[The novel in vivo platelet activation marker, soluble CLEC-2].

Unlike for anticoagulants, no good monitoring methods exist for antiplatelet agents, and their monitoring is considered unnecessary. In the platelet aggregation test, which is the standard test for platelet function, aggregation is evaluated by adding a platelet activator to platelets in a cuvette. Therefore, it provides information on the maximum potential of platelets to induce aggregation, but not the actual in vivo degree of platelet activation. In vivo platelet activation markers are not widely used because they require a special blood collection method, although some tests are covered by insurance. My colleagues and I identified the platelet activation receptor C-type lectin-like receptor 2 (CLEC-2) and found that CLEC-2 is cleaved and released during platelet activation. We established a plasma-soluble CLEC-2 (sCLEC-2) assay system with the aim of using it as a marker of in vivo platelet activation. Elevation of sCLEC-2 has been reported in various thrombotic diseases. The multi-center prospective cohort study CLECSTRO is now underway to investigate the usefulness of sCLEC-2 for diagnosis, typing, and prognostic estimation in acute ischemic stroke.

Development of an automated chemiluminescence enzyme immunoassay for the measurement of soluble C-type lectin-like receptor 2 (sCLEC-2) and molecular forms of sCLEC-2 measured in patient plasma.

Soluble CLEC-2 is anticipated to have various clinical applications as a novel biomarker for in vivo platelet activation, assessable using plasma obtained through routine sampling procedures. While sCLEC-2 has been measured using ELISA, we have developed a highly sensitive chemiluminescence enzyme immunoassay (CLEIA) reagent that can yield results in approximately 19 minutes. This study aims to assess its fundamental performance and explore the molecular forms of sCLEC-2 measured in patient samples. We examined the sensitivity, precision, linearity, influence of endogenous substances, residual platelets, as well as the correlation with the ELISA method, for the sCLEC-2 CLEIA reagent. The CLEIA method demonstrated sufficient sensitivity for levels observed in healthy donors, and its basic performance was satisfactory. It exhibited a strong correlation with the previously described ELISA method, with reference ranges that did not significantly differ from the ELISA data. The sCLEC-2 reference range for males was significantly higher than that for females. Since it is known that sCLEC-2 exists in shed form and microparticle form, we investigated molecular forms of sCLEC-2 measured by the CLEIA in in vitro-activated platelets and in patients' plasma using gel filtration. It is considered that the CLEIA method shows significantly stronger reactivity with the shed form compared to the microparticle form. Studies using gel filtration of patient samples also suggest that the shed form is being primarily measured. The sCLEC-2 CLEIA reagent exhibits robust performance and is promising for clinical applications.

Macrophage activation markers are associated with infection and mortality in patients with acute liver failure.

BACKGROUND AND AIMS: Acute liver failure is a multisystem disorder with a high mortality and frequent need for emergency liver transplantation. Following massive innate immune system activation, soluble markers of macrophage activation are released during liver damage and their association with disease severity and prognosis requires exploration. METHODS: Patients ALF from the United States Acute Liver Failure Study Group (USALFSG, n = 224) and King's College Hospital (n = 40) together with healthy controls (HC, n = 50) were recruited. Serum from early (Days 1-3) and late (>Day 3) time points were analysed for MAMs by enzyme-linked immunosorbent assay correlated to markers of illness severity and 21-day spontaneous survival. Surface expression phenotyping was performed via Flow Cytometry on CD14+ monocytes. RESULTS: All MAMs serum concentrations were significantly higher in ALF compared to controls (p < .0001). sCD206 concentration was higher in early and late stages of the disease in patients with bacteraemia (p = .002) and infection in general (p = .006). In MELD-adjusted multivariate modelling, sCD206 and sCD163 were independently associated with mortality. CD14+ monocyte expression of CD206 (p < .001) was higher in patients with ALF compared with controls and correlated with SOFA score (p = .018). sCD206 was independently validated as a predictor of infection in an external cohort. CONCLUSIONS: sCD206 is increased in serum of ALF patients with infections and poor outcome and is upregulated on CD14+ monocytes. Later measurements of sCD163 and sCD206 during the evolution of ALF have potential as mechanistic predictors of mortality. sCD206 should be explored as a biomarker of sepsis and mortality in ALF.

Early recognition of sepsis-induced coagulopathy using the C2PAC index: a ratio of soluble type C lectin-like receptor 2 (sCLEC-2) level and platelet count.

C-type lectin-like receptor 2 (CLEC-2) is a platelet-activated receptor expressed on the surface of platelet membranes. Soluble CLEC-2 (sCLEC-2) has been receiving attention as a predictive marker for thrombotic predisposition. The present study examined the relationship between sCLEC-2 level and degree of coagulation disorder in septic patients. Seventy septic patients were divided into the sepsis-induced disseminated intravascular coagulation (DIC) (SID) group (n = 44) and non-SID group (n = 26). The sCLEC-2 levels were compared between the two groups. Because we suspected that the sCLEC-2 level was affected by the platelet count, we calculated the sCLEC-2/platelet count ratio (C2PAC index). We further divided septic patients into four groups using the Japanese Association for Acute Medicine (JAAM) DIC scoring system (DIC scores: 0-1, 2-3, 4-5, and 6-8). The C2PAC index was significantly higher in the SID group (2.6 ± 1.7) compared with the non-SID group (1.2 ± 0.5) (P < .001). The C2PAC indexes in the four JAAM DIC score groups were 0.9 ± 0.3, 1.1 ± 0.3, 1.7 ± 0.7, and 3.6 ± 1.0, respectively, and this index increased significantly as the DIC score increased (P < .001). According to the receiver-operating curve analysis, the area under the curve (AUC) and optimal cutoff value for the diagnosis of SID were 0.8051 and 1.4 (sensitivity, 75.0%; specificity, 76.9%), respectively. When the C2PAC index and D-dimer level, one of the main fibrinolytic markers, were selected as predictive markers for SID diagnosis in stepwise multiple logistic regression analysis, it was possible to diagnose SID with a high probability (AUC, 0.9528; sensitivity, 0.9545; specificity, 0.8846). The C2PAC index is a useful predictor of SID progression and diagnosis in septic patients.

Endothelial dysfunction contributes to severe COVID-19 in combination with dysregulated lymphocyte responses and cytokine networks.

The systemic processes involved in the manifestation of life-threatening COVID-19 and in disease recovery are still incompletely understood, despite investigations focusing on the dysregulation of immune responses after SARS-CoV-2 infection. To define hallmarks of severe COVID-19 in acute disease (n = 58) and in disease recovery in convalescent patients (n = 28) from Hannover Medical School, we used flow cytometry and proteomics data with unsupervised clustering analyses. In our observational study, we combined analyses of immune cells and cytokine/chemokine networks with endothelial activation and injury. ICU patients displayed an altered immune signature with prolonged lymphopenia but the expansion of granulocytes and plasmablasts along with activated and terminally differentiated T and NK cells and high levels of SARS-CoV-2-specific antibodies. The core signature of seven plasma proteins revealed a highly inflammatory microenvironment in addition to endothelial injury in severe COVID-19. Changes within this signature were associated with either disease progression or recovery. In summary, our data suggest that besides a strong inflammatory response, severe COVID-19 is driven by endothelial activation and barrier disruption, whereby recovery depends on the regeneration of the endothelial integrity.

Proteomic profiling identifies CLEC4C expression as a novel biomarker of primary graft dysfunction after heart transplantation.

PURPOSE: Clinical models to identify patients at high risk of primary graft dysfunction (PGD) after heart transplantation (HT) are limited, and the underlying pathophysiology of this common post-transplant complication remains poorly understood. We sought to identify whether pre-transplant levels of circulating proteins reporting on immune activation and inflammation are associated with incident PGD. METHODS: The study population consisted of 219 adult heart transplant recipients identified between 2016 and 2020 at Duke University Medical Center, randomly divided into derivation (n = 131) and validation (n = 88) sets. PGD was defined using modified ISHLT criteria. Proteomic profiling was performed using Olink panels (n = 354 proteins) with serum samples collected immediately prior to transplantation. Association between normalized relative protein expression and PGD was tested using univariate and multivariable (recipient age, creatinine, mechanical circulatory support, and sex; donor age; ischemic time) models. Significant proteins identified in the derivation set (p < 0.05 in univariate models), were then tested in the validation set. Pathway enrichment analysis was used to test candidate biological processes. The predictive performance of proteins was compared to that of the RADIAL score. RESULTS: Nine proteins were associated with PGD in univariate models in the derivation set. Of these, only CLEC4C remained associated with PGD in the validation set after Bonferroni correction (OR [95% CI] = 3.04 [1.74,5.82], p = 2.8 × 10-4). Patterns of association were consistent for CLEC4C in analyses stratified by biventricular/left ventricular and isolated right ventricular PGD. Pathway analysis identified interferon-alpha response and C-type lectin signaling as significantly enriched biologic processes. The RADIAL score was a poor predictor of PGD (AUC = 0.55). CLEC4C alone (AUC = 0.66, p = 0.048) and in combination with the clinical covariates from the multivariable model (AUC = 0.69, p = 0.018) improved discrimination for the primary outcome. CONCLUSIONS: Pre-transplantation circulating levels of CLEC4C, a protein marker of plasmacytoid dendritic cells (pDCs), may identify HT recipients at risk for PGD. Further studies are needed to better understand the potential role pDCs and the innate immune response in PGD.

The Effects of Aerobic-Resistance Training and Broccoli Supplementation on Plasma Dectin-1 and Insulin Resistance in Males with Type 2 Diabetes.

BACKGROUND: This study aimed to evaluate the effects of a combination of aerobic-resistance training (CARET) and broccoli supplementation on dectin-1 levels and insulin resistance in men with type 2 diabetes mellitus (T2D). METHODS: Forty-four males with T2D were randomly allocated to four groups (n = 11 each group): CARET + broccoli supplement (TS), CARET + placebo (TP), control + broccoli supplement (S), and control + placebo (CP). CARET was performed three days per week for 12 weeks. TS and S groups received 10 g of broccoli supplement per day for 12 weeks. All variables were assessed at baseline and 12 weeks. RESULTS: Plasma dectin-1 levels were decreased in TS and TP groups compared with the CP group (p < 0.05). Cardiometabolic risk factors showed significant reductions in TP and TS groups compared to S and CP groups (p < 0.05). CONCLUSION: The combination of CARET and broccoli supplementation produced the largest improvements in insulin resistance and dectin-1 and other complications of T2D.

The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin.

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor+ (LepR+) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

Markers of T Cell Exhaustion and Senescence and Their Relationship to Plasma TGF-ß Levels in Treated HIV+ Immune Non-responders.

Background: Immune non-responders (INR) are HIV+, ART-controlled (>2 yrs) people who fail to reconstitute their CD4 T cell numbers. Systemic inflammation and markers of T cell senescence and exhaustion are observed in INR. This study aims to investigate T cell senescence and exhaustion and their possible association with soluble immune mediators and to understand the immune profile of HIV-infected INR. Selected participants were <50 years old to control for the confounder of older age. Methods: Plasma levels of IL-6, IP10, sCD14, sCD163, and TGF-ß and markers of T cell exhaustion (PD-1, TIGIT) and senescence (CD57, KLRG-1) were measured in ART-treated, HIV+ participants grouped by CD4 T cell counts (n = 63). Immune parameters were also measured in HIV-uninfected, age distribution-matched controls (HC; n = 30). Associations between T cell markers of exhaustion and senescence and plasma levels of immune mediators were examined by Spearman rank order statistics. Results: Proportions of CD4 T cell subsets expressing markers of exhaustion (PD-1, TIGIT) and senescence (CD57, KLRG-1) were elevated in HIV+ participants. When comparing proportions between INR and IR, INR had higher proportions of CD4 memory PD-1+, EM CD57+, TEM TIGIT+ and CD8 EM and TEM TIGIT+ cells. Plasma levels of IL-6, IP10, and sCD14 were elevated during HIV infection. IP10 was higher in INR. Plasma TGF-ß levels and CD4 cycling proportions of T regulatory cells were lower in INR. Proportions of CD4 T cells expressing TIGIT, PD-1, and CD57 positively correlated with plasma levels of IL-6. Plasma levels of TGF-ß negatively correlated with proportions of TIGIT+ and PD-1+ T cell subsets. Conclusions: INR have lower levels of TGF-ß and decreased proportions of cycling CD4 T regulatory cells and may have difficulty controlling inflammation. IP10 is elevated in INR and is linked to higher proportions of T cell exhaustion and senescence seen in INR.

Heat-Inactivation of Human Serum Destroys C1 Inhibitor, Pro-motes Immune Complex Formation, and Improves Human T Cell Function.

Heat-inactivation of sera is used to reduce possible disturbing effects of complement factors in cell-culture experiments, but it is controversially discussed whether this procedure is appropriate or could be neglected. Here, we report a strong impact of heat-inactivation of human sera on the activation and effector functions of human CD4+ T cells. While T cells cultured with native sera were characterized by a higher proliferation rate and higher expression of CD28, heat-inactivated sera shaped T cells towards on-blast formation, higher cytokine secretion (interferon γ, tumor necrosis factor, and interleukin-17), stronger CD69 and PD-1 expression, and increased metabolic activity. Heat-inactivated sera contained reduced amounts of complement factors and regulators like C1 inhibitor, but increased concentrations of circulating immune complexes. Substitution of C1 inhibitor reduced the beneficial effect of heat-inactivation in terms of cytokine release, whereas surface-molecule expression was affected by the addition of complex forming anti-C1q antibody. Our data clearly demonstrate a beneficial effect of heat-inactivation of human sera for T cell experiments but indicate that beside complement regulators and immune complexes other components might be relevant. Beyond that, this study further underpins the strong impact of the complement system on T cell function.

Measurement of multiple cytokines for discrimination and risk stratification in patients with Chagas' disease and idiopathic dilated cardiomyopathy.

Chagas' disease (CD), caused by the hemoflagellate protozoan, Trypanosoma cruzi, is endemic in most countries of Latin America. Heart failure (HF) is often a late manifestation of chronic CD, and is associated with high morbidity and mortality. Inflammatory processes mediated by cytokines play a key role in the pathogenesis and progression of CD. Keeping in view the inflammatory nature of CD, this study investigated the possible role of 21 different inflammatory cytokines as biomarkers for prediction and prognosis of CD. The plasma concentration of these cytokines was measured in a group of patients with CD (n = 94), and then compared with those measured in patients with dilated cardiomyopathy (DCM) from idiopathic causes (n = 48), and with control subjects (n = 25). Monovariately, plasma levels of cytokines such as stem cell growth factor beta (SCGF beta), hepatocyte growth factor (HGF), monokine induced by interferon gamma (CXCL9), and macrophage inhibitory factor (MIF) were significantly increased in CD patients with advanced HF compared to control group. None of the cytokines could demonstrate any prognostic potency in CD patients, and only MIF and stromal derived factor-1 alpha (CXCL12) showed significance in predicting mortality and necessity for heart transplant in DCM patients. However, multivariate analysis prognosticated a large proportion of CD and DCM patients. In CD patients, HGF and Interleukin-12p40 (IL-12p40) together separated 81.9% of 3-year survivors from the deceased, while in DCM patients, CXCL12, stem cell factor (SCF), and CXCL9 together discriminated 77.1% of survivors from the deceased. The significant increase in plasma concentrations of cytokines such as HGF and CXCL9 in CD patients, and the ability of these cytokines to prognosticate a large proportion of CD and DCM patients multivariately, encourages further studies to clarify the diagnostic and prognostic potential of cytokines in such patients.

Adipogenic function of tetranectin mediated by enhancing mitotic clonal expansion via ERK signaling.

Tetranectin (TN), an adipogenic serum protein, enhances adipocyte differentiation, however, its functional mechanism has yet to be elucidated. In the present study, we investigated the adipogenic function of TN by using medium containing TN-depleted fetal bovine serum (TN-del-FBS) and recombinant mouse TN (mTN). The adipocyte differentiation of 3T3-L1 cells was significantly enhanced by mTN supplementation essentially at differentiation induction, which indicated a potential role of the protein in the early differentiation phase. The adipogenic effect of mTN was more significant with insulin in the differentiation induction cocktail, implicating their close functional relationship. mTN enhanced not only the proliferation of growing cells, but also mitotic clonal expansion (MCE) that is a prerequisite for adipocyte differentiation in the early phase. Consistently, mTN increased the phosphorylation of ERK in the early phase of adipocyte differentiation. Results of this study demonstrate that the adipogenic function of mTN is mediated by enhancing MCE via ERK signaling. [BMB Reports 2021; 54(7): 374-379].

C-Type Lectin-Like Molecule-1 as a Biomarker for Diagnosis and Prognosis in Acute Myeloid Leukemia: A Preliminary Study.

OBJECTIVE: AML is a heterogeneous disease both in genomic and proteomic backgrounds, and variable outcomes may appear in the same cytogenetic risk group. Therefore, it is still necessary to identify new antigens that contribute to diagnostic information and to refine the current risk stratification. METHODS: The expression of C-type lectin-like molecule-1 (CLL-1) in AML blasts was examined in 52 patients with newly diagnosed or relapsed/refractory AML and was compared with two other classic markers CD33 and CD34 in AML, in order to assess the value of CLL-1 as an independent biomarker or in combination with other markers for diagnosis in AML. Subsequently, the value of CLL-1 as a biomarker for prognosis was assessed in this malignant tumor. RESULTS: The results showed that CLL-1 was expressed on the cell surface of the majority of AML blasts (78.8%) and also expressed on leukemic stem cells in varying degree but absent on normal hematopoietic stem cells. Notably, CLL-1 was able to complement the classic markers CD33 or CD34. After dividing the cases into CLL-1high and CLL-1low groups according to cutoff 59.0%, we discovered that event-free survival and overall survival (OS) of the CLL-1low group were significantly lower than that of the CLL-1high group, and low CLL-1 expression seems to be independently associated with shorter OS. CONCLUSIONS: These preliminary observations identified CLL-1 as a biomarker for diagnosis and prognosis of AML.

Polymorphisms in the DC-SIGN gene and their association with the severity of hand, foot, and mouth disease caused by enterovirus 71.

Severe hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) infection is associated with high mortality and disability. DC-SIGN, a receptor for EV71, is widely distributed in dendritic cells and may influence the severity of HFMD caused by EV71 infection. This observational study attempts to explore whether single-nucleotide polymorphisms (SNPs) in DC-SIGN are related to the severity of EV71-associated HFMD. Based on linkage disequilibrium and functional predictions, two DC-SIGN SNPs were selected and tested to explore their potential association with the severity of HFMD caused by EV71 infection. Two hundred sixteen Han Chinese children with HFMD caused by EV71 were enrolled to obtain clinical data, including the severity of HFMD, serum DC-SIGN levels, and DC-SIGN SNPs. We found a significant association between the rs7248637 polymorphism (A vs. G: OR = 0.644, 95% CI = 0.515-0.806) and the severity of HFMD caused by EV71 infection, as well as the rs4804800 polymorphism (A vs. G: OR = 1.539, 95% CI =1.229-1.928). These two DC-SIGN SNPs may have an effect on the severity of HFMD caused by EV71 infection.

A Novel Strategy to Identify Haematology Patients at High Risk of Developing Aspergillosis.

Invasive Aspergillosis (IA), typically caused by the fungus Aspergillus fumigatus, is a leading cause of morbidity and mortality in immunocompromised patients. IA remains a significant burden in haematology patients, despite improvements in the diagnosis and treatment of Aspergillus infection. Diagnosing IA is challenging, requiring multiple factors to classify patients into possible, probable and proven IA cohorts. Given the low incidence of IA, using negative results as exclusion criteria is optimal. However, frequent false positives and severe IA mortality rates in haematology patients have led to the empirical use of toxic, drug-interactive and often ineffective anti-fungal therapeutics. Improvements in IA diagnosis are needed to reduce unnecessary anti-fungal therapy. Early IA diagnosis is vital for positive patient outcomes; therefore, a pre-emptive approach is required. In this study, we examined the sequence and expression of four C-type Lectin-like receptors (Dectin-1, Dectin-2, Mincle, Mcl) from 42 haematology patients and investigated each patient's anti-Aspergillus immune response (IL-6, TNF). Correlation analysis revealed novel IA disease risk factors which we used to develop a pre-emptive patient stratification protocol to identify haematopoietic stem cell transplant patients at high and low risk of developing IA. This stratification protocol has the potential to enhance the identification of high-risk patients whilst reducing unnecessary treatment, minimizing the development of anti-fungal resistance, and prioritising primary disease treatment for low-risk patients.

Changes in NK Cell Subsets and Receptor Expressions in HIV-1 Infected Chronic Patients and HIV Controllers.

Natural killer (NK) cells are major effectors of the innate immune response and purported to play an influential role in the spontaneous control of HIV infection. In the present study, we compared the phenotypes of NK cells in the peripheral blood of three groups of subjects with chronic HIV-1 infection, HIV controllers, and healthy donors. The results showed that CD56+/CD16- NK cell subsets decreased in chronic patients and remained unchanged in controllers. Notably, we found that people living with chronic HIV-1 infection had suppressed NKp80, NKp46, and NKG2D expressions on NK cells compared to healthy donors, while HIV controllers remained unchanged. In contrast, NKG2D expression was substantially higher in controllers than in chronic patients (M=97.67, p<0.001). There were no significant differences in inhibitory receptors KIR3DL1 and KIR2DL1 expressions. In addition, plasma cytokine IFN-γ, TNF-α and IL-12showed higher levels in HIV controllers compared to chronic patients. Overall, our study revealed that, as compared to chronic patients, HIV controllers show an increased activating receptors expression and higher number ofCD56+/CD16-NK cell subset, with increased expression levels of plasma cytokines, suggesting that higher immune activation in controllers may have a key role in killing and suppressing HIV.

Comparison between two newborn screening strategies for cystic fibrosis in Argentina: IRT/IRT versus IRT/PAP.

BACKGROUND: The benefits of early cystic fibrosis (CF) detection using newborn screening (NBS) has led to widespread use in NBS programs. Since 2002, a two-stage immunoreactive trypsinogen (IRT/IRT) screening strategy has been used as a CFNBS method in all public maternity units in the City of Buenos Aires, Argentina. However, novel screening strategies may be more efficient. The aim of this study is to prospectively compare two CFNBS strategies: IRT/IRT and IRT/PAP (pancreatitis-associated protein). METHODS: A two-year prospective study was performed. IRT was measured in dried blood samples collected 48-72 h after birth. When an IRT value was abnormal, PAP was determined, and a second visit was scheduled to obtain another sample for IRT before 25 days of life. Newborns with a positive CFNBS were referred for a confirmatory sweat test. RESULTS: There were 69,827 births in the City of Buenos Aires during the period studied; 918 (1.31%) had an abnormal IRT. A total of 207 children (22.5%) failed to return for the second IRT, but only two PAP (0.2%) were not performed. IRT/IRT was more likely to lead to a referral for sweat testing than IRT/PAP (odds ratio 2.3 [95% confidence interval 1.8-2.9], p < .001). Sensitivity and specificity were: 80% and 100% and 86.5% and 82.6% for IRT/IRT and IRT/PAP strategies, respectively. CONCLUSION: The IRT/PAP strategy is more sensitive than IRT/IRT and has similar specificity; it avoids a second visit and unnecessary sweat testing, and it reduces loss to follow-up in our population.

Evaluating the Timeliness and Specificity of CD69, CD64, and CD25 as Biomarkers of Sepsis in Mice.

ABSTRACT: Sepsis occurs when an infection induces a dysregulated immune response, and is most commonly bacterial in origin. This condition requires rapid treatment for successful patient outcomes. However, the current method to confirm infection (blood culture) requires up to 48 h for a positive result and many true cases remain culture-negative. Therefore, new diagnostic tests are urgently needed. Recent clinical studies suggest that CD69, CD64, and CD25 may serve as useful biomarkers of sepsis. In this study, we evaluated the cecal ligation and puncture and cecal slurry mouse models as tools to study these biomarkers in young and aged mice, and elucidate the timeliness and specificity of sepsis diagnosis. Fluorescence-activated cell sorting analysis revealed that all three biomarkers were elevated on blood leukocytes during sepsis. CD69 was specifically upregulated during sepsis, while CD64 and CD25 were also transiently upregulated in response to sham surgery. The optimal biomarker, or combination of biomarkers, depended on the timing of detection, mouse age, and presence of surgery. CD69 demonstrated an excellent capacity to distinguish sepsis, and in some scenarios the diagnostic performance was enhanced by combining CD69 with CD64. We also analyzed biomarker expression levels on specific cell populations (lymphocytes, monocytes, and neutrophils) and determined the cell types that upregulate each biomarker. Elevations in blood biomarkers were also detected via microfluidic analyses; in this case CD64 distinguished septic mice from naive controls. Our results suggest that CD69 and CD64 are valuable biomarkers to rapidly detect sepsis, and that mouse models are useful to study and validate sepsis biomarkers.

Improved prediction of mortality by combinations of inflammatory markers and standard clinical scores in patients with acute-on-chronic liver failure and acute decompensation.

BACKGROUND AND AIM: Acute-on-chronic liver failure (ACLF) is a sinister prognosis, and there is a need for accurate biomarkers and scoring systems to better characterize ACLF patients and predict prognosis. Systemic inflammation and renal failure are hallmarks in ACLF disease development and progression. We hypothesized that the combination of specific inflammatory markers in combination with clinical scores are better predictors of survival than the originally developed CLIF-C acute decompensation (AD) and CLIF-C ACLF scores. METHODS: We reevaluated all previously measured inflammatory markers in 522 patients from the CANONIC study, 342 without and 180 with ACLF. We used the Harrell's C-index to determine the best marker alone or in combination with the original scores and calculated new scores for prediction of mortality in the original CANONIC cohort. RESULTS: The best markers to predict 90-day mortality in patients without ACLF were the plasma macrophage activation markers soluble (s)CD163 and mannose receptor (sMR). Urinary neutrophil gelatinase associated lipocalin (UNGAL) and sCD163 were predictors for 28-day mortality in patients with ACLF. The newly developed CLIF-C AD + sMR score in patients without ACLF improved 90-day mortality prediction compared with the original CLIF-C AD score (C-index 0.82 [0.78-0.86] vs 0.74 [0.70-0.78, P = 0.004]). Further, the new CLIF-C ACLF + sCD163 + UNGAL improved the original CLIF-C ACLF score for 28-day mortality (0.85 [0.79-0.91] vs 0.75 [0.70-0.80], P = 0.039). CONCLUSIONS: The capability of these inflammatory markers to improve the original prognostic scores in cirrhosis patients without and with ACLF points to a key role of macrophage activation and inflammation in the development and progression of AD and ACLF.

Phenotypical and functional alteration of unconventional T cells in severe COVID-19 patients.

COVID-19 includes lung infection ranging from mild pneumonia to life-threatening acute respiratory distress syndrome (ARDS). Dysregulated host immune response in the lung is a key feature in ARDS pathophysiology. However, cellular actors involved in COVID-19-driven ARDS are poorly understood. Here, in blood and airways of severe COVID-19 patients, we serially analyzed unconventional T cells, a heterogeneous class of T lymphocytes (MAIT, γδT, and iNKT cells) with potent antimicrobial and regulatory functions. Circulating unconventional T cells of COVID-19 patients presented with a profound and persistent phenotypic alteration. In the airways, highly activated unconventional T cells were detected, suggesting a potential contribution in the regulation of local inflammation. Finally, expression of the CD69 activation marker on blood iNKT and MAIT cells of COVID-19 patients on admission was predictive of clinical course and disease severity. Thus, COVID-19 patients present with an altered unconventional T cell biology, and further investigations will be required to precisely assess their functions during SARS-CoV-2-driven ARDS.