Regulatory T Cell Biomarkers Identify Patients at Risk of Developing Acute Cellular Rejection in the First Year Following Heart Transplantation.

BACKGROUND: Acute cellular rejection (ACR), an alloimmune response involving CD4+ and CD8+ T cells, occurs in up to 20% of patients within the first year following heart transplantation. The balance between a conventional versus regulatory CD4+ T cell alloimmune response is believed to contribute to developing ACR. Therefore, tracking these cells may elucidate whether changes in these cell populations could signal ACR risk. METHODS: We used a CD4+ T cell gene signature (TGS) panel that tracks CD4+ conventional T cells (Tconv) and regulatory T cells (Treg) on longitudinal samples from 94 adult heart transplant recipients. We evaluated combined diagnostic performance of the TGS panel with a previously developed biomarker panel for ACR diagnosis, HEARTBiT, while also investigating TGS' prognostic utility. RESULTS: Compared with nonrejection samples, rejection samples showed decreased Treg- and increased Tconv-gene expression. The TGS panel was able to discriminate between ACR and nonrejection samples and, when combined with HEARTBiT, showed improved specificity compared with either model alone. Furthermore, the increased risk of ACR in the TGS model was associated with lower expression of Treg genes in patients who later developed ACR. Reduced Treg gene expression was positively associated with younger recipient age and higher intrapatient tacrolimus variability. CONCLUSIONS: We demonstrated that expression of genes associated with CD4+ Tconv and Treg could identify patients at risk of ACR. In our post hoc analysis, complementing HEARTBiT with TGS resulted in an improved classification of ACR. Our study suggests that HEARTBiT and TGS may serve as useful tools for further research and test development.

Viral Heart Disease: Diagnosis, Management, and Mechanisms.

"Viral heart disease" is a term encompassing numerous virus-triggered heart conditions, wherein cardiac myocytes are injured, causing contractile dysfunction, cell death, or both. Cardiotropic viruses may also damage interstitial cells and vascular cells. Clinical presentation of the disorder varies widely. In most cases, patients are asymptomatic. Presentation includes-but is not limited to-flu-like symptoms, chest pain, cardiac arrhythmias, heart failure, cardiogenic shock, and sudden cardiac death. Laboratory studies, including blood-based heart injury indicators and cardiac imaging, may be needed. Management of viral heart disease requires a graded approach. Watchful observation at home may be the first step. Closer observation, with additional testing such as echocardiography in the clinic or hospital is less common yet may inform the use of cardiac magnetic resonance imaging. Intensive care may be indicated in severe acute illness. Viral heart disease mechanisms are complex. Initially, damage is predominantly virus mediated, whereas, in the second week, immune responses bring unintended obverse consequences for the myocardium. Innate immunity is largely beneficial in initial attempts to quell viral replication, whereas adaptive immunity brings helpful and antigen-specific mechanisms to fight the pathogen but also introduces the capability of autoimmunity. Each cardiotropic virus family has its own pathogenesis signature, including attack on myocytes, vascular cells, and other constitutive cells of myocardial interstitium. The stage of disease and preponderant viral pathways lend opportunities for potential intervention but also the likelihood of uncertainty about management. Overall, this review provides a novel glimpse into the depth of and need for solutions in viral heart disease.

Characterization of COVID-19-associated cardiac injury: evidence for a multifactorial disease in an autopsy cohort.

As the coronavirus disease 2019 (COVID-19) pandemic evolves, much evidence implicates the heart as a critical target of injury in patients. The mechanism(s) of cardiac involvement has not been fully elucidated, although evidence of direct virus-mediated injury, thromboembolism with ischemic complications, and cytokine storm has been reported. We examined suggested mechanisms of COVID-19-associated heart failure in 21 COVID-19-positive decedents, obtained through standard autopsy procedure, compared to clinically matched controls and patients with various etiologies of viral myocarditis. We developed a custom tissue microarray using regions of pathological interest and interrogated tissues via immunohistochemistry and in situ hybridization. Severe acute respiratory syndrome coronavirus 2 was detected in 16/21 patients, in cardiomyocytes, the endothelium, interstitial spaces, and percolating adipocytes within the myocardium. Virus detection typically corresponded with troponin depletion and increased cleaved caspase-3. Indirect mechanisms of injury-venous and arterial thromboses with associated vasculitis including a mixed inflammatory infiltrate-were also observed. Neutrophil extracellular traps (NETs) were present in the myocardium of all COVID-19 patients, regardless of injury degree. Borderline myocarditis (inflammation without associated myocyte injury) was observed in 19/21 patients, characterized by a predominantly mononuclear inflammatory infiltrate. Edema, inflammation of percolating adipocytes, lymphocytic aggregates, and large septal masses of inflammatory cells and platelets were observed as defining features, and myofibrillar damage was evident in all patients. Collectively, COVID-19-associated cardiac injury was multifactorial, with elevated levels of NETs and von Willebrand factor as defining features of direct and indirect viral injury.

COVID-19-positivity in a heart transplant recipient-antibody-mediated rejection or SARS-CoV-2-associated cardiac injury?

Through the ongoing and heightening coronavirus disease 2019 (COVID-19) pandemic, the heart has been implicated as a central target of injury associated with significantly increased morbidity and mortality. Correspondingly, heart transplant recipients are a vulnerable population for which insufficient research has been conducted. Pathologic antibody-mediated rejection (pAMR) of cardiac allografts shares many characteristics with COVID-19-associated cardiac injury. In this case study, we investigate a 57-year-old female who contracted COVID-19 11 days postheart transplant and was observed to have pAMR while positive for laboratory-confirmed COVID-19, resulting in a diagnostic conundrum.

Advanced detection strategies for cardiotropic virus infection in a cohort study of heart failure patients.

The prevalence and contribution of cardiotropic viruses to various expressions of heart failure are increasing, yet primarily underappreciated and underreported due to variable clinical syndromes, a lack of consensus diagnostic standards and insufficient clinical laboratory tools. In this study, we developed an advanced methodology for identifying viruses across a spectrum of heart failure patients. We designed a custom tissue microarray from 78 patients with conditions commonly associated with virus-related heart failure, conditions where viral contribution is typically uncertain, or conditions for which the etiological agent remains suspect but elusive. Subsequently, we employed advanced, highly sensitive in situ hybridization to probe for common cardiotropic viruses: adenovirus 2, coxsackievirus B3, cytomegalovirus, Epstein-Barr virus, hepatitis C and E, influenza B and parvovirus B19. Viral RNA was detected in 46.4% (32/69) of heart failure patients, with 50% of virus-positive samples containing more than one virus. Adenovirus 2 was the most prevalent, detected in 27.5% (19/69) of heart failure patients, while in contrast to previous reports, parvovirus B19 was detected in only 4.3% (3/69). As anticipated, viruses were detected in 77.8% (7/9) of patients with viral myocarditis and 37.5% (6/16) with dilated cardiomyopathy. Additionally, viruses were detected in 50% of patients with coronary artery disease (3/6) and hypertrophic cardiomyopathy (2/4) and in 28.6% (2/7) of transplant rejection cases. We also report for the first time viral detection within a granulomatous lesion of cardiac sarcoidosis and in giant cell myocarditis, conditions for which etiological agents remain unknown. Our study has revealed a higher than anticipated prevalence of cardiotropic viruses within cardiac muscle tissue in a spectrum of heart failure conditions, including those not previously associated with a viral trigger or exacerbating role. Our work forges a path towards a deeper understanding of viruses in heart failure pathogenesis and opens possibilities for personalized patient therapeutic approaches.

Aortic Dimensions, Biophysical Properties, and Plasma Biomarkers in Children and Adults with Marfan or Loeys-Dietz Syndrome.

BACKGROUND: Aortic dilation, stiffening, and dissection are common and potentially lethal complications of Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS), which involve abnormal transforming growth factor beta (TGF-ß) signalling. The relation of aortic dimensions, stiffness, and biomarker levels is unknown. The objective of this study was to measure aortic dimensions, stiffness, TGF-ß and matrix metalloproteinase (MMP) levels, and endothelial function in patients with MFS, and to compare TGF-ß levels in patients with MFS receiving different therapeutic regimens. METHODS: This was a cohort study of 40 MFS and 4 LDS patients and 87 control participants. Aortic dimension and stiffness indexes, including pulse wave velocity (PWV), were measured using echocardiography and Doppler. Total and free TGF-ß and MMP blood levels were measured using Quantikine (R&D Systems, Inc, Minneapolis, MN) and Quanterix (Billerica, MA) kits. Endothelial function was measured using brachial artery flow-mediated dilation. RESULTS: PWV was increased in patients with MFS. There were increased MMP-2 levels in those with MFS but no increase in free or total TGF-ß or MMP-9 levels compared with control participants. There was no difference in TGF-ß levels between MFS patients receiving no medications, angiotensin receptor blockers, and ß-blockers. PWV correlated most strongly with age. Endothelial function showed premature gradual decline in patients with MFS. CONCLUSIONS: Despite the increased PWV, monitoring aortic stiffness or TGF-ß levels would not be helpful in patients with MFS. TGF-ß levels were not increased and the increased MMP-2 levels suggest consideration of a different therapeutic target.

CONTEXTE: La dilatation, la rigidification et la dissection de l'aorte sont des complications fréquentes et parfois mortelles du syndrome de Marfan (SM) et du syndrome de Loeys-Dietz (SLD), qui sont tous deux dûs à une anomalie de la voie de signalisation du facteur de croissance transformant bêta (TGF-ß). On ne connaît pas la relation entre les dimensions et la rigidité de l'aorte et la présence de biomarqueurs. Notre étude visait à mesurer les dimensions et la rigidité de l'aorte, les taux de TGF-ß et de métalloprotéases matricielles (MMP) et la fonction endothéliale chez des patients atteints du SM, et à les comparer aux taux de TGF-ß observés chez des patients également atteints de SM, mais recevant un autre traitement. MÉTHODOLOGIE: Il s'agissait d'une étude de cohorte menée auprès de 40 patients atteints du SM et de quatre patients atteints du SLD, ainsi que de 87 témoins. Les indices des dimensions et de la rigidité aortiques, y compris la vitesse d'onde de pouls (VOP), ont été mesurés par échocardiographie et par échographie Doppler. Les taux sanguins de TGF-ß et de MMP totaux et libres ont été mesurés à l'aide de trousses Quantikine (R&D Systems, Inc, Minneapolis, MN) et Quanterix (Billerica, MA). La fonction endothéliale a été mesurée par dilatation liée au flux dans l'artère brachiale. RÉSULTATS: La VOP était plus élevée chez les patients atteints du SM. On a aussi observé une hausse des taux de MMP-2 chez les patients atteints de SM, mais aucune augmentation des taux de TGF-ß ou de MMP-9 libres ou totaux comparativement aux témoins. Il n'y avait pas de différence entre les taux de TGF-ß chez les patients atteints de SM ne recevant aucun traitement, ceux qui prenaient un antagoniste des récepteurs de l'angiotensine et ceux qui prenaient un bêtabloquant. La VOP été plus fortement corrélée avec l'âge. La fonction endothéliale a affiché un déclin progressif prématuré chez les patients atteints du SM. CONCLUSIONS: Malgré l'augmentation de la VOP, il ne semble pas utile de surveiller la rigidité aortique ni les taux de TGF-ß en cas de SM. Les taux de TGF-ß n'étaient pas plus élevés chez les patients atteints du SM, et la hausse des taux de MMP-2 indique qu'il conviendrait de choisir une autre cible thérapeutique.

SARS-CoV-2 infection of human iPSC-derived cardiac cells reflects cytopathic features in hearts of patients with COVID-19.

Although coronavirus disease 2019 (COVID-19) causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human induced pluripotent stem cell (iPSC)-derived heart cells to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural genes corroborates adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and nuclear disruption. Human autopsy specimens from patients with COVID-19 reflected similar alterations, particularly sarcomeric fragmentation. These notable cytopathic features in cardiomyocytes provide insights into SARS-CoV-2-induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise concerns about the long-term consequences of COVID-19 in asymptomatic and severe cases.

Overlapping and distinct biological effects of IL-6 classic and trans-signaling in vascular endothelial cells.

IL-6 affects tissue protective/reparative and inflammatory properties of vascular endothelial cells (ECs). This cytokine can signal to cells through classic and trans-signaling mechanisms, which are differentiated based on the expression of IL-6 receptor (IL-6R) on the surface of target cells. The biological effects of these IL-6-signaling mechanisms are distinct and have implications for vascular pathologies. We have directly compared IL-6 classic and trans-signaling in ECs. Human ECs expressed IL-6R in culture and in situ in coronary arteries from heart transplants. Stimulation of human ECs with IL-6, to model classic signaling, triggered the activation of phosphatidylinositol 3-kinase (PI3K)-Akt and ERK1/2 signaling pathways, whereas stimulation with IL-6 + sIL-6R, to model trans-signaling, triggered activation of STAT3, PI3K-Akt, and ERK1/2 pathways. IL-6 classic signaling reduced persistent injury of ECs in an allograft model of vascular rejection and inhibited cell death induced by growth factor withdrawal. When inflammatory effects were examined, IL-6 classic signaling did not induce ICAM or CCL2 expression but was sufficient to induce secretion of CXCL8 and support transmigration of neutrophil-like cells. IL-6 trans-signaling induced all inflammatory effects studied. Our findings show that IL-6 classic and trans-signaling have overlapping but distinct properties in controlling EC survival and inflammatory activation. This has implications for understanding the effects of IL-6 receptor-blocking therapies as well as for vascular responses in inflammatory and immune conditions.

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

Analytical Validation of HEARTBiT: A Blood-Based Multiplex Gene Expression Profiling Assay for Exclusionary Diagnosis of Acute Cellular Rejection in Heart Transplant Patients.

BACKGROUND: HEARTBiT is a whole blood-based gene profiling assay using the nucleic acid counting NanoString technology for the exclusionary diagnosis of acute cellular rejection in heart transplant patients. The HEARTBiT score measures the risk of acute cellular rejection in the first year following heart transplant, distinguishing patients with stable grafts from those at risk for acute cellular rejection. Here, we provide the analytical performance characteristics of the HEARTBiT assay and the results on pilot clinical validation. METHODS: We used purified RNA collected from PAXgene blood samples to evaluate the characteristics of a 12-gene panel HEARTBiT assay, for its linearity range, quantitative bias, precision, and reproducibility. These parameters were estimated either from serial dilutions of individual samples or from repeated runs on pooled samples. RESULTS: We found that all 12 genes showed linear behavior within the recommended assay input range of 125 ng to 500 ng of purified RNA, with most genes showing 3% or lower quantitative bias and around 5% coefficient of variation. Total variation resulting from unique operators, reagent lots, and runs was less than 0.02 units standard deviation (SD). The performance of the analytically validated assay (AUC = 0.75) was equivalent to what we observed in the signature development dataset. CONCLUSION: The analytical performance of the assay within the specification input range demonstrated reliable quantification of the HEARTBiT score within 0.02 SD units, measured on a 0 to 1 unit scale. This assay may therefore be of high utility in clinical validation of HEARTBiT in future biomarker observational trials.

HEARTBiT: A Transcriptomic Signature for Excluding Acute Cellular Rejection in Adult Heart Allograft Patients.

BACKGROUND: Nine mRNA transcripts associated with acute cellular rejection (ACR) in previous microarray studies were ported to the clinically amenable NanoString nCounter platform. Here we report the diagnostic performance of the resulting blood test to exclude ACR in heart allograft recipients: HEARTBiT. METHODS: Blood samples for transcriptomic profiling were collected during routine post-transplantation monitoring in 8 Canadian transplant centres participating in the Biomarkers in Transplantation initiative, a large (n = 1622) prospective observational study conducted between 2009 and 2014. All adult cardiac transplant patients were invited to participate (median age = 56 [17 to 71]). The reference standard for rejection status was histopathology grading of tissue from endomyocardial biopsy (EMB). All locally graded ISHLT ≥ 2R rejection samples were selected for analysis (n = 36). ISHLT 1R (n = 38) and 0R (n = 86) samples were randomly selected to create a cohort approximately matched for site, age, sex, and days post-transplantation, with a focus on early time points (median days post-transplant = 42 [7 to 506]). RESULTS: ISHLT ≥ 2R rejection was confirmed by EMB in 18 and excluded in 92 samples in the test set. HEARTBiT achieved 47% specificity (95% confidence interval [CI], 36%-57%) given ≥ 90% sensitivity, with a corresponding area under the receiver operating characteristic curve of 0.69 (95% CI, 0.56-0.81). CONCLUSIONS: HEARTBiT's diagnostic performance compares favourably to the only currently approved minimally invasive diagnostic test to rule out ACR, AlloMap (CareDx, Brisbane, CA) and may be used to inform care decisions in the first 2 months post-transplantation, when AlloMap is not approved, and most ACR episodes occur.

Deficiency of Circulating Monocytes Ameliorates the Progression of Myxomatous Valve Degeneration in Marfan Syndrome.

BACKGROUND: Myxomatous valve degeneration (MVD) involves the progressive thickening and degeneration of the heart valves, leading to valve prolapse, regurgitant blood flow, and impaired cardiac function. Leukocytes composed primarily of macrophages have recently been detected in myxomatous valves, but the timing of the presence and the contributions of these cells in MVD progression are not known. METHODS: We examined MVD progression, macrophages, and the valve microenvironment in the context of Marfan syndrome (MFS) using mitral valves from MFS mice (Fbn1C1039G/+), gene-edited MFS pigs (FBN1Glu433AsnfsX98/+), and patients with MFS. Additional histological and transcriptomic evaluation was performed by using nonsyndromic human and canine myxomatous valves, respectively. Macrophage ontogeny was determined using MFS mice transplanted with mTomato+ bone marrow or MFS mice harboring RFP (red fluorescent protein)-tagged C-C chemokine receptor type 2 (CCR2) monocytes. Mice deficient in recruited macrophages (Fbn1C1039G/+;Ccr2RFP/RFP) were generated to determine the requirements of recruited macrophages to MVD progression. RESULTS: MFS mice recapitulated histopathological features of myxomatous valve disease by 2 months of age, including mitral valve thickening, increased leaflet cellularity, and extracellular matrix abnormalities characterized by proteoglycan accumulation and collagen fragmentation. Diseased mitral valves of MFS mice concurrently exhibited a marked increase of infiltrating (MHCII+, CCR2+) and resident macrophages (CD206+, CCR2-), along with increased chemokine activity and inflammatory extracellular matrix modification. Likewise, mitral valve specimens obtained from gene-edited MFS pigs and human patients with MFS exhibited increased monocytes and macrophages (CD14+, CD64+, CD68+, CD163+) detected by immunofluorescence. In addition, comparative transcriptomic evaluation of both genetic (MFS mice) and acquired forms of MVD (humans and dogs) unveiled a shared upregulated inflammatory response in diseased valves. Remarkably, the deficiency of monocytes was protective against MVD progression, resulting in a significant reduction of MHCII macrophages, minimal leaflet thickening, and preserved mitral valve integrity. CONCLUSIONS: All together, our results suggest sterile inflammation as a novel paradigm to disease progression, and we identify, for the first time, monocytes as a viable candidate for targeted therapy in MVD.

Blood biomarkers to predict short-term pulmonary exacerbation risk in children and adolescents with CF: A pilot study.

In CF, pulmonary exacerbations (PEx) can lead to permanent loss in lung function and thus should be prevented. Previously, we identified a blood protein biosignature consisting of 6 proteins capable of predicting short-term PEx events in CF adults. In this study, we utilized blood samples from the placebo arm of a randomized controlled trial to assess whether this candidate protein biosignature was also capable of predicting short-term PEx events in CF children and adolescents. This pilot study provides preliminary evidence that blood inflammation can be monitored to predict short-term PEx risk in CF children and adolescents.

Cleavage and Sub-Cellular Redistribution of Nuclear Pore Protein 98 by Coxsackievirus B3 Protease 2A Impairs Cardioprotection.

Myocarditis, inflammation of the heart muscle, affects all demographics and is a major cause of sudden and unexpected death in young people. It is most commonly caused by viral infections of the heart, with coxsackievirus B3 (CVB3) being among the most prevalent pathogens. To understand the molecular pathogenesis of CVB3 infection and provide strategies for developing treatments, we examined the role of a key nuclear pore protein 98 (NUP98) in the setting of viral myocarditis. NUP98 was cleaved as early as 2 h post-CVB3 infection. This cleavage was further verified through both the ectopic expression of viral proteases and in vitro using purified recombinant CVB3 proteases (2A and 3C), which demonstrated that CVB3 2A but not 3C is responsible for this cleavage. By immunostaining and confocal imaging, we observed that cleavage resulted in the redistribution of NUP98 to punctate structures in the cytoplasm. Targeted siRNA knockdown of NUP98 during infection further increased viral protein expression and viral titer, and reduced cell viability, suggesting a potential antiviral role of NUP98. Moreover, we discovered that expression levels of neuregulin-1 (NRG1), a cardioprotective gene, and presenilin-1 (PSEN1), a cellular protease processing the tyrosine kinase receptor ERBB4 of NRG1, were reliant upon NUP98 and were downregulated during CVB3 infection. In addition, expression of these NUP98 target genes in myocardium tissue not only occurred at an earlier phase of infection, but also appeared in areas away from the initial inflammatory regions. Collectively, CVB3-induced cleavage of NUP98 and subsequent impairment of the cardioprotective NRG1-ERBB4/PSEN1 signaling cascade may contribute to increased myocardial damage in the context of CVB3-induced myocarditis. To our knowledge, this is the first study to demonstrate the link between NUP98 and the NRG1 signaling pathway in viral myocarditis.

Effect of short-term oral prednisone therapy on blood gene expression: a randomised controlled clinical trial.

BACKGROUND: Effects of systemic corticosteroids on blood gene expression are largely unknown. This study determined gene expression signature associated with short-term oral prednisone therapy in patients with chronic obstructive pulmonary disease (COPD) and its relationship to 1-year mortality following an acute exacerbation of COPD (AECOPD). METHODS: Gene expression in whole blood was profiled using the Affymetrix Human Gene 1.1 ST microarray chips from two cohorts: 1) a prednisone cohort with 37 stable COPD patients randomly assigned to prednisone 30 mg/d + standard therapy for 4 days or standard therapy alone and 2) the Rapid Transition Program (RTP) cohort with 218 COPD patients who experienced AECOPD and were treated with systemic corticosteroids. All gene expression data were adjusted for the total number of white blood cells and their differential cell counts. RESULTS: In the prednisone cohort, 51 genes were differentially expressed between prednisone and standard therapy group at a false discovery rate of < 0.05. The top 3 genes with the largest fold-changes were KLRF1, GZMH and ADGRG1; and 21 genes were significantly enriched in immune system pathways including the natural killer cell mediated cytotoxicity. In the RTP cohort, 27 patients (12.4%) died within 1 year after hospitalisation of AECOPD; 32 of 51 genes differentially expressed in the prednisone cohort significantly changed from AECOPD to the convalescent state and were enriched in similar cellular immune pathways to that in the prednisone cohort. Of these, 10 genes including CX3CR1, KLRD1, S1PR5 and PRF1 were significantly associated with 1-year mortality. CONCLUSIONS: Short-term daily prednisone therapy produces a distinct blood gene signature that may be used to determine and monitor treatment responses to prednisone in COPD patients during AECOPD. TRIAL REGISTRATION: The prednisone cohort was registered at clinicalTrials.gov ( NCT02534402 ) and the RTP cohort was registered at ClinicalTrials.gov ( NCT02050022 ).

Ensembling Electrical and Proteogenomics Biomarkers for Improved Prediction of Cardiac-Related 3-Month Hospitalizations: A Pilot Study.

BACKGROUND: Many risk models for predicting mortality, hospitalizations, or both in patients with heart failure have been developed but do not have sufficient discriminatory ability. The purpose of this study was to identify predictive biomarkers of hospitalizations in heart failure patients using omics-based technologies applied to blood and electrical monitoring of the heart. METHODS: Blood samples were collected from 58 heart failure patients during enrollment into this study. Each patient wore a 48-hour Holter monitor that recorded the electrical activity of their heart. The blood samples were profiled for gene expression using microarrays and protein levels using multiple reaction monitoring. Statistical deconvolution was used to estimate cellular frequencies of common blood cells. Classification models were developed using clinical variables, Holter variables, cell types, gene transcripts, and proteins to predict hospitalization status. RESULTS: Of the 58 patients recruited, 13 were hospitalized within 3 months after enrollment. These patients had lower diastolic and systolic blood pressures, higher brain natriuretic peptide levels, most had higher blood creatinine levels, and had been diagnosed with heart failure for a longer time period. The best-performing clinical model had an area under the receiver operating characteristic curve of 0.76. An ensemble biomarker panel consisting of Holter variables, cell types, gene transcripts, and proteins had an area under the receiver operating characteristic curve of 0.88. CONCLUSIONS: Molecular-based analyses as well as sensory data might provide sensitive biomarkers for the prediction of hospitalizations in heart failure patients. These approaches may be combined with traditional clinical models for the development of improved risk prediction models for heart failure.

Phenotyping and outcomes of hospitalized COPD patients using rapid molecular diagnostics on sputum samples.

BACKGROUND: Etiologies of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are heterogeneous. We phenotyped severe AECOPD based on molecular pathogen detection of sputum samples collected at hospitalization of COPD patients and determined their outcomes. METHODS: We phenotyped 72 sputum samples of COPD patients who were hospitalized with a primary diagnosis of AECOPD using a molecular array that detected common bacterial and viral respiratory pathogens. Based on these results, the patients were classified into positive or negative pathogen groups. The pathogen-positive group was further divided into virus or bacteria subgroups. Admission day 1 blood samples were assayed for N-terminal prohormone brain natriuretic peptide, CRP, and complete blood counts. RESULTS: A total of 52 patients had a positive result on the array, while 20 patients had no pathogens detected. The most common bacterial pathogen detected was Haemophilus influenzae and the most common virus was rhinovirus. The pathogen-negative group had the worse outcomes with longer hospital stays (median 6.5 vs 5 days for bacteria-positive group, P=0.02) and a trend toward increased 1-year mortality (P=0.052). The bacteria-positive group had the best prognosis, whereas the virus-positive group had outcomes somewhere in between the bacteria-positive and pathogen-negative groups. CONCLUSION: Molecular diagnostics on sputum can rapidly phenotype serious AECOPD into bacteria-, virus-, or pathogen-negative groups. The bacteria-positive group appears to have the best prognosis, while pathogen-negative group has the worst. These data suggest that AECOPD is a heterogeneous event and that accurate phenotyping of AECOPD may lead to novel management strategies that are personalized and more precise.