key: cord-0706817-rb55qcyk authors: Brunetta, E.; Folci, M.; Bottazzi, B.; De Santis, M.; Protti, A.; Mapelli, S.; Leone, R.; My, I.; Bacci, M.; Zanon, V.; Spata, G.; Gianatti, A.; Sironi, M.; Angelini, C.; Garlanda, C.; Ciccarelli, M.; Cecconi, M.; Mantovani, A. title: Macropahge expression and prognostic significance of the long pentraxin PTX3 in COVID-19 date: 2020-06-29 journal: nan DOI: 10.1101/2020.06.26.20139923 sha: 6f20b20caef73473780189d1484fda82061f2299 doc_id: 706817 cord_uid: rb55qcyk PTX3 is an essential component of humoral innate immunity, involved in resistance to selected pathogens and in the regulation of inflammation. PTX3 plasma levels are associated with poor outcome in systemic inflammatory conditions and vascular pathology. The present study was designed to assess expression and significance of PTX3 in COVID-19. By bioinformatics analysis of public databases PTX3 expression was detected in lung respiratory cell lines exposed to SARS-CoV-2. By analysis at single cell level of COVID-19 circulating mononuclear cells, we found that PTX3 was selectively expressed by monocytes among circulating leukocytes. Moreover, in lung bronchoalveolar lavage fluid, single cell analysis revealed selective expression of PTX3 in neutrophils and macrophages, which play a major role in the pathogenesis of the disease. By immunohistochemistry, PTX3 was expressed by lung myelomocytic cells, type 2 pneumocytes and vascular endothelial cells. PTX3 plasma levels were determined by ELISA in 96 consecutive patients with a laboratory-confirmed diagnosis of COVID-19. Higher PTX3 plasma levels were observed in 52 (54.2%) patients admitted in ICU (median 21.0ng/mL, IQT 15.5-46.3 vs 12.4ng/mL IQT 6.1-20.2 in ward patients; p=0.0017) and in 22 (23%) patients died by 28 days (39.8ng/mL, IQT 20.2-75.7 vs 15.7ng/mL, IQT 8.2-21.6 in survivors; p=0.0001). After determining an optimal PTX3 cut-off for the primary outcome, the Kaplan-Meier curve showed an increased mortality in patients with PTX3>22.25ng/mL (Log-rank tests p<0.0001). In Cox regression model, PTX3>22.25ng/mL showed an adjusted Hazard Ratio (aHR) of 7.6 (95%CI2.45-23.76) in predicting mortality. Performing a multivariate logistic regression including all inflammatory markers (PTX3, ferritin, D-Dimer, IL-6, and CRP), PTX3 was the only marker significantly associated with death (aHR 1.13;95%CI1.02-1.24; p=0.021). The results reported here suggest that circulating and lung myelomonocytic cells are a major source of PTX3 and that PTX3 plasma levels can serve as a strong prognostic indicator of short-term mortality in COVID-19. Highly pathogenic betacoronaviruses, causing Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and the currently pandemic COVID-19, affect the lower respiratory tract leading to critical acute respiratory distress syndrome (ARDS) and fatality in a high percentage of cases [1] [2] [3] [4] . SARS-CoV-2 infection is characterized by variable clinical forms with symptoms including fever, cough, and general malaise in mild and moderate cases 5 , which progress to severe pneumonia or ARDS, shock and/or multiple organ failure, requiring hospitalization in Intensive Care Units (ICU), in more severe cases. The high morbidity and mortality observed in COVID-19 pandemics are caused by alveolar damage and pneumonia 6, 7 , cardiovascular complications, and multiorgan failure 8 . SARS-CoV-2 interacts with ACE2 expressed by pneumocytes in the alveolar lining, leading to lung injury. ACE2 is also widely expressed on endothelial cells, thus possibly explaining the evidence of direct viral infection of the endothelium, diffuse endothelial inflammation, and widespread microvascular dysfunction, leading to organ ischaemia, inflammation, edema, and a procoagulant state 4, 9, 10 . In addition, uncontrolled activation of innate and adaptive immunity in response to the infection results in hyperinflammatory responses, as demonstrated by the cytokine storm and activation of macrophages and neutrophils in COVID-19 patients, which, by affecting lung tissue and blood vessels, contribute to ARDS pathogenesis, shock and multiorgan failure 4, 7, 11 . PTX3 is a key component of humoral innate immunity, belonging to the family of pentraxins 12 . In contrast with its relative, the short pentraxin C reactive protein (CRP), essentially produced by The similarity with CRP prompted investigations as to the usefulness of PTX3 as a marker in diverse human conditions of infective or inflammatory origin. The local production by different cell types at inflammatory sites and the release of the preformed protein by neutrophils in response to primary proinflammatory cytokines or microbial recognition accounts for the rapidity of PTX3 increase in these conditions. Increased PTX3 plasma concentrations were described in infections of fungal, bacterial and viral origin [14] [15] [16] [17] , severe inflammatory response syndrome (SIRS), sepsis [18] [19] [20] [21] , and cardiovascular diseases [22] [23] [24] . In different pathological conditions high PTX3 plasma levels were associated with disease severity and mortality (e.g. [18] [19] [20] . Moreover, PTX3 has been shown to serve as a biomarker of disease activity in inflammatory conditions involving the vascular bed, ranging from atherosclerosis to vasculitis (e.g. 22,25-30 ). Previous findings on the prognostic significance of PTX3 in systemic inflammatory conditions as well as in vascular pathology 18, 29 prompted the present investigation which was designed to assess PTX3 expression and plasma levels in COVID-19. Here we report that, based on a bioinformatic analysis on public databases, PTX3 was strongly induced by SARS-COV-2 in respiratory tract epithelial cells. At single cell level, COVID-19 monocytes and lung macrophages expressed PTX3. Finally, PTX3 plasma levels were a strong predictor of 28-day mortality in hospitalized COVID-19 patients. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint Data relative to the transcriptional response to SARS-CoV-2 infection were derived from datasets deposited within the Gene Expression Omnibus (GEO). Data relative to bulk transcription in human normal bronchial (NHBE) and malignant cell lines (Calu-3 and A549) upon SARS-CoV-2 infection, were derived from the experiments within the series GSE147507 31 . Raw bulk RNA-Seq reads were quality inspected with the software "FastQC" (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and aligned with STAR (version 2.6.1) 32 on the GRCh38 genome guided by GENCODE annotation (version 33). Gene summarized counts were processed in R, genes whose expression was major than 2 reads were filtered and vst normalized with the R package DESeq2 33 . Significantly changing genes upon SARS- were explored with the portal cellxgene (https://chanzuckerberg.github.io/cellxgene/) and obtained from "The COVID19 Cell Atlas portal" (https://www.covid19cellatlas.org/#wilk20). Sparse count matrices or Seurat objects were obtained as released and processed with the R package "Seurat" 37 and confirmed with the published pipelines shared by respective authors. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint Classification of clusters was performed according to the authors' parameters and annotations were performed with the Blueprint-Encode signatures. The distribution of PTX3 expression was obtained after imputation with the "Rmagic" package 38 . Immunohistochemical staining was performed as follows: 3-µm-thick sections were prepared from formalin-fixed paraffin-embedded autoptic tissue lung blocks from a COVID-19 patient and were dried in a 60°C for 20'. The sections were placed in a BOND-III Automated Immunohistochemistry Vision Biosystem (Leica Microsystems GmbH, Wetzlar, Germany) according to the following protocol. First, tissues were deparaffinized and pre-treated with the Epitope Retrieval Solution 1 (Citrate-buffer pH 5.9-6.1) at 100°C for 10 min. After washing steps, peroxidase blocking was carried out for 5 min using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH). Tissues were again washed and then incubated with the primary antibody (affinity purified rabbit IgG anti-human PTX3) 39 for 15 min. Subsequently, tissues were incubated with polymer for 8 min and developed with DAB-Chromogen for 10 min. This cohort study analyzed a cohort of 96 patients. We included all males and non-pregnant females, 18 years of age or older, admitted to Humanitas Clinical and Research Center (Rozzano, Milan, Italy) between March 4th and May 16rd, 2020 (data cutoff on May 13rd) with a laboratory-confirmed diagnosis of COVID-19. Hospital admission criteria were based on a positive assay for SARS-CoV-2 associated with respiratory failure requiring oxygen therapy, or radiological evidence of significant pulmonary infiltrates on chest computed tomography (CT) scan, or reduction in respiratory/cardiopulmonary reserve as assessed by 6 minutes walking test, or due to frailty related with patient comorbidity. We assessed an outcome of death. 52 patients of 96 (54%) were transferred to ICU because requiring invasive ventilation or non-invasive mechanical ventilation with oxygen All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint fraction over 60%. Patients with continuous positive airway pressure therapy (CPAP) were followed up by ICU outreach team and ward physicians in COVID-19 wards. Acute respiratory syndrome (ARDS) was defined according to the Berlin definition 40 . Laboratory testing at hospital admission included: complete blood count, renal and liver function (transaminase, total/direct/indirect bilirubin, gamma-glutamyl transferase, alkaline phosphatase), creatinine kinase, lactate dehydrogenase, myocardial enzymes, electrolytes and triglycerides. A panel of acute phase reactants including interleukin-6 (IL-6), serum ferritin, D-dimer, C-reactive protein (CRP), fibrinogen, and procalcitonin (PCT) was performed. Body temperature, blood pressure, heart rate, peripheral saturation, and respiratory rate were measured in all patients. Chest CT scan and arterial blood gas analysis were performed in the emergency department. In all patients PTX3 was measured within the first few days after the admission date (mean 2.1+1.6 days). Pneumococcal and Legionella urinary antigen tests were routinely performed. Nasopharyngeal swab for influenza A, B and H1N1 was also routinely performed to exclude co-infections. Additional microbiological tests were performed to exclude other pathogens as possible etiological agents when suggested by clinical conditions (bacterial cultures of sputum, blood and urine). We obtained a comprehensive present and past medical history from patients. Positivity was assessed on the basis of reverse-transcriptasepolymerase-chain-reaction (RT-PCR) assay for SARS-CoV-2 on a respiratory tract sample tested by our laboratory, in accordance with the protocol established by the WHO ( https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratoryguidance ). Due to the high false negative rate of RT-PCR from pharyngeal swab, two different swabs were performed in every patient to increase the detection rate 41 . In cases of negative assay in throatswab specimens, but with suggestive clinical manifestations, presence of contact history or suggestive radiological evidence for COVID-19, the detection was performed on bronchoalveolar lavage fluid (BAL) or endotracheal aspirate, which has higher diagnostic accuracy. All demographics, medical All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint history and laboratory tests were extracted from electronic medical records and were checked by a team of three expert physicians. The study was approved by the local Ethical Committee (authorization 233/20), and the requirement for informed consent was waived. Venous blood samples were collected during the first 5 days after hospital admission (mean 2.1± standard deviation 1.6 days), centrifuged, and EDTA plasma was stored at -80°C until use. PTX3 plasma levels were measured, as previously described 20 , by a sandwich ELISA (detection limit 0.1 ng/mL, inter-assay variability from 8 to 10%) developed in-house, by personnel blind to patients' characteristics. In each analytical session a sample obtained from a pool of plasma from healthy donors was used as internal control. The mean PTX3 concentration measured in this sample was 1.88+0.6 ng/mL. Demographic, clinical, laboratory, and outcome data were obtained from electronic medical records and patient chart notes using a standardized data collection form. Descriptive statistics included means with standard deviations (SD) and medians with interquartile ranges (IQR) for continuous variables, and frequency analyses (percentages) for categorical variables. Wilcoxon rank-sum tests were applied to continuous variables and two-tailed Fisher's exact tests were used for categorical variables. Linearity of continuous variables was checked by comparing models with the linear term to the model with restricted cubic splines. The optimal cut-off levels of PTX3 for predicting 28-day outcome of death have been investigated using receiver operating characteristic (ROC) curves. The correlation between variables was evaluated by Spearman's rank correlation coefficient (rho). To identify the association between PTX3 levels and the outcome in hospitalized COVID-19 patients, we used time-to-event (survival) methods for censored observations. The composite study endpoint was "death" within 28 days from hospital admission. Time to event was defined as the time from All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint hospital admission until the date of event or censoring. Patients discharged early and alive from the hospital were considered event-free through day 28 42 . May 13rd 2020 was considered as data cutoff. Kaplan-Meier estimates were used to draw the cumulative incidence curves, compared by log-rank tests. Furthermore, multivariable Cox proportional hazards (PH) models of prognostic factors were used. The analyses were based on non-missing data (missing data not imputed). Confounders were selected according to a review of the literature, statistical relevance, and consensus opinion by an expert group of physicians and methodologists. After fitting the model, the PH assumption was examined on the basis of Schoenfeld residuals. The hazard ratios (HR) were presented with their 95% confidence intervals (CI) and the respective p-values. A ratio higher than 1.0 implies a higher probability of event compared to the reference group. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint We conducted an in silico bioinformatic analysis of the expression of PTX3 using public databases. As shown in Figure immunostaining was also observed in multinucleated macrophages ( Figure 2A ). Interestingly, PTX3 staining was particularly intense in the adventitia and endothelium of small vessels occluded by a thrombus ( Figure 2B ). Based on previous studies on the prognostic significance of PTX3 in inflammatory conditions and on transcript expression mainly in myelomonocytic cells, which play a key role in pathogenesis, we assessed PTX3 plasma levels in 96 consecutive COVID-19 patients admitted to the Humanitas Clinical and Research Center. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint Demographic, clinical, and laboratory features of patients were shown in Table 1 . All variables were also stratified by clinical outcome, dead versus alive (Table 1) Table 1 and Figure 3 panel A); moreover, higher PTX3 levels were also found in ICU patients compared to ward patients (mean 32.4+27.7 ng/mL; median 21.0 ng/mL, IQT 15.6-46.3 ng/mL versus mean 24.3+31.8 ng/mL; median 12.4 ng/mL, IQT 6.12-20.2 ng/mL, respectively; Figure 3 panel B). The best AUC value for the prediction of death at 28 days was calculated for PTX3 (0.75, 95% CI 0.64-0.86). The most valid cut-off level to predict 28-day mortality was PTX3 = 22.25 ng/mL (sensitivity 0.73; specificity 0.78; Figure 4 ). The Kaplan-Meier curve showed an overall 28-day event-free survival of 0.94+0.03 (95% CI, 0.83 to 0.97) in low PTX3 group (<22.25 ng/mL) and 0.52+0.09 (95% CI, 0.34 to 0.67) in high PTX3 group (>22.25 ng/mL; Log-ranks test p<0.0001; Figure 5 ). In the univariate COX analysis, PTX3 was a strong predictor of mortality in COVID-19 patients (unadjusted HR for ≥22.25 versus <22.25 ng/mL, 10.25; 95% CI, 3.41 to 30.75; p<0.0001). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Correlations between PTX3 and other inflammatory markers were initially assessed with Spearman test and were reported in Table 3 . PTX3 was significantly correlated to CRP, procalcitonin, IL-6, ferritin, and D-dimer, but also with other COVID-19 poor prognostic factors, such as LDH, troponin-I, lymphocyte count (Table 3) . PTX3, CRP, ferritin, IL-6, and D-dimer were also analyzed as continuous values for predicting mortality (univariate Cox regression; Table 4 ). PTX3 was confirmed to be significantly associated to mortality; moreover, IL-6 and D-dimer were also significantly, but weakly, associated with mortality at the unadjusted analysis. Performing a multivariate logistic regression including all these markers (PTX3, ferritin, D-Dimer, IL-6, and CRP), PTX3 was the only inflammatory marker significantly associated with death (adjusted HR for 1 ng/mL increase, 1.13; 95% CI, 1.02 to 1.24; p=0.021; Table 5 ). To investigate if PTX3 could be also considered a biomarker of COVID-19 severity at the time of hospital admission, identified by basal SOFA score >3, we performed a multivariate logistic regression including PTX3, CRP, IL-6, and D-dimer. CRP was the only factor significantly associated to SOFA score >3 (Table 6) . As of May, 25 th the case fatality rate of COVID-19 in Italy is reported to be 14.3% with an average ICU admission rate of more than 20.4% 45 . Elevated levels of CRP, cytokines, and chemokines 8,44,46 together with low lymphocyte and eosinophil counts characterize patients with severe disease 47 . However, a reliable biomarker of poor outcome in COVID-19 is still lacking. The early and accurate triaging of the patients may contribute to better patient management and All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint stratification in clinical trials. The present study was designed to investigate expression and clinical significance of the fluid phase pattern recognition receptor PTX3 in COVID-19. We found that PTX3 was induced by SARS-CoV-2 in respiratory tract epithelial cells. In COVID-19 patients, PTX3 analyzed at single cell level was selectively expressed by monocytes among circulating cells and by lung macrophages. High PTX3 plasma levels (> 22.25 ng/mL) were a strong independent indicator of short term 28-day mortality with an adjusted Hazard Ratio of 7.8 (95% CI 2.5-24). In this patient cohort, PTX3 fared definitely better than other known prognostic markers including CRP, IL-6, ferritin and D-dimer. PTX3 blood levels above the normal value (<2 ng/mL 48 ) can be found in the subclinical inflammatory status of cardiovascular diseases 49 as well as in infections and sepsis, with increasing median values when moving to more severe conditions. In a large study conducted in 1326 unselected hospitalized subjects (14% with infectious diseases), PTX3 above 95th percentile of healthy nonhospitalized subjects (>6.4 ng/mL) was significantly associated to higher mortality in the short term, independently of hospitalization causes (adjusted HR 5, 95%CI 2.9-8.8) 50 . Elevated PTX3 serum levels are indeed not related to a specific diagnosis rather predict severe cases or poor prognosis in different contexts characterized by a systemic inflammatory response 50 . In a recent, prospective, observational study including 547 ICU patients (42.4% with infections), a PTX3 cut off similar to that identified in our study was reported to predict mortality: PTX3 serum level above the median cohort value of 20.9 ng/mL was independently associated to 28-day mortality when adjusted for age, sex, chronic diseases, and immunosuppression (HR 1.87, 95% CI 1.41-2.48) 51 . In another recent paper conducted on 281 sepsis patients, serum PTX3 >26 ng/mL was associated to mortality 52 . Taken together, these findings and our results suggest that circulating PTX3 levels ten-fold above the normal value reflect a severe systemic inflammatory involvement with ominous outcome. PTX3 has been shown to be produced by diverse cell types including myelomonocytic cells, lung epithelial cells and endothelial cells. In the present study, we found that SARS-CoV-2 induced gene expression of PTX3 in respiratory tract epithelial cells. Peripheral blood mononuclear cells All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint represent the easily accessible cellular source in patients. By bioinformatic analysis at single cell level, we found that PTX3 was selectively expressed by monocytes among circulating leukocytes. Moreover, in lung bronchoalveolar lavage fluid, single cell analysis revealed selective expression of PTX3 in neutrophils and macrophages, which play a major role in the pathogenesis of the disease 3,4,53 . The PTX3 gene was originally cloned in endothelial cells 12 PTX3 is a fluid phase pattern recognition molecule which binds selected viruses and play a role in anti-microbial immunity 12 . Moreover, PTX3 has a regulatory role in inflammation by interfering with selectin-dependent neutrophils recruitment and by regulating the complement cascade 59, 60 . It is tempting to speculate that high levels of PTX3 in COVID-19 reflect failed negative regulation of uncontrolled inflammation. The actual role of PTX3 and, more in general, of humoral innate immunity in resistance against SARS-CoV-2 and in disease pathogenesis deserve further investigation. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint are strongly associated with unfavorable COVID-19 disease progression, defined as 28-day mortality, and may serve as a useful prognostic biomarker to decide intensity of care based on the predicted individual risk of death. PTX3 fared better than other classic biomarkers including CRP and IL-6. Given the relatively small sample size (96 patients) this finding should be interpreted with caution. With this caveat, it is tempting to speculate that PTX3 plasma levels may better reflect local tissue disruptive inflammation including the involvement of myelomonocytic cells and the vascular bed. The significance of PTX3 as a biomarker in COVID-19 patient management and stratification and its role in the virus-host interaction deserve further studies. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint Figure 5 . Kaplan-Meier curves by level of PTX3. High level was defined as > of 22.2ng/mL, low level as < 22.2ng/mL. The 28-day event-free survival was 0.94±0.03 (95% CI, 0.83 to 0.97) in low PTX3 group and 0.52±0.08 (95% CI, 0.34 to 0.67) in high PTX3 group. CI: confidence interval. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.26.20139923 doi: medRxiv preprint A new coronavirus associated with human respiratory disease in China A Novel Coronavirus from Patients with Pneumonia in Covid-19 -Navigating the Uncharted Ten things we learned about COVID-19 Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study Virological assessment of hospitalized patients with COVID-2019 Pathological findings of COVID-19 associated with acute respiratory distress syndrome Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19 PTX3, a Humoral Pattern Recognition Molecule, in Innate Immunity, Tissue Repair, and Cancer The Pentraxins Genetic PTX3 deficiency and aspergillosis in stem-cell transplantation Elevated plasma levels of the long pentraxin, pentraxin 3, in severe dengue virus infections Pentraxin 3 and C-reactive protein in severe meningococcal disease Long pentraxin PTX3 is associated with mortality and disease severity in severe Leptospirosis Pentraxin 3 in patients with severe sepsis or shock: the ALBIOS trial Circulating levels of the long pentraxin PTX3 correlate with severity of infection in critically ill patients Persisting high levels of plasma pentraxin 3 over the first days after severe sepsis and septic shock onset are associated with mortality COVID-19 Mortality and ICU Admission: The Italian Experience Using IL-2R/lymphocytes for predicting the clinical progression of patients with COVID-19 Longitudinal hematologic and immunologic variations associated with the progression of COVID-19 patients in China Determination of physiological plasma pentraxin 3 (PTX3) levels in healthy populations Pentraxin 3 in Cardiovascular Disease Pentraxin-3 level at admission is a strong predictor of short-term mortality in a community-based hospital setting Complement related pattern recognition molecules as markers of short-term mortality in intensive care patients Biomarker combination and SOFA score for the prediction of mortality in sepsis and septic shock: A prospective observational study according to the Sepsis-3 definitions Kawasaki-like disease: emerging complication during the COVID-19 pandemic Clinical Characteristics of 58 Children With a Pediatric Inflammatory Multisystem Syndrome Temporally Associated With SARS-CoV-2 Detection of viral SARS-CoV-2 genomes and histopathological changes in endomyocardial biopsies Venous and arterial thromboembolic complications in COVID-19 patients admitted to an academic hospital in Regulation of leukocyte recruitment by the long pentraxin PTX3 Binding of the Long Pentraxin PTX3 to Factor H µg/mL; normal range 0.2-0.35) 2136 ± 6583 Wilcoxon rank-sum test and two-tailed Fisher's exact test between death and alive patients: * p < 0 Fibrinogen (mg/dL; normal range 160-400) 507 ± 169 Triglycerides (mg/dL; normal range 10-150) 150 ± 78 We are grateful to Prof. Alessandro Rambaldi and Dr. Giovanni Gritti (Azienda Ospedaliera ASST Papa Giovanni XXIII, Bergamo, Italy) for helpful discussion. We also acknowledge the work and contribution of Monica Rimoldi, Paolo Tentorio and Sonia Valentino (Humanitas Clinical and Research Center, Rozzano, Milano, Italy) for their tireless work on the collection of biological samples from COVID-19 patients.