Peripheral Blood Research Articles

Integrated multi-omics predictive analysis of atherosclerosis: a sub-study from the Mineralocorticoid Receptor Antagonism in Diabetic Atherosclerosis (MAGMA) trial

Abstract Background/Introduction Mineralocorticoid receptor (MR) antagonists (MRA) are beneficial in cardiorenal outcomes in randomized controlled trials but the mechanisms are unclear. MAGMA was an NHLBI sponsored randomized, double-blind, placebo controlled, 12-month trial comparing Spironolactone (n = 37) vs. placebo (n = 42) in Type 2 diabetics with CKD stages 3-4 on maximal renin-angiotensin system (RAS) blockade and a prior atherosclerotic event and/or left ventricular (LV) hypertrophy. The primary outcome of percent (%) change in total aortic wall volume (TWV) at 12 months, measured by magnetic resonance imaging (MRI), was significantly reduced by Spironolactone. Purpose/Originality The purpose of this analysis was to understand mechanistic pathways of MRAs using a multi-omics approach that could help tease out molecular mechanisms of benefit. Revealing for the first time which of these integrated pathways are predictive of the primary outcome will help design treatments for targeted interventions. Methods Plasma and peripheral blood mononuclear cells from patients randomized to Spironolactone or placebo at baseline and 3-months were measured for aptamer-based proteomic biomarkers (7,596 proteins) and 10-X platform-based single-cell RNA-sequencing (scRNAseq), respectively. Pre-selected candidate predictors were used as inputs of a predictive model of changes of TWV. We fit a Mixed-Multivariate Random Forest (RF) model, a variation of the RF supervised tree-based machine learning method to take the experimental design into account in the regression formulation. The two sources of "omics predictors" were integrated into a supervised multi-omics model to jointly explain the outcome using an extension of Sparse Generalized Canonical Correlation Analysis (SGCCA). Integrated multiome functional analyses with graphical visualizations were carried out by statistical enrichment analysis using Over Representation Analysis (ORA) and Gene Set Enrichment Analysis (GSEA) against databases of gene ontologies, biological pathways, putative regulatory motifs, proteins, or disease annotations. Results The plasma proteome in response to Spironolactone revealed downregulation of MR targets including fibrosis, immune activation/inflammation, leukocyte activation, proliferation and pathways involved in cytokine stimulation. scRNAseq pathways revealed negative regulation of cytokines production such as IL-2 and redistribution of multiple cell types. Predictors of plaque progression involved cytokine-receptor, complement-coagulation, cell adhesion and axonal guidance targets. Multiome integrated functional analyses results of predictive pathways will be presented. Conclusions The changes in plasma proteomic profile with Spironolactone were consistent with the phenotype of reduced atherosclerosis and downregulation of multiple inflammatory, immune response and profibrotic pathways.

Sex differences in the expression of metabolic and pro-inflammatory markers in the initial post-COVID-19 phase

Abstract Cardiovascular complications are a common manifestation of post-acute COVID-19 infection. Post-COVID-19 syndrome is often associated with the female sex and exacerbated inflammation; however, the underlying molecular mechanisms of this sex difference remain poorly understood. In the present study, we aimed to investigate several metabolic, mitochondrial, autophagy, oxidative stress, and inflammatory markers in peripheral blood mononuclear cells (PBMCs) and plasma isolated from older (>75) male and female patients at the initial phase (1-2 weeks) after COVID-19 pneumonia. Age-matched non-infected women and men were used as a control group. We found a significantly higher plasma TNF-α in post-COVID-19 women, but not in men, compared to the control group. Similarly, a significant elevation of IL-1β and IL-18 mRNA in PBMCs from post-COVID-19 women, but not men, was observed. These effects were associated with marked activation of AMPK (defined as pAMPK/AMPK ratio), suggesting potential metabolic stress in female post-COVID-19 PBMCs. Further analysis revealed increased expression of mitochondrial genes, which may be due to AMPK activation as a compensatory reaction to metabolic stress. Analysis of autophagy or oxidative stress (serum 8-OHdG) markers did not reveal any differences between the groups investigated. We conclude that women in the initial phase of post-COVID-19 have elevated expression of inflammatory markers in plasma and in immune cells, which is associated with metabolic stress. This may contribute to the cardiovascular complications in female post-COVID-19 patients.

Successful eculizumab treatment as an adjunctive therapy to desensitization in ABO-incompatible living donor kidney transplantation and its molecular phenotypes

IntroductionABO-incompatible (ABOi) kidney transplantation (KT) has become an important option to overcome organ shortage. Plasmapheresis/rituximab-based desensitization therapy has successfully reduced anti-ABO antibody levels and suppressed antibody-mediated rejection (AMR) in ABOi KT. However, high titers of anti-ABO antibodies in some patients are refractory to standard desensitization, leading to loss of KT opportunities or AMR.MethodsEculizumab treatment was used an adjunctive therapy to rescue high-titer ABOi KT patients refractory to plasmapheresis/rituximab-based desensitization. Molecular phenotypes of allograft biopsies and cellular phenotypes of peripheral blood mononuclear cells of eculizumab group were compared with those of control groups using the Banff Human Organ Transplant gene panel and flow-cytometric analysis, respectively.ResultsThe initial titers of anti-ABO antibodies in the two patients were 1:512 and >1:1024; the final pre-transplant titers after desensitization were 1:128 and 1:64. Both patients received eculizumab from KT day to two or four weeks post-KT and maintained stable renal function up to one-year post-transplantation without overt infection, despite early episodes of probable AMR or borderline T cell-mediated rejection. Molecular phenotype analysis revealed that gene expression patterns in the ABOi KT with eculizumab group overlapped with those in the ABOi KT with AMR group more than in the ABOi KT without AMR group, except for complement pathway-related gene expression. Anti-ABO antibody titers decreased to low levels 1–3 months post-transplant in the eculizumab group in parallel with decreasing anti-B-specific B cells.ConclusionsShort-term eculizumab therapy is promising for rescuing ABOi KT recipients with high anti-ABO antibody titers refractory to plasmapheresis-based desensitization therapy.

Changes in blood metabolomes as potential biomarkers for severity and prognosis in doxorubicin-induced heart failure: a study in HER2-positive and HER2-negative breast cancer patients

Abstract Background The use of doxorubicin for breast cancer treatment is often limited due to cardiotoxicity. Doxorubicin was shown to cause the alterations of cardiac metabolome levels in doxorubicin-treated rats. Interestingly, these changes were robustly associated with the development of doxorubicin-induced heart failure. Unfortunately, measurements of cardiac metabolome levels are rarely possible in humans. Hence, non-invasive biomarkers as representatives of doxorubicin-induced heart failure are required in clinical research and practice. Human epidermal growth factor receptor 2 (HER2) is a point of interest in breast cancer treatment. HER2 is associated with tumor aggressiveness. However, it was observed that HER2 protected against systemic oxidative stress and dilated cardiomyopathy. Therefore, we hypothesized that the alterations of blood metabolome levels and cardiac functions following doxorubicin treatment were different between HER2-positive and HER2-negative breast cancer patients. Purpose We determined the potential roles of changes in blood metabolomes as severity and prognostic markers of doxorubicin-induced heart failure. In addition, the changes in blood metabolomes and cardiac parameters following doxorubicin treatment in HER2-positive and HER2-negative breast cancer patients were compared. Methods HER2-positive (n=37) and HER2-negative (n=37) breast cancer patients were enrolled. Echocardiography, heart rate variability, and blood collection were performed at baseline and 2 weeks after completion of doxorubicin treatment in all patients, as well as at 3 months after completion of doxorubicin treatment in HER2-negative breast cancer patients. Blood obtained at all 3 time points was processed for measuring cardiac injury markers. Blood obtained at baseline and 2 weeks after completion of doxorubicin treatment was also processed for measuring oxidative stress in peripheral blood mononuclear cells using flow cytometry and 85 metabolome levels in plasma using mass spectrometry-based targeted metabolomics. Results Cardiac injury and systolic dysfunction at 2 weeks after completion of doxorubicin treatment were comparable between HER2-positive and HER2-negative breast cancer patients. However, only HER2-negative breast cancer patients exhibited increased systemic oxidative stress and cardiac autonomic dysfunction at this time point. Moreover, 33 and 29 blood metabolomes were altered at 2 weeks after completion of doxorubicin treatment in HER2-positive and HER2-negative breast cancer patients, respectively (Figure). Interestingly, the changes in most of these metabolomes were correlated with the changes in cardiac parameters, both at 2 weeks and 3 months after completion of doxorubicin treatment (Figure). Conclusions The changes in blood metabolomes following doxorubicin treatment were dependent on HER2 status, which might be served as severity and prognostic markers of doxorubicin-induced heart failure.

Deciphering postoperative hemodynamics in pediatric intensive care: insights from patient-specific computational fluid dynamics models for congenital heart disease

Abstract Purpose Precise hemodynamic control is imperative for optimizing outcomes in children undergoing cardiac surgery, particularly in the pediatric intensive care unit (PICU). Computational fluid dynamic (CFD) models tailored to individual pediatric patients following definitive repairs for congenital heart disease hold the potential to discern and analyze the intricacies of their hemodynamic systems. This study aims to explore the applicability of patient-specific CFD models in determining optimal treatment strategies to enhance patient hemodynamics. Methods Ten postoperative pediatric patients (6 females and 4 males, median age 4.5±3.6 months, median height 62.5±5.3 cm, and median weight 6.2±1.3 kg) diagnosed with ventricular septal defect (VSD, n=5), atrial septal defect (ASD, n=2), and atrioventricular septal defect (AVSD, n=3) were enrolled. Continuous radial arterial waveforms and sporadic arterial blood draws, including lactate levels, were recorded in the PICU postoperatively. Fluid dynamics equations were employed to model major and peripheral blood vessels, utilizing a 0–1-dimensional hemodynamic model validated in previous studies and refined for this investigation. Sensitivity analysis based on correlation coefficients identified significant parameters, adjusted for replicating pressure waveforms. Comparison and re-approximation was made with actual radial artery pressure waveforms from 78 arterial waveforms and blood draws. Updated parameters were then correlated with postoperative time and lactate levels in the ICU. Results Replicated waveforms demonstrated high agreement with original arterial waveforms, with mean, maximal, and minimal blood pressure percentages of 96.7±1.8%, 96.7±2.4%, and 95.8±2.4%, respectively. Notably, systemic vascular resistance and compliance exhibited a substantial relationship with time elapsed after admission to the PICU, with correlation coefficients (R) of 0.26 and 0.43, respectively. Lactate levels demonstrated a decrease with reduced peripheral vascular resistance and an increase in vascular compliance, with R of 0.23 and 0.32, respectively, aligning with expected clinical improvement and stability. Conclusion This study successfully attained precise replication of radial artery blood pressures and intricate pressure waveforms using patient-specific CFD models in pediatric patients following congenital heart disease repairs. The insights gained shed light on the pivotal hemodynamic parameters that impact both stability and instability. This research sets the stage for theoretically informed treatments by amalgamating real-time biological information with patient-specific CFD models.Re-approximation of arterial waveformCorrelation of hemodynamic parameters

Circulating levels of neutrophil extracellular traps reflects local neutrophil activity but not fibrinolytic activity in coronary thrombi from patients with ST-elevation myocardial infarction

Abstract Introduction Neutrophil extracellular traps (NETs) are associated with impaired fibrinolysis in ischemic stroke, while NETs degradation enhances lysis of coronary thrombi in ex vivo experiments. Whether circulating NET markers are associated with local fibrinolytic activity in patients with ST-elevation myocardial infarction (STEMI) has not been reported. Purpose We investigated associations between circulating NET markers and gene expression of fibrinolytic markers in aspirated coronary thrombi from patients with STEMI. Furthermore, we explored whether local upregulation of NET formation by peptidylarginine deiminase 4 (PAD4) was reflected in the circulation. Methods Coronary thrombi from 35 STEMI patients undergoing primary percutaneous coronary intervention (PCI) were aspirated and promptly snap-frozen to -80°C. Peripheral blood samples were collected simultaneously. Median time from start of symptoms to PCI was 152 min. Thrombus gene expression of tissue Plasminogen Activator (tPA), urinary-type Plasminogen Activator (uPA), plasminogen-activation inhibitor type 1 (PAI-1) and PAD4 were quantified with RT-PCR. Gene expression of PAD4 was also measured in circulating leukocytes. Circulating NET markers were measured by a fluorescent nucleic acid stain using fluorometry (dsDNA), an in-house ELISA technique (MPO-DNA) and commercial ELISA (H3Cit). Correlations were tested using Spearman’s rho. Results There were no correlations between any of the circulating NET markers and gene expression of the fibrinolytic markers tPA, uPA and PAI-1 in the thrombus. Of the circulating NET markers, dsDNA correlated with thrombus gene expression of PAD4 (rs=0.491 p=0.006), whereas none of the NET markers correlated with gene expression of PAD4 in circulating leukocytes. Conclusion Circulating NET markers were not associated with gene expression of fibrinolytic activity in thrombi from patients with STEMI. Local NET formation in the thrombi, measured as PAD4 expression, was associated with the circulating NET marker dsDNA. These findings suggest that circulating NET markers have limited utility for assessing local fibrinolytic activity, but might reflect PAD4-dependent NET formation in coronary thrombi.

Selenopolysaccharide Isolated from Lentinula edodes Mycelium Affects Human T-Cell Function

Lentinula edodes polysaccharides are natural immunomodulators. SeLe30, analyzed in this study, is a new mixture of selenium-enriched linear 1,4-α-glucans and 1,3-β- and 1,6-β-glucans isolated from L. edodes mycelium. In the present study, we evaluated its immunomodulatory properties in human T cells. Peripheral blood mononuclear cells (PBMCs) and T cells were isolated from healthy donors’ buffy coats. The effects of SeLe30 on CD25, CD366, and CD279 expression, the subsets of CD8+ T cells, and IFN-γ, IL-6, and TNF-α production were analyzed. SeLe30 downregulated CD25, CD279, and CD366 expression on T cells stimulated by the anti-CD3 antibody (Ab) and upregulated in unstimulated and anti-CD3/CD28-Abs-stimulated T cells. It increased the percentage of central memory CD8+ T cells in unstimulated PBMCs and naïve and central memory T cells in anti-CD3-Ab-stimulated PBMCs. SeLe30 decreased the number of central memory and naïve CD8+ T cells in anti-CD3/CD28-stimulated T cells, whereas, in PBMCs, it reduced the percentage of effector memory CD8+ T cells. Moreover, SeLe30 upregulated cytokine production. SeLe30 exhibits context-dependent effects on T cells. It acts on unstimulated T cells, affecting their activation while increasing the expression of immune checkpoints, which sensitizes them to inhibitory signals that can silence this activation. In the case of a lack of costimulation, SeLe30 exhibits an inhibitory effect, reducing T-cell activation. In cells stimulated by dual signals, its effect is further enhanced, again increasing the “safety brake” of CD366 and CD279. However, the final SeLe30 effect is mediated by its indirect impacts by altering interactions with other immune cells.

SNORD3A: a new possible circulating biomarker of human heart failure

Abstract Background Heart failure (HF) is a complex clinical syndrome and the leading cause of mortality, morbidity and hospitalization in industrialized countries. Human cardiac biopsies are invaluable resources for identifying cardiac signaling pathways, however blood fractions and peripheral blood mononuclear cells (PBMCs) could be used as a source of surrogate biomarkers of signaling pathway activation in human heart failure. Purpose In the present study we aimed to identify novel circulating biomarkers mirroring human myocardial signaling in HF and to study the role played by SNORD3A in cardiomyocyte survival and death. Methods Cardiac left ventricle samples and PBLs from 8 HF patients undergoing heart transplantation and 2 control patients (CTRL) were analyzed by RNA sequencing (RNASeq) to identify transcripts that were regulated in both hearts and PBLs. Five identified transcripts, KCNQOT1, MIAT, SCRN1, MALAT1 and SNORD3A, were then validated by quantitative real-time PCR in PBMCs and in LVs. Next, we analyzed the expression of the Small Nucleolar RNA (snoRNA) SNORD3A in LVs and PBMCs from 8-week-old wild type C57BL/6 mice with pressure overload-induced HF by transverse aortic constriction (TAC). Sham-operated mice (sham) were used as controls. To further investigate the role of SNORD3A in cardiomyocyte function and survival, SNORD3A antisense oligonucleotides (SNOaso) and scramble control sequences (GFPaso) were synthesized and tested in cardiomyoblasts H9C2 cells under normoxic or hypoxic conditions to evaluate SNORD3A transcription levels, cell death and protein synthesis. Results SNORD3A expression was significantly increased in both LVs and PBMCs from HF patients compared to control subjects. Consistent with these results, SNORD3A levels were increased both in PBMCs and LVs from TAC mice compared to sham. In vitro hypoxia significantly increased SNORD3A expression levels in H9C2 cells, compared to normoxia. After SNOaso transfection, SNORD3A transcripts were downregulated, and they were further reduced following 3-hour-hypoxia. Depletion of SNORD3A levels increased cardiomyocyte death and reduced protein synthesis in H9C2 cells. Conclusions Our data suggest that SNORD3A might be involved in the regulation of cardiomyocyte survival in HF and could be useful for diagnostic and prognostic applications in HF.

Treatment of Post-COVID-19 POTS by inhibiting FcRn: a phase 2 randomized, placebo controlled, double-blind, proof of concept study with efgartigimod

Abstract Background While the underlying pathophysiology is unknown, post–COVID-19 postural orthostatic tachycardia syndrome (POTS) may be related to immune dysfunction and autoimmunity precipitated by SARS-CoV-2 infection. Various autoantibodies, including those targeting G-protein coupled receptors (GPCRs), have been observed in patients with POTS. Binding of these autoantibodies to adrenergic receptors, which are prototypical GPCRs, has been hypothesized to modulate receptor activity, increase sympathetic tone, and cause tachycardia. Moreover, IgG anti-GPCR autoantibodies, acting as partial agonists, are thought to decrease the effect of peripheral vasomotor control, leading to an increased sympathetic response to upright posture that can result in postural tachycardia in the absence of hypotension. Purpose We hypothesize that a reduction in autoantibody levels by efgartigimod, a FcRn-antagonist, will ameliorate the underlying immune-mediated pathogenesis and lead to clinical improvements in patients who developed POTS following COVID-19 infection. We propose a phase II multicenter, randomized, placebo-controlled, double-blind, proof-of-mechanism study initiated to evaluate the safety and efficacy of efgartigimod in adults with post–COVID-19 POTS. Methods Adult patients diagnosed with new-onset of POTS following SARS-CoV-2 infection are eligible for participation in the study. Prior COVID-19 must be confirmed by documentation of historical PCR test, and POTS diagnosis must meet consensus criteria. Study subjects must have moderate to severe autonomic symptoms (COMPASS-31 score ≥ 35 points at screening). Approximately 42 patients will be randomly allocated (2:1) to receive weekly intravenous efgartigimod or placebo for 24 weeks. At the end of the treatment period, participants who complete the study may roll over into an open-label extension (OLE). The co-primary endpoints are change from baseline to week 24 in the COMPASS-31 and Malmo POTS Symptom Score as well as safety and tolerability outcomes. Key secondary endpoints include assessments of disease activity, fatigue, cognitive function, walking capacity and quantitative autonomic testing. Additional exploratory endpoints include intraepidermal small fiber densities (optional), sudomotor innervation (optional) and peripheral blood biomarkers to define histopathological and immune determinants associated with treatment response. Conclusions This phase II study of efgartigimod will evaluate the effect of FcRn inhibition on disease pathology and the potential for therapeutic benefit in patients with post–COVID-19 POTS.

Artemisia Ordosica Polysaccharides Enhance Antioxidant Capacity of Peripheral Blood Lymphocytes in Poultry Through Nrf2/Keap1 and TLR4/NF-κB Signal Pathway

Artemisia ordosica polysaccharides (AOP) can promote animal growth, improve intestinal morphology, regulate immunity, and enhance antioxidant capacity. To investigate the antioxidant capacity of AOP, three experiments were conducted. (1) Different concentrations of AOP (0, 50, 100, 150, 200, and 250 μg/mL) and 1 µg/mL VA on peripheral blood lymphocytes (PBLs) treated with/without lipopolysaccharides (LPS) were investigated to select the optimum concentration. The results showed that 150 μg/mL AOP had significant antioxidation activity. (2) The PBLs was randomly divided into eight treatments with six replicates, namely CON, AOP, LPS, ML385 (Nrf2 inhibitor), AOP + LPS, AOP + ML385, LPS + ML385 and LPS + ML385 + AOP. The results showed that under a normal condition or stress condition, AOP presented antioxidation activity via upregulating Nrf2/Keap1 pathway-related gene expression. (3) The PBLs was randomly divided into eight treatments with six replicates, namely CON, AOP, LPS, PDTC (NF-κB inhibitor), AOP + LPS, AOP + PDTC, LPS + PDTC and LPS + PDTC + AOP. The results showed that under a normal condition, AOP presented antioxidation activity via increasing TLR4/NF-κB pathway-related gene expression; under a stress condition, AOP alleviated oxidative damage caused by LPS via suppressing TLR4/NF-κB pathway-related gene expression.

Exome sequencing identifies a novel FBN1 variant in a Chinese family with Marfan syndrome that includes aberrant right subclavian artery

Abstract Background Marfan syndrome (MFS) is an inherited autosomal dominant disorder that affects connective tissue with an incidence of about 1 in 5,000 to 10,000 people. Ninety percent of MFS is caused by mutations in the fibrillin-1 (fbn1) gene. We recruited a family with MFS phenotype in South China and identified a novel variant in fbn1 by whole exome sequencing (WES). The zebrafish share high genetic similarity to humans. This study aimed to generate this novel mutation in the fbn1 gene of zebrafish using the CRISPR/Cas9 technology and researched whether this variant is pathogenic. Methods A three-generation consanguineous family was recruited in this study, which was visited and interviewed for the clinical data and family history. Peripheral blood samples were collected from family members for DNA isolation and WES. The 3D structure of the protein was predicted by Alphafold. CRISPR/Cas9 was applied to generate an fbn1 nonsense mutation (fbn1+/−) in zebrafish. Morphological abnormalities were assessed in F2 fbn1+/− zebrafish by comparing with the wild-type (WT) controls at different development stages. Results Our study recruited a family with MFS phenotype in South China and identified a novel variant [NM_000138.5; c.7764C>G: p.(Y2588*)] in fbn1. Through Sanger sequencing, we found that family members Ⅲ-1 (son) and Ⅲ-2 (daughter) had the fbn1 variant segregated with MFS in the family. The p.Y2588* mutation resulted in the absence of 283 amino acids, thereby leading to the deficiency of 17 β-folded structures, 4 α-helix structures, and various loop structures. Compared with WT zebrafish, F2 fbn1+/− zebrafish exhibited aortic arches bleeding, abnormal angiogenesis, decreased cardiac volume, cardiac function defects, curly tail, and cartilage malformations. Conclusion A novel variant [NM_000138.5; c.7764C>G: p.(Y2588*)] was identified in fbn1 gene, which has not been reported in previous literature. The variant caused loss of function through premature protein truncation or nonsense-mediated mRNA decay. In zebrafish, we identified functional evidence of the detected nonsense mutation, which aligns with the clinical significance, suggesting a pathogenic variant of fbn1. Zebrafish as a novel, efficient tool to allow for the quick classification of unknown variants in fbn1 and improve the clinical diagnosis and treatment of MFS. It has brought the implementation of personal and precision medicine in the clinic within reach and made it possible to find patient-specific treatments.Marfanoid symptoms of the patient.Phenotypic Spectrum of fbn1+/− Zebrafish

Drug eluting stents trigger the secretion of inflammatory proteins in an experimental in vitro study

Abstract Introduction Ischemic cardiovascular diseases commonly appear following the occlusion of one or more of the coronary arteries. Drug eluting stents (DES) constitute the gold standard to help to keep open the arteries and maintain the blood flow. After their placement into the coronary arteries, they stay in direct contact with the blood stream, disturbing the physiological conditions and interacting with several cells and proteins within the surrounding blood and vessel wall. Besides the beneficial effects of DES, their clinical application is also associated with complications, such as (late) stent thromboses. Previous studies have shown that thrombosis and inflammation are tightly linked with each other by activating inflammatory cells and secreting cytokines and chemokines to further promote an inflammatory surrounding. However, the influence of DES and their components on the inflammatory cascade and their corresponding players was not investigated yet. Methods To investigate the inflammatory secretome in response to DES, we performed an in vitro study with isolated human peripheral blood mononuclear cells (PBMCs). Therefore, PBMCs were isolated from the whole blood of four healthy volunteers. In total, 63 stents were used in the study, including Medtronic Resolute Onyx (n=37), Abbott XIENCE Sierra (n=5), Boston Scientific SYNERGY (n=4), Terumo Ultimaster (n=3), and Biotronik Orsiro (n=14). We measured the secretion of cytokines and chemokines, including IL-1β, IL-6, TNF-α, IL-8, and IL-12, as well as ICAM-1, VCAM-1, and MCP-1 by commercially available enzyme-linked immunosorbent assay (ELISA). Results Our results showed that DES significantly increase the secretion of the cytokines IL-1β (p=0.0005), and IL-6 (p=0.0002), as well as the chemokine IL-8 (p=0.0010). Additionally, we also detected an elevation of the reaction product ICAM-1 (p=0.0003). Further analyses resulted in a reduced secretion of IL-1β, IL-6, TNF-α, IL-8, and ICAM-1 into the cells’ supernatants in response to Resolute Onyx stents, in comparison to the other stents. Moreover, multiple regression analysis revealed a high dependency of the inflammatory reaction on the elution drug, the polymer type and the stent’s length. Additionally, the material of the stent platform, as well as the polymer thickness further have impact on the cytokine and chemokine response of PBMCs. Conclusion Our study indicate that DES by themselves trigger an inflammatory reaction of blood cells by the secretion of cytokines and chemokines. Furthermore, we provided first insights into the composition of the inflammosome in response to DES. A triggered inflammation may increase the vulnerability for complications and the failure of the DES. As these complications were already observed in clinical studies, our results would potentially provide predictions about the application of certain DES designs and may help to choose the appropriate stents in the clinical setting.

Hypertension and global DNA methylation: a population-based study in rural, Punjab, India

Hypertension is a significant public health concern and a modifiable risk factor for increased mortality worldwide. It is reported to be influenced by gene-environment interactions and micronutrient intake. This study aims to understand the relationship between global DNA methylation levels and hypertension, independently and in the context of micronutrient status, among rural population in Punjab, India. A total of 2300 individuals, aged 30–75 years, (54.9% females) were screened for blood pressure. Of 2300 screened individuals, 900 (age sex matched 450 cases and 450 controls of hypertension) individuals were selected to examine the relationship between hypertension, global DNA methylation (5mC), and biochemicals (Folate, Vitamin B12, and Homocysteine). Folate, vitamin B12, and homocysteine levels were estimated using chemiluminescence technique. The ELISA-based colorimetric technique was used for performing peripheral blood leucocyte (PBL) global DNA methylation (5mC). Statistical analyses were performed using SPSS version 22.0. Hypertensives were found to have significantly lower levels of global DNA methylation than normotensives (0.65 vs. 0.72 respectively; p-value = 0.01*). Individuals in the 1st quartile of 5mC were at significantly (OR: 1.671; 95% CI: 1.206–2.315; p-value = 0.01*) increased risk for hypertension in comparison to those in the 4th quartile (reference). Further hypertensives on medication with controlled blood pressure (BP) were significantly hypermethylated as compared to hypertensives on medication with uncontrolled BP (0.70 vs. 0.62 respectively; p-value = 0.04*). Folate appeared to mediate global DNA methylation among hypertensives on medication-controlled BP. Further hypertension driven hypomethylation hints towards accelerated biological aging among younger hypertensives. Hypertension may be associated with Global DNA hypomethylation in the studied rural population of Punjab, India. Folate sufficiency may prove to be an aid in improving the methylation levels among the cases of hypertension who were on medication and had controlled BP.

Characterizing Non-T2 Asthma: Key Pathways and Molecular Implications Indicative of Attenuated Th2 Response.

Non-Type 2 (non-T2) asthma is characterized by a lack of allergic sensitization and normal to low total IgE levels. We aimed to explore molecular mechanisms and pathways differentiating non-T2 from T2-high pediatric asthma. We analyzed peripheral blood RNA samples from 11 non-T2 and 17 T2-high pediatric asthma patients using bulk RNA sequencing. Differentially expressed genes (DEGs) were identified, followed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, and Protein-Protein Interaction (PPI) network construction. Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) were employed to explore significance of these DEGs. We utilized independent public datasets GSE145505 to validate our findings. We investigated Th cytokine profiles in an independent cohort of pediatric patients with non-T2 asthma (n = 38) and T2-high asthma (n = 64). We demonstrated that the total serum IgE levels of children with non-T2 asthma (128.4 ± 159.5IU/mL) was significantly lower than that of those with T2-high asthma (405.8 ± 252.1IU/mL). Our analysis revealed 136 DEGs distinguishing non-T2 from T2-high asthma. IPA identified predicted inhibition of IgE-FcεRI signaling pathways in non-T2 asthma. Our DEG data showed the expression of IGHV4-39, IGLV1-40, IGLV1-47, IGLV1-44, IGHV1-69, IGLV6-57, IGLV3-19, IGLV3-1, and IGLC7 were downregulated in our non-T2 asthma patient. The non-T2 group exhibited significantly higher concentrations of IL-2, IFN-γ, IL-6, and IL-17A compared to the T2-high group. Our integrated analysis differentiated non-T2 from T2-high asthma by revealing downregulation of specific immunoglobulin genes influencing FcεRI signaling, elevated Th1 cytokines and Th17 cytokines might affect IgE associated sensitization and alter Th2 allergic response.

Gut microbiota predict retinopathy in patients with diabetes: A longitudinal cohort study

The gut microbiota has emerged as an independent risk factor for diabetes and its complications. This research aimed to delve into the intricate relationship between the gut microbiome and diabetic retinopathy (DR) through a dual approach of cross-sectional and prospective cohort studies. In our cross-sectional study cross-sectional investigation involving ninety-nine individuals with diabetes, distinct microbial signatures associated with DR were identified. Specifically, gut microbiome profiling revealed decreased levels of Butyricicoccus and Ruminococcus torques group, alongside upregulated methanogenesis pathways among DR patients. These individuals concurrently exhibited lower concentrations of short-chain fatty acids in their plasma. Leveraging machine learning models, including random forest classifiers, we constructed a panel of microbial genera and genes that robustly differentiated DR cases. Importantly, these genera also demonstrated significant correlations with dietary patterns and the molecular profiles of peripheral blood mononuclear cells. Building upon these findings, our prospective cohort study followed 62 diabetes patients over a 2-year period to assess the predictive value of these microbial markers. The results underlined the panel’s efficacy in predicting DR incidence. By stratifying patients based on the predictive genera and metabolites identified in the cross-sectional phase, we established significant associations between reduced levels of Butyricicoccus, plasma acetate, and increased susceptibility to DR. This investigation not only deepens our understanding of how gut microbiota influences DR but also underscores the potential of microbial markers as early indicators of disease risk. These insights hold promise for developing targeted interventions aimed at mitigating the impact of diabetic complications.Key points• Microbial signatures are differed in diabetic patients with and without retinopathy• DR-related taxa are linked to dietary habits and transcriptomic profiles• Lower abundances of Butyricicoccus and acetate were prospectively associated with DR

Immunomodulatory effects of cysteamine and its potential use as a host-directed therapy for tuberculosis

ObjectiveCysteamine, a drug approved to treat cystinosis, has been proposed as a host-directed therapy for M. tuberculosis (Mtb) and SARS-CoV-2. The impact of cysteamine on the immune responses has not been fully investigated. We aimed to in vitro evaluate the immunomodulatory effects of cysteamine on peripheral blood mononuclear cells (PBMCs) using the purified protein derivative (PPD) as a recall antigen, and an unspecific stimulus as staphylococcal enterotoxin B (SEB).MethodsPBMCs isolated from subjects with tuberculosis infection (TBI), those with tuberculosis disease (TB), and healthy controls (HC) were in vitro stimulated with PPD or SEB and treated or not with cysteamine at different concentrations (50 µM–400 µM) for 6 hours (h) and 24 h. We evaluated the T helper1 (Th1) and T cytotoxic1 (Tc1) cell cytokine production by flow cytometry and immune-enzymatic assays. In HC, we also evaluated apoptosis and/or necrosis by flow cytometry.ResultsWe observed an immunomodulatory effect of cysteamine at 400 µM in PBMCs from TB and TBI subjects. It significantly reduced PPD-specific Th1 responses at 24 h and at 6 h (p=0.0004 and p=0.0009, respectively), and a similar non-significant trend was observed with cysteamine at 200 µM (p=0.06 at 24 h and p=0.14 at 6 h). Moreover, cysteamine at both 400 µM (p<0.0001 and p=0.0187 at 24 h, respectively, and p<0.0001 at 6 h for both) and 200 µM (p=0.0119 and p=0.0028 at 24 h and p=0.0028 and p=0.0003 at 6 h, respectively) significantly reduced SEB-induced Th1 and Tc1 responses. Furthermore, we found that cysteamine induced morphological lymphocyte changes and significantly reduced the lymphocyte percentage in a dose- and time-dependent manner. Cysteamine at 400 µM induced 8% late apoptosis and 1.6% necrosis (p<0.05) at 24 h. In contrast, despite significant differences from untreated conditions (p<0.05), cysteamine at 400 µM for 6 h induced approximately 1% late apoptosis and 0.1% necrosis in the cells.ConclusionsHigh doses of cysteamine in vitro reduce the percentages of PPD- and SEB-induced Th1 and Tc1 cells and induce late apoptosis and necrosis. Differently, cysteamine at lower doses retains the immunomodulatory effect without affecting cell viability. These findings suggest cysteamine as a potential adjunct to antimicrobial regimens as in the TB or COVID-19 field, for its ability to reduce the inflammatory status.

BRAF V600E-Mutant Acute Myeloid Leukemia: A Case Series and Literature Review of a Rare Entity

Background: Although BRAF V600E mutations are common in solid tumors and select hematologic neoplasms, they are reported less frequently in myeloid malignancies. Of the cases of BRAF V600E-mutant acute myeloid leukemia (AML) that have been described, most display monocytic morphology and concurrent KMT2A rearrangement. Strikingly, all cases have been associated with poor survival. Case Presentation: Here, we report two cases of AML, one diagnosed in an elderly male with metastatic lung adenocarcinoma and hepatocellular carcinoma and the other diagnosed in a young boy previously treated for B-cell acute lymphoblastic leukemia. Peripheral blood NGS revealed oncogenic mutations in BRAF p.V600E (VAF = 33%), TET2 p.M508Cfs*25 (VAF = 48%), TET2 p.C211* (VAF = 49%), ZRSR2 p.R295* (VAF = 71%), BRAF p.N581S (VAF = 6%), and EZH2 c.118-2A>G, p.? (VAF = 4%) in case 1 and BRAF p.V600E (VAF = 1%) and KRAS p.G12A (VAF = 28%) in case 2. Cytogenetic workup revealed a complex karyotype in case 1 and an abnormal karyotype with non-clonal aberrations and KMT2A (MLL) rearrangement in case 2. Morphologically, both patients were found to have AML with monocytic features. The post-mortem examination of case 2 also revealed extensive solid organ infiltration, consistent with a monocytic leukemia. Both patients died within days of diagnosis, demonstrating the lethality of this molecular subgroup of AML. Conclusions: Our cases add to the literature, highlighting the poor prognosis of patients diagnosed with BRAF-mutant AML. Although it is uncertain whether the complex karyotype and somatic mutations observed in case 1 and KMT2A rearrangement and variants identified in case 2 may have either independently or cooperatively conferred a poor prognosis, we contend that additional comprehensive studies are needed to further understand the pathophysiology and prognosis of BRAF mutations in AML. We further posit whether patients with BRAF V600E-mutant AML may benefit from the combined use of BRAF inhibitors and/or RAS-pathway-targeting regimens, which are currently FDA-approved for the treatment of BRAF V600-mutant solid tumors and BRAF-mutant histiocytic neoplasms.

Deciphering the Multifaceted Immune Landscape of Unresectable Primary Liver Cancer to Predict Immunotherapy Response.

Immunotherapies employing PD-1/PD-L1 immune checkpoint inhibitors (ICIs) are vital for primary liver cancer (PLC), but response rates remain unsatisfying. Accurate differentiation of responders from non-responders to immunotherapy is imperative. Here, single-cell-scaled mass cytometry analysis on sequential peripheral blood mononuclear cells (PBMCs) from ICI-treated PLC patients is conducted, and tissue residence of immune subpopulations is assessed via multiplex immunohistochemistry. In the discovery cohort (n = 24), responders have lower baseline B cell and HLA-DR+CD8+T cell, and higher CD14+CD16- classical monocyte (CM) proportions. CMs decrease more in responders PBMCs, while HLA-DR+CD8+T cells conformably amplify after ICI-exposure. Responsive individuals display upregulated exhaustion and activation markers in peripheral immune lineages. In the expanded cohort of 77 patients, the augment of the B cells in non-responders is re-confirmed. Responders demonstrate much higher enrichment of B cells or tertiary lymphoid structures in tumor compared to non-responders. A prospective model that excelled in early discrimination of responders is developed using generalized linear model and achieves a satisfactory AUC over 0.9 in all three independent cohorts. Integratedly, the study unveils dynamic immune landscapes in PLC patients undergoing ICI-based therapy, aiding in PLC patient stratification for ICI-based treatment and fostering new response monitoring strategies.

HIV DNA Levels in Persons Who Acquired HIV in the Setting of Long-Acting Cabotegravir for HIV Prevention: Analysis of Cases from HPTN 083 and 084.

We evaluated HIV DNA levels in individuals who received long-acting cabotegravir (CAB-LA) or tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) pre-exposure prophylaxis in the HPTN 083 and 084 trials and had HIV DNA testing performed to help determine HIV status. HIV DNA testing was performed using peripheral blood mononuclear cell (PBMC) samples collected after a reactive HIV test was obtained at a study site. DNA was quantified using droplet digital PCR (lower limit of detection [LLOD]: 4.09 copies/million PBMCs). Final HIV status and the timing of the first HIV-positive visit were determined by an independent adjudication committee based on HIV test results from real-time site testing and retrospective testing at a centralized laboratory. HIV DNA testing was performed for 133 participants [21 HIV-positive (7 CAB-LA arm, 14 TDF/FTC arm) and 112 HIV-negative; 1-6 tests/person]. For persons with HIV, the time between the first HIV-positive visit and collection of the first sample for DNA testing was a median of 81 days for those receiving CAB-LA (range 41-246) and 11 days for those receiving TDF/FTC (range 3-127). Four (57.1%) of the seven CAB-LA cases with infection had a low initial DNA result [three detected <LLOD; one near the LLOD (4.2 copies/106 PBMCs); in 2/4 cases, the DNA level was still <10 copies/106 PBMCs ≥40 weeks after the first HIV-positive visit. In contrast, only 3/14 (21.4%) of the TDF/FTC cases had a low or negative initial DNA test result (one not detected; two <10 copies/106 PBMCs). In this study, the time between the first HIV-positive visit and the first DNA test was longer in the CAB-LA cases than the TDF/FTC cases. Despite this difference, low or undetectable DNA levels were more frequently observed in the CAB-LA cases. This suggests that CAB-LA exposure may limit seeding of the HIV reservoir in early infection.

Bidirectional roles of extracellular and intracellular HMGB2 in the development of atherosclerosis

Abstract Rationale: HMGB family plays an important role in homeostasis of immunity and regulation of inflammation. However, the role of HMGB2 in atherosclerosis remains uncharacterized. Objective The current study aimed to ascertain the relation of HMGB2 levels in macrophages of atherosclerotic plaques and in circulatory monocytes/macrophages with coronary artery disease (CAD), and to investigate the influence of HMGB2 on atherogenesis in mice and inflammatory reaction in vitro. Methods/Results We evaluated HMGB2 levels in macrophages of atherosclerotic plaques in coronary endarterectomy tissue from patients with angiographically documented severe coronary artery disease (CAD)(n=23), and in coronary artery specimens from autopsy cases with no or mild atherosclerosis (n=11) by immunohistochemistry. HMGB2 levels were significantly decreased in macrophages of progressive atherosclerotic plaques, as compared with those from mild plaques. Moreover, we also analyzed HMGB2 levels in peripheral blood monocytes from patients with CAD (n=44) or age- and sex-matched non-CAD controls (n=44). The results showed that decreased intracellular HMGB2 protein levels in monocytes were associated with CAD. In experiments, HMGB2 deficiency significantly promoted atherosclerosis in ApoE-/- mice, whereas such increment of atherosclerosis was markedly attenuated after transplantation of bone marrow derived macrophages from ApoE-/-mice. Mechanistically, intracellular HMGB2 repressed IL-1 transcription and induced autophagic degradation of NLRP3, thus suppressing NLRP3 inflammasome priming and activation. Extracellular HMGB2 levels increased in progressive atherosclerotic plaques with macrophage apoptosis. Recombinant HMGB2 protein promoted inflammation in vitro via RAGE and intraperitoneal administration of this protein exacerbated atherosclerosis in mice. Conclusion This study indicates that decreased HMGB2 level in macrophage is associated with inflammation and atherosclerosis in patients with CAD. Intracellular HMGB2 inhibits NLRP3 inflammasome and atherosclerosis in mice, whereas released HMGB2 promotes inflammation and atherosclerosis in mice.