Validation of circulating histone detection by mass spectrometry for early diagnosis, prognosis, and management of critically ill septic patients.

BACKGROUND: As leading contributors to worldwide morbidity and mortality, sepsis and septic shock are considered a major global health concern. Proactive biomarker identification in patients with sepsis suspicion at any time remains a daunting challenge for hospitals. Despite great progress in the understanding of clinical and molecular aspects of sepsis, its definition, diagnosis, and treatment remain challenging, highlighting a need for new biomarkers with potential to improve critically ill patient management. In this study we validate a quantitative mass spectrometry method to measure circulating histone levels in plasma samples for the diagnosis and prognosis of sepsis and septic shock patients. METHODS: We used the mass spectrometry technique of multiple reaction monitoring to quantify circulating histones H2B and H3 in plasma from a monocenter cohort of critically ill patients admitted to an Intensive Care Unit (ICU) and evaluated its performance for the diagnosis and prognosis of sepsis and septic shock (SS). RESULTS: Our results highlight the potential of our test for early diagnosis of sepsis and SS. H2B levels above 121.40 ng/mL (IQR 446.70) were indicative of SS. The value of blood circulating histones to identify a subset of SS patients in a more severe stage with associated organ failure was also tested, revealing circulating levels of histones H2B above 435.61 ng/ml (IQR 2407.10) and H3 above 300.61 ng/ml (IQR 912.77) in septic shock patients with organ failure requiring invasive organ support therapies. Importantly, we found levels of H2B and H3 above 400.44 ng/mL (IQR 1335.54) and 258.25 (IQR 470.44), respectively in those patients who debut with disseminated intravascular coagulation (DIC). Finally, a receiver operating characteristic curve (ROC curve) demonstrated the prognostic value of circulating histone H3 to predict fatal outcomes and found for histone H3 an area under the curve (AUC) of 0.720 (CI 0.546-0.895) p < 0.016 on a positive test cut-off point at 486.84 ng/mL, showing a sensitivity of 66.7% and specificity of 73.9%. CONCLUSIONS: Circulating histones analyzed by MS can be used to diagnose SS and identify patients at high risk of suffering DIC and fatal outcome.

The Intricate Role of Non-Coding RNAs in Sepsis-Associated Disseminated Intravascular Coagulation.

Disseminated Intravascular Coagulation (DIC) is a type of tissue and organ dysregulation in sepsis, due mainly to the effect of the inflammation on the coagulation system. Unfortunately, the underlying molecular mechanisms that lead to this disorder are not fully understood. Moreover, current biomarkers for DIC, including biological and clinical parameters, generally provide a poor diagnosis and prognosis. In recent years, non-coding RNAs have been studied as promising and robust biomarkers for a variety of diseases. Thus, their potential in the diagnosis and prognosis of DIC should be further studied. Specifically, the relationship between the coagulation cascade and non-coding RNAs should be established. In this review, microRNAs, long non-coding RNAs, and circular RNAs are studied in relation to DIC. Specifically, the axis between these non-coding RNAs and the corresponding affected pathway has been identified, including inflammation, alteration of the coagulation cascade, and endothelial damage. The main affected pathway identified is PI3K/AKT/mTOR axis, where several ncRNAs participate in its regulation, including miR-122-5p which is sponged by circ_0005963, ciRS-122, and circPTN, and miR-19a-3p which is modulated by circ_0000096 and circ_0063425. Additionally, both miR-223 and miR-24 were found to affect the PI3K/AKT pathway and were regulated by lncGAS5 and lncKCNQ1OT1, respectively. Thus, this work provides a useful pipeline of inter-connected ncRNAs that future research on their impact on DIC can further explore.

Alterations in leukocyte DNA methylome are associated to immunosuppression in severe clinical phenotypes of septic patients.

Introduction: Sepsis patients experience a complex interplay of host pro- and anti-inflammatory processes which compromise the clinical outcome. Despite considering the latest clinical and scientific research, our comprehension of the immunosuppressive events in septic episodes remains incomplete. Additionally, a lack of data exists regarding the role of epigenetics in modulating immunosuppression, subsequently impacting patient survival. Methods: To advance the current understanding of the mechanisms underlying immunosuppression, in this study we explored the dynamics of DNA methylation using the Infinium Methylation EPIC v1.0 BeadChip Kit in leukocytes from patients suffering from sepsis, septic shock, and critically ill patients as controls, within the first 24 h after admission in the Intensive Care Unit of a tertiary hospital. Results and discussion: Employing two distinct analysis approaches (DMRcate and mCSEA) in comparing septic shock and critically ill patients, we identified 1,256 differentially methylated regions (DMRs) intricately linked to critical immune system pathways. The examination of the top 100 differentially methylated positions (DMPs) between septic shock and critically ill patients facilitated a clear demarcation among the three patient groups. Notably, the top 6,657 DMPs exhibited associations with organ dysfunction and lactate levels. Among the individual genes displaying significant differential methylation, IL10, TREM1, IL1B, and TNFAIP8 emerged with the most pronounced methylation alterations across the diverse patient groups when subjected to DNA bisulfite pyrosequencing analysis. These findings underscore the dynamic nature of DNA methylation profiles, highlighting the most pronounced alterations in patients with septic shock, and revealing their close association with the disease.

Histone Citrullination Mediates a Protective Role in Endothelium and Modulates Inflammation.

NETosis is a key host immune process against a pathogenic infection during innate immune activation, consisting of a neutrophil "explosion" and, consequently, NET formation, containing mainly DNA, histones, and other nuclear proteins. During sepsis, an exacerbated immune host response to an infection occurs, activating the innate immunity and NETosis events, which requires histone H3 citrullination. Our group compared the circulating histone levels with those citrullinated H3 levels in plasma samples of septic patients. In addition, we demonstrated that citrullinated histones were less cytotoxic for endothelial cells than histones without this post-translational modification. Citrullinated histones did not affect cell viability and did not activate oxidative stress. Nevertheless, citrullinated histones induced an inflammatory response, as well as regulatory endothelial mechanisms. Furthermore, septic patients showed elevated levels of circulating citrullinated histone H3, indicating that the histone citrullination is produced during the first stages of sepsis, probably due to the NETosis process.

Extracellular Histones Activate Endothelial NLRP3 Inflammasome and are Associated with a Severe Sepsis Phenotype.

Introduction: Circulating extracellular histones acquire relevance as cytotoxic mediators in sepsis. Extracellular histones act as damage-associated molecular patterns (DAMPs), which induce oxidative stress and NLRP3 inflammasome activation. Inflammasome mediates pyroptosis, a programmed cell death mechanism that produces inflammation. Despite evidence for inflammasome activation in immune cells during sepsis, it was unknown whether extracellular histones can produce endothelial inflammasomes activation. Methods: We used human umbilical vein endothelial cells (HUVEC) to explore the activation of pyroptosis, endothelial function and inflammation by extracellular histones. We evaluated pyroptosis by flow cytometry, caspase-1 activity assay, and gene and protein expression analysis by RT-qPCR and Western blot, respectively. The upstream molecular responses involved in pyroptosis activation by extracellular histones were validated by means of using antioxidant glutathione ethyl ester and NLRP3 inflammasome inhibitors. Finally, using mass spectrometry, we measured circulating histones in blood from critically-ill patients and demonstrated that circulating histone levels correlated with the expression of pyroptosis-related cytokines, the release of endothelial adhesion factors and septic shock severity. Results: We found that extracellular histones mediate the activation of NLRP3 inflammasome and pyroptosis in endothelial cells by contributing to endothelial dysfunction and the dysregulation of the immune response mediated by endothelium. Likewise, we demonstrated how the hyperacetylation of extracellular histones or the use of antioxidants decreased pyroptosis. In addition, we showed that pyroptosis is a feasible process occurring in septic shock patients. Discussion: Circulating histone levels correlated with the expression of pro-inflammatory and pyroptosis-related cytokines, the release of endothelial adhesion factors and septic shock severity. We propose to block histone-mediated pyroptosis as a feasible therapeutic strategy in sepsis.

Use of Circular RNAs in Diagnosis, Prognosis and Therapeutics of Renal Cell Carcinoma.

Renal cell carcinoma is the most common type of kidney cancer, representing 90% of kidney cancer diagnoses, and the deadliest urological cancer. While the incidence and mortality rates by renal cell carcinoma are higher in men compared to women, in both sexes the clinical characteristics are the same, and usually unspecific, thereby hindering and delaying the diagnostic process and increasing the metastatic potential. Regarding treatment, surgical resection remains the main therapeutic strategy. However, even after radical nephrectomy, metastasis may still occur in some patients, with most metastatic renal cell carcinomas being resistant to chemotherapy and radiotherapy. Therefore, the identification of new biomarkers to help clinicians in the early detection, and treatment of renal cell carcinoma is essential. In this review, we describe circRNAs related to renal cell carcinoma processes reported to date and propose the use of some in therapeutic strategies for renal cell carcinoma treatment.

Characterization of Early Peripheral Immune Responses in Patients with Sepsis and Septic Shock.

(1) Background: Sepsis is a life-threatening condition caused by an abnormal host response to infection that produces altered physiological responses causing tissue damage and can result in organ dysfunction and, in some cases, death. Although sepsis is characterized by a malfunction of the immune system leading to an altered immune response and immunosuppression, the high complexity of the pathophysiology of sepsis requires further investigation to characterize the immune response in sepsis and septic shock. (2) Methods: This study analyzes the immune-related responses occurring during the early stages of sepsis by comparing the amounts of cytokines, immune modulators and other endothelial mediators of a control group and three types of severe patients: critically ill non-septic patients, septic and septic shock patients. (3) Results: We showed that in the early stages of sepsis the innate immune system attempts to counteract infection, probably via neutrophils. Conversely, the adaptive immune system is not yet fully activated, either in septic or in septic shock patients. In addition, immunosuppressive responses and pro-coagulation signals are active in patients with septic shock. (4) Conclusions: The highest levels of IL-6 and pyroptosis-related cytokines (IL-18 and IL-1α) were found in septic shock patients, which correlated with D-dimer. Moreover, endothelial function may be affected as shown by the overexpression of adhesion molecules such as s-ICAM1 and E-Selectin during septic shock.

Role of non-coding RNAs as biomarkers of deleterious cardiovascular effects in sepsis.

The mechanisms occurring during sepsis that produce an increased risk of cardiovascular (CV) disease (CVD) are poorly understood. Even less information exists regarding CV dysfunction as a complication of sepsis, particularly for sepsis-induced cardiomyopathy. However, recent research has demonstrated that non-coding RNAs, including microRNAs, long non-coding RNAs, and circular RNAs, play a crucial role in genetic reprogramming, gene regulation, and inflammation during the development of CVD. Here we describe experimental findings showing the importance of non-coding RNAs mediating relevant mechanisms underlying CV dysfunction after sepsis, so contributing to sepsis-induced cardiomyopathy. Importantly, non-coding RNAs are critical novel regulators of CVD risk factors. Thus, they are potential candidates to improve diagnostics and prognosis of sepsis-induced cardiomyopathy and other CVD events occurring after sepsis and set the basis to design novel therapeutic strategies.

Role of microRNAs As Biomarkers in Sepsis-Associated Encephalopathy.

Sepsis-associated encephalopathy (SAE) is a neurological complication of sepsis, characterized by brain dysfunction without any direct central nervous system infection. The diagnosis of SAE is currently a challenge. In fact, problems in making a diagnosis of SAE cause a great variability of incidence that can reach up to 70% of all septic patients. Even more, despite SAE is the most frequent type of encephalopathy occurring in critically ill patients, the molecular mechanisms that guide its progression have not been completely elucidated. On the other hand, miRNAs have proven to be excellent biomarkers for both diagnosis and prognosis, especially in brain pathologies because of their small size they can cross the blood-brain barrier easier than other biomolecules. The identification of new miRNAs as biomarkers may help to improve SAE diagnosis and prognosis and also to design new therapies for this clinical manifestation that produces diffuse cerebral dysfunction. This review is focused on SAE physiopathology and the need to have clear criteria for its diagnosis; thus, this work postulates some miRNA candidates to be used for SAE biomarkers because of their role in both, neurological damage and sepsis.

DNA Methylation Analysis to Unravel Altered Genetic Pathways Underlying Early Onset and Late Onset Neonatal Sepsis. A Pilot Study.

Background: Neonatal sepsis is a systemic condition widely affecting preterm infants and characterized by pro-inflammatory and anti-inflammatory responses. However, its pathophysiology is not yet fully understood. Epigenetics regulates the immune system, and its alteration leads to the impaired immune response underlying sepsis. DNA methylation may contribute to sepsis-induced immunosuppression which, if persistent, will cause long-term adverse effects in neonates. Objective: To analyze the methylome of preterm infants in order to determine whether there are DNA methylation marks that may shed light on the pathophysiology of neonatal sepsis. Design: Prospective observational cohort study performed in the neonatal intensive care unit (NICU) of a tertiary care center. Patients: Eligible infants were premature ≤32 weeks admitted to the NICU with clinical suspicion of sepsis. The methylome analysis was performed in DNA from blood using Infinium Human Methylation EPIC microarrays to uncover methylation marks. Results: Methylation differential analysis revealed an alteration of methylation levels in genomic regions involved in inflammatory pathways which participate in both the innate and the adaptive immune response. Moreover, differences between early and late onset sepsis as compared to normal controls were assessed. Conclusions: DNA methylation marks can serve as a biomarker for neonatal sepsis and even contribute to differentiating between early and late onset sepsis.

Oxidative Stress and Inflammation in COVID-19-Associated Sepsis: The Potential Role of Anti-Oxidant Therapy in Avoiding Disease Progression.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak emerged, countless efforts are being made worldwide to understand the molecular mechanisms underlying the coronavirus disease 2019 (COVID-19) in an attempt to identify the specific clinical characteristics of critically ill COVID-19 patients involved in its pathogenesis and provide therapeutic alternatives to minimize COVID-19 severity. Recently, COVID-19 has been closely related to sepsis, which suggests that most deceases in intensive care units (ICU) may be a direct consequence of SARS-CoV-2 infection-induced sepsis. Understanding oxidative stress and the molecular inflammation mechanisms contributing to COVID-19 progression to severe phenotypes such as sepsis is a current clinical need in the effort to improve therapies in SARS-CoV-2 infected patients. This article aims to review the molecular pathogenesis of SARS-CoV-2 and its relationship with oxidative stress and inflammation, which can contribute to sepsis progression. We also provide an overview of potential antioxidant therapies and active clinical trials that might prevent disease progression or reduce its severity.

Sepsis and Coronavirus Disease 2019: Common Features and Anti-Inflammatory Therapeutic Approaches.

Great efforts are being made worldwide to identify the specific clinical characteristics of infected critically ill patients that mediate the associated pathogenesis, including vascular dysfunction, thrombosis, dysregulated inflammation, and respiratory complications. Recently, coronavirus disease 2019 has been closely related to sepsis, which suggests that most deaths in ICUs in infected patients are produced by viral sepsis. Understanding the physiopathology of the disease that lead to sepsis after severe acute respiratory syndrome coronavirus 2 infection is a current clinical need to improve intensive care-applied therapies applied to critically ill patients. Although the whole representative data characterizing the immune and inflammatory status in coronavirus disease 2019 patients are not completely known, it is clear that hyperinflammation and coagulopathy contribute to disease severity. Here, we present some common features shared by severe coronavirus disease 2019 patients and sepsis and describe proposed anti-inflammatory therapies for coronavirus disease 2019 which have been previously evaluated in sepsis.

Circular RNAs in Sepsis: Biogenesis, Function, and Clinical Significance.

Sepsis is a life-threatening condition that occurs when the body responds to an infection that damages it is own tissues. The major problem in sepsis is rapid, vital status deterioration in patients, which can progress to septic shock with multiple organ failure if not properly treated. As there are no specific treatments, early diagnosis is mandatory to reduce high mortality. Despite more than 170 different biomarkers being postulated, early sepsis diagnosis and prognosis remain a challenge for clinicians. Recent findings propose that circular RNAs (circRNAs) may play a prominent role in regulating the patients' immune system against different pathogens, including bacteria and viruses. Mounting evidence also suggests that the misregulation of circRNAs is an early event in a wide range of diseases, including sepsis. Despite circRNA levels being altered in sepsis, the specific mechanisms controlling the dysregulation of these noncoding RNAs are not completely elucidated, although many factors are known to affect circRNA biogenesis. Therefore, there is a need to explore the molecular pathways that lead to this disorder. This review describes the role of this new class of regulatory RNAs in sepsis and the feasibility of using circRNAs as diagnostic biomarkers for sepsis, opening up new avenues for circRNA-based medicine.

Epigenetic biomarkers for human sepsis and septic shock: insights from immunosuppression.

Sepsis is a life-threatening condition that occurs when the body responds to an infection damaging its own tissues. Sepsis survivors sometimes suffer from immunosuppression increasing the risk of death. To our best knowledge, there is no 'gold standard' for defining immunosuppression except for a composite clinical end point. As the immune system is exposed to epigenetic changes during and after sepsis, research that focuses on identifying new biomarkers to detect septic patients with immunoparalysis could offer new epigenetic-based strategies to predict short- and long-term pathological events related to this life-threatening state. This review describes the most relevant epigenetic mechanisms underlying alterations in the innate and adaptive immune responses described in sepsis and septic shock, and their consequences for immunosuppression states, providing several candidates to become epigenetic biomarkers that could improve sepsis management and help predict immunosuppression in postseptic patients.

Epigenetic Regulation in the Pathogenesis of Sjögren Syndrome and Rheumatoid Arthritis.

Autoimmune rheumatic diseases, such as Sjögren syndrome (SS) and rheumatoid arthritis (RA), are characterized by chronic inflammation and autoimmunity, which cause joint tissue damage and destruction by triggering reduced mobility and debilitation in patients with these diseases. Initiation and maintenance of chronic inflammatory stages account for several mechanisms that involve immune cells as key players and the interaction of the immune cells with other tissues. Indeed, the overlapping of certain clinical and serologic manifestations between SS and RA may indicate that numerous immunologic-related mechanisms are involved in the physiopathology of both these diseases. It is widely accepted that epigenetic pathways play an essential role in the development and function of the immune system. Although many published studies have attempted to elucidate the relation between epigenetic modifications (e.g. DNA methylation, histone post-translational modifications, miRNAs) and autoimmune disorders, the contribution of epigenetic regulation to the pathogenesis of SS and RA is at present poorly understood. This review attempts to shed light from a critical point of view on the identification of the most relevant epigenetic mechanisms related to RA and SS by explaining intricate regulatory processes and phenotypic features of both autoimmune diseases. Moreover, we point out some epigenetic markers which can be used to monitor the inflammation status and the dysregulated immunity in SS and RA. Finally, we discuss the inconvenience of using epigenetic data obtained from bulk immune cell populations instead specific immune cell subpopulations.

Epigenetic IVD Tests for Personalized Precision Medicine in Cancer.

Epigenetic alterations play a key role in the initiation and progression of cancer. Therefore, it is possible to use epigenetic marks as biomarkers for predictive and precision medicine in cancer. Precision medicine is poised to impact clinical practice, patients, and healthcare systems. The objective of this review is to provide an overview of the epigenetic testing landscape in cancer by examining commercially available epigenetic-based in vitro diagnostic tests for colon, breast, cervical, glioblastoma, lung cancers, and for cancers of unknown origin. We compile current commercial epigenetic tests based on epigenetic biomarkers (i.e., DNA methylation, miRNAs, and histones) that can actually be implemented into clinical practice.