Characterization of a proteomic profile associated with organ dysfunction and mortality of sepsis and septic shock

Proteomic profile; Organ dysfunction; Septic shockPerfil proteòmic; Disfunció orgànica; Xoc sèpticPerfil proteómico; Disfunción orgánica; Choque sépticoIntroduction The search for new biomarkers that allow an early diagnosis in sepsis and predict its evolution has become a necessity in medicine. The objective of this study is to identify, through omics techniques, potential protein biomarkers that are expressed in patients with sepsis and their relationship with organ dysfunction and mortality. Methods Prospective, observational and single-center study that included adult patients (≥ 18 years) who were admitted to a tertiary hospital and who met the criteria for sepsis. A mass spectrometry-based approach was used to analyze the plasma proteins in the enrolled subjects. Subsequently, using recursive feature elimination classification and cross-validation with a vector classifier, an association of these proteins with mortality and organ dysfunction was established. The protein-protein interaction network was analyzed with String software. Results 141 patients were enrolled in this study. Mass spectrometry identified 177 proteins. Of all of them, and by recursive feature elimination, nine proteins (GPX3, APOB, ORM1, SERPINF1, LYZ, C8A, CD14, APOC3 and C1QC) were associated with organ dysfunction (SOFA > 6) with an accuracy of 0.82 ± 0.06, precision of 0.85 ± 0.093, sensitivity 0.81 ± 0.10, specificity 0.84 ± 0.10 and AUC 0.82 ± 0.06. Twenty-two proteins (CLU, LUM, APOL1, SAA1, CLEBC3B, C8A, ITIH4, KNG1, AGT, C7, SAA2, APOH, HRG, AFM, APOE, APOC1, C1S, SERPINC1, IGFALS, KLKB1, CFB and BTD) were associated with mortality with an accuracy of 0.86 ± 0.05, a precision of 0.91 ± 0.05, a sensitivity of 0.91 ± 0.05, a specificity of 0.72 ± 0.17, and an area under the curve (AUC) of 0.81 ± 0.08 with a confidence interval of 95%. Conclusion In sepsis there are proteomic patterns associated with organ dysfunction and mortality.To Toni del Pino, Rosa Ras and Pol Herrero from the Proteomics and Metabolomics Area of the Center for Omic Sciences (COS), a Joint between Rovira I Virgili University and Eurecat (Reus, Spain), for their contribution to the proteomics analysis. Samples and data from patients included in this study were provided by the Vall d’Hebron University Hospital Biobank (PT20/00107), integrated in the Spanish National Biobanks Network, and they were processed following standard operating procedures with the appropriate approval of the Ethical and Scientific Committees. The authors kindly appreciate the generous donation of samples and clinical data of the donors of the Sepsis Bank of HUVH Biobank

JAK2-STAT Epigenetically Regulates Tolerized Genes in Monocytes in the First Encounter With Gram-Negative Bacterial Endotoxins in Sepsis

Metilación del ADN; Tolerancia a la endotoxina; MonocitosDNA methylation; Endotoxin tolerance; MonocytesMetilació de l'ADN; Tolerància a l'endotoxina; MonòcitsMicrobial challenges, such as widespread bacterial infection in sepsis, induce endotoxin tolerance, a state of hyporesponsiveness to subsequent infections. The participation of DNA methylation in this process is poorly known. In this study, we perform integrated analysis of DNA methylation and transcriptional changes following in vitro exposure to gram-negative bacterial lipopolysaccharide, together with analysis of ex vivo monocytes from septic patients. We identify TET2-mediated demethylation and transcriptional activation of inflammation-related genes that is specific to toll-like receptor stimulation. Changes also involve phosphorylation of STAT1, STAT3 and STAT5, elements of the JAK2 pathway. JAK2 pathway inhibition impairs the activation of tolerized genes on the first encounter with lipopolysaccharide. We then confirm the implication of the JAK2-STAT pathway in the aberrant DNA methylome of patients with sepsis caused by gram-negative bacteria. Finally, JAK2 inhibition in monocytes partially recapitulates the expression changes produced in the immunosuppressive cellular state acquired by monocytes from gram-negative sepsis, as described by single cell-RNA-sequencing. Our study evidences both the crucial role the JAK2-STAT pathway in epigenetic regulation and initial response of the tolerized genes to gram-negative bacterial endotoxins and provides a pharmacological target to prevent exacerbated responses.EB was funded by the Spanish Ministry of Science, Innovation and Universities (grant numbers SAF2017-88086-R & PID2020-117212RB-I00), and was cofunded by FEDER funds/European Regional Development Fund (ERDF) - a way to build Europe. OM-P holds an i-PFIS PhD fellowship (grant number IFI17/00034) from Acción Estratégica en Salud 2013-2016 ISCIII, cofinanced by the Fondo Social Europeo

Inflammatory cytokines and organ dysfunction associate with the aberrant DNA methylome of monocytes in sepsis

Background: Sepsis, a life-threatening organ dysfunction caused by a dysregulated systemic immune response to infection, associates with reduced responsiveness to subsequent infections. How such tolerance is acquired is not well understood but is known to involve epigenetic and transcriptional dysregulation. Methods: Bead arrays were used to compare global DNA methylation changes in patients with sepsis, noninfectious systemic inflammatory response syndrome, and healthy controls. Bioinformatic analyses were performed to dissect functional reprogramming and signaling pathways related to the acquisition of these specific DNA methylation alterations. Finally, in vitro experiments using human monocytes were performed to test the induction of similar DNA methylation reprogramming. Results: Here, we focused on DNA methylation changes associated with sepsis, given their potential role in stabilizing altered phenotypes. Tolerized monocytes from patients with sepsis display changes in their DNA methylomes with respect to those from healthy controls, affecting critical monocyte-related genes. DNA methylation profiles correlate with IL-10 and IL-6 levels, significantly increased in monocytes in sepsis, as well as with the Sequential Organ Failure Assessment score; the observed changes associate with TFs and pathways downstream to toll-like receptors and inflammatory cytokines. In fact, in vitro stimulation of toll-like receptors in monocytes results in similar gains and losses of methylation together with the acquisition of tolerance. Conclusion: We have identified a DNA methylation signature associated with sepsis that is downstream to the response of monocytes to inflammatory signals associated with the acquisition of a tolerized phenotype and organic dysfunction

Inflammatory cytokines and organ dysfunction associate with the aberrant DNA methylome of monocytes in sepsis

Sepsis, a life-threatening organ dysfunction caused by a dysregulated systemic immune response to infection, associates with reduced responsiveness to subsequent infections. How such tolerance is acquired is not well understood but is known to involve epigenetic and transcriptional dysregulation. Bead arrays were used to compare global DNA methylation changes in patients with sepsis, non-infectious systemic inflammatory response syndrome, and healthy controls. Bioinformatic analyses were performed to dissect functional reprogramming and signaling pathways related to the acquisition of these specific DNA methylation alterations. Finally, in vitro experiments using human monocytes were performed to test the induction of similar DNA methylation reprogramming. Here, we focused on DNA methylation changes associated with sepsis, given their potential role in stabilizing altered phenotypes. Tolerized monocytes from patients with sepsis display changes in their DNA methylomes with respect to those from healthy controls, affecting critical monocyte-related genes. DNA methylation profiles correlate with IL-10 and IL-6 levels, significantly increased in monocytes in sepsis, as well as with the Sequential Organ Failure Assessment score; the observed changes associate with TFs and pathways downstream to toll-like receptors and inflammatory cytokines. In fact, in vitro stimulation of toll-like receptors in monocytes results in similar gains and losses of methylation together with the acquisition of tolerance. We have identified a DNA methylation signature associated with sepsis that is downstream to the response of monocytes to inflammatory signals associated with the acquisition of a tolerized phenotype and organic dysfunction

Label-free plasmonic biosensor for rapid, quantitative, and highly sensitive COVID-19 serology: implementation and clinical validation

Serological tests are essential for the control and management of COVID-19 pandemic, not only for current and historical diagnostics but especially for surveillance, epidemiological, and acquired immunity studies. Clinical COVID-19 serology is routinely performed by enzymatic or chemiluminescence immunoassays (i.e., ELISA or CLIA), which provide good sensitivities at the expense of relatively long turnaround times and specialized laboratory settings. Rapid serological tests, based on lateral flow assays, have also been developed and widely commercialized, but they suffer from limited reliability due to relatively low sensitivity and specificity. We have developed and validated a direct serological biosensor assay employing proprietary technology based on Surface Plasmon Resonance (SPR). The biosensor offers a rapid -less than 15 min- identification and quantification of SARS-CoV-2 antibodies directly in clinical samples, without the need of any signal amplification. The portable plasmonic biosensor device employs a custom-designed multi-antigen sensor biochip, combining the two main viral antigens (RBD peptide and N protein), for simultaneous detection of human antibodies targeting both antigens. The SPR serology assay reaches detection limits in the low ng mL-1 range employing polyclonal antibodies as standard, which are well below the commonly detected antibody levels in COVID-19 patients. The assay has also been implemented employing the first WHO approved anti-SARS-CoV-2 immunoglobulin standard. We have carried out a clinical validation with COVID-19 positive and negative samples (n=120) that demonstrates the excellent diagnostic sensitivity (99%) and specificity (100%). This positions our biosensor device as an accurate, robust, and easy-to-use diagnostics tool for rapid and reliable COVID-19 serology to be employed both at laboratory and decentralized settings for the management of COVID-19 patients and for the evaluation of immunological status during vaccination, treatment or in front of emerging variants.H2020 Research and Innovation Programme of the European Commission Project, No. 101003544 Spanish Research Agency (AEI, grant no. SEV-2017-0706AEI, grant no. SEV-2017-0706) Spanish Ministry of Science and Innovation and the Spanish Research Agency and the European Social Fund (ESF)BES-2017-080527 GENCAT-DGRIS COVID EU H2020 Programme (644956) Plan Nacional de I+D+i 2013-2016 ISCIII- Ministerio de Ciencia e Innovación, Vall d’Hebron University Hospital Biobank PT17/0015/0047 European Virus Archive GLOBAL (EVA-GLOBAL) EU Horizon 2020 (grant agreement No. 871029) Fundació Glòria Soler for COVIDBANK collection Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0003)N

Supporting information Label-Free Plasmonic Biosensor for Rapid, Quantitative, and Highly Sensitive COVID-19 Serology: Implementation and Clinical Validation

15 pages. -- Content: 1. Supplementary text: 1.1.Chemical and biological reagents; 1.2.SPR biosensor device; 1.3.Plasmonic sensor chip preparation; 1.4.Clinical samples collection; 1.5.Stratification of convalescent COVID patients. Samples collection from Clinic Hospital (Barcelona); 1.6. Standard analytical techniques (ELISA, CLIA and LFA); 1.7.Data analysis; 1.8.Diagnostic sensitivity and specificity. -- 2. Figures. -- Tables S1-S3. -- References.Serological tests are essential for the control and management of COVID-19 pandemic (diagnostics and surveillance, and epidemiological and immunity studies). We introduce a direct serological biosensor assay employing proprietary technology based on plasmonics, which offers rapid (<15 min) identification and quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in clinical samples, without signal amplification. The portable plasmonic device employs a custom-designed multiantigen (RBD peptide and N protein) sensor biochip and reaches detection limits in the low ng mL–1 range employing polyclonal antibodies. It has also been implemented employing the WHO-approved anti-SARS-CoV-2 immunoglobulin standard. A clinical validation with COVID-19 positive and negative samples (n = 120) demonstrates its excellent diagnostic sensitivity (99%) and specificity (100%). This positions our biosensor as an accurate and easy-to-use diagnostics tool for rapid and reliable COVID-19 serology to be employed both at laboratory and decentralized settings for the disease management and for the evaluation of immunological status during vaccination or treatment.Peer reviewe

Shap values graphics.

A) blue = proteins expressed in patients with SOFA ≤ 6, red = proteins expressed in patients with SOFA > 6. B) blue = proteins expressed in patients survivors, red = proteins expressed in patients non-survivors.</p

Proteins analyzed and their relationship with mortality /organ dysfunction.

Proteins analyzed and their relationship with mortality /organ dysfunction.</p

Characteristics of the study population.

Characteristics of the study population.</p

Fig 1 -

Relationship between proteins (strings): A) organ dysfunction, B) Mortality (https://string-db.org/).</p