of the data prior to imputation indicated that covariates  were associated with key study variables of Pre-Feeding Delay Distress, Mid-Feeding Delay Distress, and/or amount consumed for at least one timepoint; for example amount consumed was positively associated with being bottlefed and consuming formula, and distress was positively associated with being bottlefed. Bivariate analyses also indicated that Pre-Feeding Delay Distress, Mid-Feeding Delay Distress, and amount consumed were all positively associated  = 22.31, p < 0.001). Infants consumed significantly more at 16 weeks compared to 8 weeks , at 16 weeks compared to 2 weeks , and at 8 weeks compared to 2 weeks . The percentage of infants who were breastfed and fed breastmilk declined across the three ages  = 15.02, p < 0.001; see post-hoc test revealed significantly lower Pre-Feeding Delay Distress at 16 weeks compared to 8 weeks  and at 16 weeks compared to 2 weeks . No significant difference was found between 8 and 2 weeks. There was also a difference in Mid-Feeding Delay Distress across ages  = 27.04, p < 0.001; see post-hoc test revealed significantly higher Mid-Feeding Delay Distress at 16 weeks compared to 2 weeks  and 8 weeks compared to 2 weeks . No significant difference was found between 16 and 8 weeks.ANOVA results revealed a difference in Pre-Feeding Delay Distress across timepoints . The association between Mid-Feeding Delay Distress and amount consumed became stronger as infants grew older. p = 0.007, 95% CI = ), formula feeding during the protocol , and time elapsed since last feeding , but not significantly associated with infant age, Pre-Feeding Delay Distress, age by Pre-Feeding Delay Distress interaction, Mid-Feeding Delay Distress, or feeding mode.Results from MLM analysis see  revealedThe current study sought to investigate infant responses hypothesized to reflect emerging eating behavior regulation in a novel protocol – the ATDG-FIT. Specifically, we tested whether distress during a Pre-Feeding and a Mid-Feeding Delay changed with infant age, and whether distress would predict the amount of milk infants consumed during the protocol across early development. First, we found that infants showed distress in the ATDG-FIT as early as 2 weeks after birth. Second, we found that Pre-Feeding Delay distress decreased from 8 to 16 weeks, whereas Mid-Feeding Delay distress increased from 2 to 8 weeks of age. Third, the association between distress during Mid-Feeding Delay – but not Pre-Feeding Delay – and amount consumed became stronger with age such that it was present at 16 weeks, but not at 2 or 8 weeks. This association was present independent of infant WLZ, feeding mode, or milk type, and time elapsed since last feeding.Despite significant research on ATDG during early childhood, little work has examined responses to food delay tasks during infancy . Prior rand have caloric need , suggesting infants may be more able to tolerate hunger while waiting for milk as they get older. Between ages 2 weeks and 3 months, infant adiposity nearly triples . Blood ric need , 58, 59;In contrast to Pre-Feeding Delay, and somewhat counter to our hypothesis, distress during Mid-Feeding Delay significantly increased between 2 and 8 weeks of age. Potential explanations for this observation could relate to GSR, AR, and Bottom-Up Food Approach. First, this finding may in part reflect the development of object permanence throughout the first several months of infancy, a fundamental cognitive achievement that underlies later GSR capacities such as delay of gratification skills . In the Additional explanations of the increase in Mid-Feeding Delay distress with age may involve AR. For example, as noted above, CCK is released in response to milk feeding, suppresses feeding, reduces seeking of feeding-related stimuli, pacifies crying, and causes sedation –28. Yet,Finally, it is also possible that distress during Mid-Feeding Delay may reflect Bottom-Up Food Approach factors such as food responsiveness . Milk feRegarding associations between ATDG-FIT distress displays and amount consumed, we found that distress during Mid-Feeding Delay associated with amount consumed only as infants grew older. Potential explanations for this observation may also reflect GSR, AR, and Bottom-Up Food Approach. Distress during Mid-Feeding Delay could indicate infants who are less able to tolerate delayed gratification  and who may become more likely to consume excess calories in response to frustration. Becoming reliant on food to soothe could establish links between behavioral distress and amount consumed and disrupt infant recognition of their own AR needs. Over time, this could translate to emotionally driven eating behaviors, such as eating in response to stress rather than hunger cues, and disrupted eating later in development . Infant AR-related explanations for this association may also operate through a few different pathways. In animal models, although feeding releases CCK from early infancy, CCK causes calming and reduces the incentive salience for food in the earliest days of infancy, but does not affect volume intake; volume intake is only affected by CCK later in early infancy , 28. TheThe association between Mid-Feeding Delay distress and amount consumed may also reflect changes in the reward value of food with development, an aspect of Bottom-Up Food Approach. Animal models have demonstrated that the components for reward-driven eating can change over time , reflectThe current study used a novel protocol and a longitudinal design to examine distress in response to a food delay and at an earlier age than has previously been considered. Such contextually specific, observational work is essential for understanding emerging eating behavior regulation and how GSR, AR, and Bottom-Up Approach may interact and underlie early individual differences in this domain. Prior research in this area has heavily relied on parent-reports, for example of general infant temperament e.g., , 23]. In. In23)].Despite the aforementioned strengths, there were several study limitations. It is possible that distress observed during the ATDG-FIT relates to dyadic processes that also shape infant response to food delays. Mothers were allowed to use any non-feeding/non-pacifier method to soothe the infant, hence the protocol likely captured variation in the mother’s ability to soothe their child without feeding or pacifier use. Given the resources required to conduct observational coding, we were unable to assess potentially confounding variables such as maternal attempts to soothe infant distress. Mothers were also instructed to complete the ATDG-FIT when they perceived their infants to be hungry, which could lead to bias, but we elected to do so to preserve ecological validity and based on findings that mothers are reasonably able to identify hunger . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.SS: contributed to drafting the initial manuscript, conducted the statistical analyses, and approved the final manuscript. HR: contributed to drafting the initial manuscript and approved the final manuscript. NK, JS, and AG-K: reviewed the statistical analyses and approved the final manuscript as submitted. KR and JL: conceptualized and designed the study, provided critical review of the manuscript, and approved the final manuscript. AG: provided critical review of the manuscript and approved the final manuscript. AM: conceptualized and designed the study, contributed to drafting the initial manuscript, provided critical review of the manuscript, and approved the final manuscript. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Myasthenia gravis (MG) is a rare autoimmune disease that is potentially threatening for patient life. Auto-antibodies targeting structures of the neuromuscular junction, particularly the acetylcholine receptor (AchR), are often found in the serum of MG patients. New-onset MG after SARS-CoV-2 vaccination has rarely been reported since the introduction of vaccination. Infections and COVID-19 infection have also been reported as possible triggers for a myasthenic crisis. We report a case of new-onset MG after receiving the mRNA COVID-19 vaccination. The patient was a 73-year-old male initially presenting with ocular symptoms and a rapid generalization. We also performed a literature revision of 26 described cases of MG after SARS-CoV-2 immunization. The patients were a majority of males with generalized late-onset MG occurring after the first dose of vaccine, similar to our patient. Only our patient showed a thymoma. Thymic mass and the positivity of AchR antibodies suggest that vaccination might have triggered a subclinical pre-existing MG with symptoms flaring. Clinicians should be aware of possible new-onset MG after COVID-19 vaccination, particularly in at-risk patients. Even though COVID-19 vaccination should be recommended in MG patients, particularly in well-compensated patients. However, more studies need to be performed in the future. Since March 2020, after the World Health Organization (WHO) declared a global pandemic caused by the novel coronavirus disease 2019 (COVID-19), an enormous number of people have been infected, resulting in morbidity and mortality , in 2022MG is a rare autoimmune disorder of the neuromuscular junction caused, in most cases, by antibodies to the acetylcholine receptor (AchR) . More raWe performed a literature review through a search of the PubMed and Google Scholar library databases through several MeSH terms: [vaccination] and [myasthenia gravis], [vaccination] and [MG] [vaccines] and [myasthenia gravis]; [vaccines] and [MG] and [COVID-19] or [Sars-Cov-2]. Studies or reports were included if published before 31 October 2022, with an English language restriction. We considered eligible cases describing patients with a new-onset MG following SARS-CoV-2 vaccination with no restriction of time from immunization. We excluded patients with pre-existing MG or with new-onset MG after SARS-CoV-2 infection .All statistical analyses were performed using GraphPad Prism 9 for Windows . We presented the continuous data as the mean and standard deviation (SD), categorical data as the median and range, and proportions as numbers with the corresponding percentage. The data distribution was preliminarily assessed, and resampling methods were not conducted. Unpaired t-test with Welch’s test, Mann–Whitney U test, and Kruskal–Wallis test were used to compare the continuous variables; the chi-squared test and Fisher test were used for the categorical variables. No particular post-hoc analysis was added. A total of 196 possible results were found. Two independent authors (E.V. and G.T.) screened the titles and abstracts. After removing redundant titles, articles not pertinent, and articles not in English, a final selection of 18 papers was included. Therefore, in addition to our case, we collected 28 patients with post-vaccinal MG until October 2022. Two of the 18 papers (corresponding to two patients) were later excluded for lack of the full-text and abstract. The results of our literature review are reported in A 73-year-old man with a history of hypertension under stable treatment (low dose ramipril 2.5 mg/day) and type II diabetes (treated only with diet) reported several intermittent episodes of ptosis in the left eye associated with diplopia. Four weeks before, he was administrated with the first dose of the Oxford-AstraZeneca ChAdOx1 vaccine. He denied any symptoms of muscular fatigue prior to the injection. His symptoms occurred mostly in the evenings watching television. In the following months, he executed a cranial MRI without any pathological alteration, with his blood exams showing an elevated glycated hemoglobin of 56 mmol/mol  and a fasting glucose of 155 mg/dL as well as a neurological evaluation. Considering the fluctuation of the symptoms, we proposed an electrophysiological examination, a Hess–Lancaster test, and the AchR antibodies serum research (dosed with ELISA) with the suspicion of a neuromuscular junction disease. He later clinically worsened with bulbar and superior limb weaknesses and was admitted to our neurology department three months after the injection. Repetitive stimulation in the facial and anconeus muscles confirmed the generalized MG clinical diagnosis. Thoracic CT demonstrated a thymic mass without infiltration of surrounding structures, later established by a PET-CT . Serum ADuring their hospitalization, treatment with pyridostigmine orally, initially at 120 mg/daily, then increased to 360 mg, was started with rapid clinical improvement. There was no need for immunoglobulin iv, and oral steroids were introduced at a lower dosage of 12.5 mg, considering the weight of 70 kg of the patient and the history of type II diabetes. At discharge after 16 days, examination of the patient revealed only mild bilateral ptosis of 20% after muscle activation. The patient was then started with oral azathioprine, 75 mg per day. Later, the thymic mass was removed, confirming the thymoma diagnosis  [Patients presented a mean age of 60.8 years old (±SD 18.6 years), but interestingly, 16/26 (61.6%) could be classified as late-onset MG (LOMG) defined as an onset ≥60 years old, whereas 9/26 (34.6%) showed adult-onset (between 18 and 59 years old), and only one patient (3.8%) displayed a pediatric onset at 13 years old a. When cMG is a rare neuromuscular disease. Diagnosis of MG AchR positive  is usually based on clinical features, electrophysiological exams to confirm defects in neuromuscular transmission, and positivity in serological tests. The complete pathophysiology of the disease is yet to be elucidated. Here, we report the first patient with new LOMG associated with thymoma within four weeks from SARS-CoV-2 vaccination. Our patient also suffered from well-controlled hypertension treated with a stable low-dose ace-inhibitor and mild type II diabetes, which have not been reported in the literature to be associated with MG, or a higher rate of COVID-19 vaccination neurological autoimmune side effects. Moreover, we also discussed 26 cases reported in the literature up until October 2022. Our findings indicate that older males may be at risk of a generalized LOMG following COVID-19 vaccination (particularly within the first week after the first dose). Usually, MG is more common in young females, and LOMG normally represents one-third of all MG patients, with a higher rate in males ,14,16. CGlobally, MG is a relatively uncommon disorder with an annual incidence of approximately seven to 23 new cases per million . The preThe pathological mechanism underlying this possible association is still debated: a possible bystander activation has been hypothesized . VaccinaOur article presents several limitations: first of all, the limited sample size of the reported patients indicates that the results may be not representative of the population, and second, all of the heterogeneity concerning the administered vaccines may have influenced our results. The large amount of missing data both regarding the clinical presentation, the diagnosis, and the follow-up of the patients is also an issue and limited the possibility of using standardized causality checklists. Finally, the statistical analysis used without bootstrapping techniques also needs to be considered. Therefore, more data must be collected to draw solid conclusions and define a causal relationship. Registry-based studies may provide the large sample sizes that are needed, however, these type of studies often lack specific information regarding the disease . Prospective studies to study a possible causal relationship between MG and COVID vaccination would need the availability of negative serum samples of the patients before immunization in a large prospective study setting, which is highly difficult to achieve. Moreover, considering that concerns about the neurological side effects of vaccines is an old discussion, we can learn from previous experiences with other vaccines in MG and other immune mediated diseases. Another approach should focus on the implementation of studies to compare the effects on the disease and general population of COVID-19 vaccines and COVID-19 infection itself as well as compare between different types of immunization.Mass vaccination represents the turning point against the pandemic. However, adverse reactions such as the possibility of autoimmune disease triggered by vaccination might lead to reduced vaccine acceptance and hesitancy. Data in the literature are still lacking for rare diseases such as MG, and at the moment, only a small number of cases of Mg following COVID-19 immunization have been reported. Therefore, solid conclusions cannot be formulated. However, given the possible risks of COVID-19 infection on already diagnosed patients, the benefits of vaccinations could possibly outweigh the risks. Nonetheless, clinicians should be aware that the presence of fatigability after SARS-CoV-2 immunization raises the suspicion of a MG diagnosis.