Universal Digital High-Resolution Melt Analysis for the Diagnosis of Bacteremia.

Fast and accurate diagnosis of bloodstream infection is necessary to inform treatment decisions for septic patients, who face hourly increases in mortality risk. Blood culture remains the gold standard test but typically requires approximately 15 hours to detect the presence of a pathogen. We, therefore, assessed the potential for universal digital high-resolution melt (U-dHRM) analysis to accomplish faster broad-based bacterial detection, load quantification, and species-level identification directly from whole blood. Analytical validation studies demonstrated strong agreement between U-dHRM load measurement and quantitative blood culture, indicating that U-dHRM detection is highly specific to intact organisms. In a pilot clinical study of 17 whole blood samples from pediatric patients undergoing simultaneous blood culture testing, U-dHRM achieved 100% concordance when compared with blood culture and 88% concordance when compared with clinical adjudication. Moreover, U-dHRM identified the causative pathogen to the species level in all cases where the organism was represented in the melt curve database. These results were achieved with a 1-mL sample input and sample-to-answer time of 6 hours. Overall, this pilot study suggests that U-dHRM may be a promising method to address the challenges of quickly and accurately diagnosing a bloodstream infection.

Universal digital high resolution melt analysis for the diagnosis of bacteremia.

Fast and accurate diagnosis of bloodstream infection is necessary to inform treatment decisions for septic patients, who face hourly increases in mortality risk. Blood culture remains the gold standard test but typically requires ∼15 hours to detect the presence of a pathogen. Here, we assess the potential for universal digital high-resolution melt (U-dHRM) analysis to accomplish faster broad-based bacterial detection, load quantification, and species-level identification directly from whole blood. Analytical validation studies demonstrated strong agreement between U-dHRM load measurement and quantitative blood culture, indicating that U-dHRM detection is highly specific to intact organisms. In a pilot clinical study of 21 whole blood samples from pediatric patients undergoing simultaneous blood culture testing, U-dHRM achieved 100% concordance when compared with blood culture and 90.5% concordance when compared with clinical adjudication. Moreover, U-dHRM identified the causative pathogen to the species level in all cases where the organism was represented in the melt curve database. These results were achieved with a 1 mL sample input and sample-to-answer time of 6 hrs. Overall, this pilot study suggests that U-dHRM may be a promising method to address the challenges of quickly and accurately diagnosing a bloodstream infection. Universal digital high resolution melt analysis for the diagnosis of bacteremia: April Aralar, Tyler Goshia, Nanda Ramchandar, Shelley M. Lawrence, Aparajita Karmakar, Ankit Sharma, Mridu Sinha, David Pride, Peiting Kuo, Khrissa Lecrone, Megan Chiu, Karen Mestan, Eniko Sajti, Michelle Vanderpool, Sarah Lazar, Melanie Crabtree, Yordanos Tesfai, Stephanie I. Fraley.

Loss of growth differentiation factor 15 exacerbates lung injury in neonatal mice.

Growth differentiation factor 15 (GDF15) is a divergent member of the transforming growth factor-ß (TGF-ß) superfamily, and its expression increases under various stress conditions, including inflammation, hyperoxia, and senescence. GDF15 expression is increased in neonatal murine bronchopulmonary dysplasia (BPD) models, and GDF15 loss exacerbates oxidative stress and decreases cellular viability in vitro. Our overall hypothesis is that the loss of GDF15 will exacerbate hyperoxic lung injury in the neonatal lung in vivo. We exposed neonatal Gdf15-/- mice and wild-type (WT) controls on a similar background to room air or hyperoxia (95% [Formula: see text]) for 5 days after birth. The mice were euthanized on postnatal day 21 (PND 21). Gdf15-/- mice had higher mortality and lower body weight than WT mice after exposure to hyperoxia. Hyperoxia exposure adversely impacted alveolarization and lung vascular development, with a greater impact in Gdf15-/- mice. Interestingly, Gdf15-/- mice showed lower macrophage count in the lungs compared with WT mice both under room air and after exposure to hyperoxia. Analysis of the lung transcriptome revealed marked divergence in gene expression and enriched biological pathways in WT and Gdf15-/- mice and differed markedly by biological sex. Notably, pathways related to macrophage activation and myeloid cell homeostasis were negatively enriched in Gdf15-/- mice. Loss of Gdf15 exacerbates mortality, lung injury, and the phenotype of the arrest of alveolarization in the developing lung with loss of female-sex advantage in Gdf15-/- mice.NEW & NOTEWORTHY We show for the first time that loss of Gdf15 exacerbates mortality, lung injury, and the phenotype of the arrest of alveolarization in the developing lung with loss of female-sex advantage in Gdf15-/- mice. We also highlight the distinct pulmonary transcriptomic response in the Gdf15-/- lung including pathways related to macrophage recruitment and activation.

Leveraging the placenta to advance neonatal care.

The impact of placental dysfunction and placental injury on the fetus and newborn infant has become a topic of growing interest in neonatal disease research. However, the use of placental pathology in directing or influencing neonatal clinical management continues to be limited for a wide range of reasons, some of which are historical and thus easily overcome today. In this review, we summarize the most recent literature linking placental function to neonatal outcomes, focusing on clinical placental pathology findings and the most common neonatal diagnoses that have been associated with placental dysfunction. We discuss how recent technological advances in neonatal and perinatal medicine may allow us to make a paradigm shift, in which valuable information provided by the placenta could be used to guide neonatal management more effectively, and to ultimately enhance neonatal care in order to improve our patient outcomes. We propose new avenues of clinical management in which the placenta could serve as a diagnostic tool toward more personalized neonatal intensive care unit management.

Remarkable sex-specific differences at single-cell resolution in neonatal hyperoxic lung injury.

Exposure to supraphysiological concentrations of oxygen (hyperoxia) predisposes to bronchopulmonary dysplasia (BPD), which is characterized by abnormal alveolarization and pulmonary vascular development, in preterm neonates. Neonatal hyperoxia exposure is used to recapitulate the phenotype of human BPD in murine models. Male sex is considered an independent predictor for the development of BPD, but the main mechanisms underlying sexually dimorphic outcomes are unknown. Our objective was to investigate sex-specific and cell-type specific transcriptional changes that drive injury in the neonatal lung exposed to hyperoxia at single-cell resolution and delineate the changes in cell-cell communication networks in the developing lung. We used single-cell RNA sequencing (scRNAseq) to generate transcriptional profiles of >35,000 cells isolated from the lungs of neonatal male and female C57BL/6 mice exposed to 95% [Formula: see text] between PND1-5 (saccular stage of lung development) or normoxia and euthanized at PND7 (alveolar stage of lung development). ScRNAseq identified 22 cell clusters with distinct populations of endothelial, epithelial, mesenchymal, and immune cells. Our data identified that the distal lung vascular endothelium (composed of aerocytes and general capillary endothelial cells) is exquisitely sensitive to hyperoxia exposure with the emergence of an intermediate capillary endothelial population with both general capillaries (gCap) and aerocytes or alveolar capillaries (aCap) markers. We also identified a myeloid-derived suppressor cell population from the lung neutrophils. Sex-specific differences were evident in all lung cell subpopulations but were striking among the lung immune cells. Finally, we identified that the specific intercellular communication networks and the ligand-receptor pairs that are impacted by neonatal hyperoxia exposure.

The Lung Microenvironment Instructs Gene Transcription in Neonatal and Adult Alveolar Macrophages.

Immaturity of alveolar macrophages (AMs) around birth contributes to the susceptibility of newborns to lung disease. However, the molecular features differentiating neonatal and mature, adult AMs are poorly understood. In this study, we identify the unique transcriptomes and enhancer landscapes of neonatal and adult AMs in mice. Although the core AM signature was similar, murine adult AMs expressed higher levels of genes involved in lipid metabolism, whereas neonatal AMs expressed a largely proinflammatory gene profile. Open enhancer regions identified by an assay for transposase-accessible chromatin followed by high-throughput sequencing (ATAC-seq) contained motifs for nuclear receptors, MITF, and STAT in adult AMs and AP-1 and NF-κB in neonatal AMs. Intranasal LPS activated a similar innate immune response in both neonatal and adult mice, with higher basal expression of inflammatory genes in neonates. The lung microenvironment drove many of the distinguishing gene expression and open chromatin characteristics of neonatal and adult AMs. Neonatal mouse AMs retained high expression of some proinflammatory genes, suggesting that the differences in neonatal AMs result from both inherent cell properties and environmental influences.

A census of the lung: CellCards from LungMAP.

The human lung plays vital roles in respiration, host defense, and basic physiology. Recent technological advancements such as single-cell RNA sequencing and genetic lineage tracing have revealed novel cell types and enriched functional properties of existing cell types in lung. The time has come to take a new census. Initiated by members of the NHLBI-funded LungMAP Consortium and aided by experts in the lung biology community, we synthesized current data into a comprehensive and practical cellular census of the lung. Identities of cell types in the normal lung are captured in individual cell cards with delineation of function, markers, developmental lineages, heterogeneity, regenerative potential, disease links, and key experimental tools. This publication will serve as the starting point of a live, up-to-date guide for lung research at https://www.lungmap.net/cell-cards/. We hope that Lung CellCards will promote the community-wide effort to establish, maintain, and restore respiratory health.

Single-cell multiomic profiling of human lungs reveals cell-type-specific and age-dynamic control of SARS-CoV2 host genes.

Respiratory failure associated with COVID-19 has placed focus on the lungs. Here, we present single-nucleus accessible chromatin profiles of 90,980 nuclei and matched single-nucleus transcriptomes of 46,500 nuclei in non-diseased lungs from donors of ~30 weeks gestation,~3 years and ~30 years. We mapped candidate cis-regulatory elements (cCREs) and linked them to putative target genes. We identified distal cCREs with age-increased activity linked to SARS-CoV-2 host entry gene TMPRSS2 in alveolar type 2 cells, which had immune regulatory signatures and harbored variants associated with respiratory traits. At the 3p21.31 COVID-19 risk locus, a candidate variant overlapped a distal cCRE linked to SLC6A20, a gene expressed in alveolar cells and with known functional association with the SARS-CoV-2 receptor ACE2. Our findings provide insight into regulatory logic underlying genes implicated in COVID-19 in individual lung cell types across age. More broadly, these datasets will facilitate interpretation of risk loci for lung diseases.

Transcriptomic and epigenetic mechanisms underlying myeloid diversity in the lung.

The lung is inhabited by resident alveolar and interstitial macrophages as well as monocytic cells that survey lung tissues. Each cell type plays distinct functional roles under homeostatic and inflammatory conditions, but mechanisms establishing their molecular identities and functional potential remain poorly understood. In the present study, systematic evaluation of transcriptomes and open chromatin of alveolar macrophages (AMs), interstitial macrophages (IMs) and lung monocytes from two mouse strains enabled inference of common and cell-specific transcriptional regulators. We provide evidence that these factors drive selection of regulatory landscapes that specify distinct phenotypes of AMs and IMs and entrain qualitatively different responses to toll-like receptor 4 signaling in vivo. These studies reveal a striking divergence in a fundamental innate immune response pathway in AMs and establish a framework for further understanding macrophage diversity in the lung.

An upstream enhancer regulates Gpihbp1 expression in a tissue-specific manner.

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1Enh/Enh). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia-even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1Enh/- mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.

An environment-dependent transcriptional network specifies human microglia identity.

Microglia play essential roles in central nervous system (CNS) homeostasis and influence diverse aspects of neuronal function. However, the transcriptional mechanisms that specify human microglia phenotypes are largely unknown. We examined the transcriptomes and epigenetic landscapes of human microglia isolated from surgically resected brain tissue ex vivo and after transition to an in vitro environment. Transfer to a tissue culture environment resulted in rapid and extensive down-regulation of microglia-specific genes that were induced in primitive mouse macrophages after migration into the fetal brain. Substantial subsets of these genes exhibited altered expression in neurodegenerative and behavioral diseases and were associated with noncoding risk variants. These findings reveal an environment-dependent transcriptional network specifying microglia-specific programs of gene expression and facilitate efforts to understand the roles of microglia in human brain diseases.

Affinity and dose of TCR engagement yield proportional enhancer and gene activity in CD4+ T cells.

Affinity and dose of T cell receptor (TCR) interaction with antigens govern the magnitude of CD4+ T cell responses, but questions remain regarding the quantitative translation of TCR engagement into downstream signals. We find that while the response of mouse CD4+ T cells to antigenic stimulation is bimodal, activated cells exhibit analog responses proportional to signal strength. Gene expression output reflects TCR signal strength, providing a signature of T cell activation. Expression changes rely on a pre-established enhancer landscape and quantitative acetylation at AP-1 binding sites. Finally, we show that graded expression of activation genes depends on ERK pathway activation, suggesting that an ERK-AP-1 axis plays an important role in translating TCR signal strength into proportional activation of enhancers and genes essential for T cell function.

Rosiglitazone preserves pulmonary vascular function in lambs with increased pulmonary blood flow.

BACKGROUND: Pulmonary vascular function is impaired with increased pulmonary blood flow (PBF). We hypothesized that a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist would mitigate this effect. METHODS: An aorta-to-pulmonary-artery shunt was placed in 11 fetal lambs. Lambs received the PPAR-γ agonist rosiglitazone (RG, 3 mg/kg/d, n = 6) or vehicle (n = 5) for 4 wk. Lung tissue from five normal 4-wk-old lambs was used for comparisons. RESULTS: At 4 wk, pulmonary artery pressure (PAP) and vascular resistance (PVR) decreased with inhaled nitric oxide (NO) in RG- and vehicle-treated shunt lambs. PAP and PVR decreased with acetylcholine (Ach) in RG-treated, but not vehicle-treated, shunt lambs. In vehicle-treated shunt lambs, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, rac1, superoxide, and 3-nitrotyrosine (3-NT) levels were increased, and Ser1177 endothelial NO synthase (eNOS) protein was decreased as compared with normal lambs. In RG-treated shunt lambs, NOx, Ser1177 eNOS protein, and eNOS activity were increased, and NADPH activity, rac1, superoxide levels, and 3-NT levels were decreased, as compared with vehicle-treated shunt lambs. PPAR-γ protein expression was lower in vehicle-treated shunt lambs than in normal and RG-treated shunt lambs. CONCLUSION: The PPAR-γ agonist RG prevents the loss of agonist-induced endothelium-dependent pulmonary vascular relaxation in lambs with increased PBF, in part, due to decreased oxidative stress and/or increased NO production.

Altered lymphatics in an ovine model of congenital heart disease with increased pulmonary blood flow.

Abnormalities of the lymphatic circulation are well recognized in patients with congenital heart defects. However, it is not known how the associated abnormal blood flow patterns, such as increased pulmonary blood flow (PBF), might affect pulmonary lymphatic function and structure. Using well-established ovine models of acute and chronic increases in PBF, we cannulated the efferent lymphatic duct of the caudal mediastinal node and collected and analyzed lymph effluent from the lungs of lambs with acutely increased PBF (n = 6), chronically increased PBF (n = 6), and age-matched normal lambs (n = 8). When normalized to PBF, we found that lymph flow was unchanged following acute increases in PBF but decreased following chronic increases in PBF. The lymph:plasma protein ratio decreased with both acute and chronic increases in PBF. Lymph bioavailable nitric oxide increased following acute increases in PBF but decreased following chronic increases in PBF. In addition, we found perturbations in the transit kinetics of contrast material through the pleural lymphatics of lambs with chronic increases in PBF. Finally, there were structural changes in the pulmonary lymphatic system in lambs with chronic increases in PBF: lymphatics from these lambs were larger and more dilated, and there were alterations in the expression of vascular endothelial growth factor-C, lymphatic vessel endothelial hyaluronan receptor-1, and Angiopoietin-2, proteins known to be important for lymphatic growth, development, and remodeling. Taken together these data suggest that chronic increases in PBF lead to both functional and structural aberrations of lung lymphatics. These findings have important therapeutic implications that warrant further study.

Nitric oxide alterations following acute ductal constriction in the fetal lamb: a role for superoxide.

Acute partial compression of the fetal ductus arteriosus (DA) results in an initial abrupt increase in pulmonary blood flow (PBF), which is followed by a significant reduction in PBF to baseline values over the ensuing 2-4 h. We have previously demonstrated that this potent vasoconstricting response is due, in part, to an endothelin-1 (ET-1)-mediated decrease in nitric oxide synthase (NOS) activity. In addition, in vitro data demonstrate that ET-1 increases superoxide levels in pulmonary arterial smooth muscle cells and that oxidative stress alters NOS activity. Therefore, the objectives of this study were to determine the potential role of superoxide in the alterations of hemodynamics and NOS activity following acute ductal constriction in the late-gestation fetal lamb. Eighteen anesthetized near-term fetal lambs were instrumented, and a lung biopsy was performed. After a 48-h recovery, acute constriction of the DA was performed by inflating a vascular occluder. Polyethylene glycol-superoxide dismutase (PEG-SOD; 1,000-1,500 units/kg, n = 7) or PEG-alone (vehicle control group, n = 5) was injected into the pulmonary artery before ductal constriction. Six animals had a sham operation. In PEG-alone-treated lambs, acute ductal constriction rapidly decreased pulmonary vascular resistance (PVR) by 88%. However, by 4 h, PVR returned to preconstriction baseline. This vasoconstriction was associated with an increase in lung superoxide levels (82%), a decrease in total NOS activity (50%), and an increase in P-eNOS-Thr495 (52%) (P < 0.05). PEG-SOD prevented the increase of superoxide after ductal constriction, attenuated the vasoconstriction, preserved NOS activity, and increased P-eNOS Ser1177 (307%, P < 0.05). Sham procedure induced no changes. These data suggest that an acute decrease in NOS activity that is mediated, in part, by increased superoxide levels, and alterations in the phosphorylation status of the endothelial NOS isoform, underlie the pulmonary vascular response to acute ductal constriction.

Tumor angiogenesis and metastasis formation are associated with individual differences in behavior of inbred Lewis rats.

There are large individual differences in cancer progression and it has been suggested that behavioral and psychological characteristics of cancer patients may contribute to the course of the disease. To get more insight in the contribution of behavioral characteristics to cancer progression, we investigated in rats, whether a stable behavioral trait characteristic is associated with NK cell activity, tumor angiogenesis, and tumor metastasis formation. Lewis rats were characterized based on locomotor activity in an open field. Rats in the upper and lower quartile were designated as high and low active rats. Low active animals had higher NK cell activity compared to their high active counterparts. In addition, we examined tumor angiogenesis by using a subcutaneous Matrigel implant containing MADB106 adenocarcinoma cells. Tumor Matrigel implants from low active animals contained significantly more hemoglobin compared to implants from high active animals, indicating a more pronounced angiogenic response in the low active animals. Finally, experimental lung metastasis formation was investigated by injecting MADB106 cells into the tail vein. Low active animals tended to develop more metastases. Moreover, low active animals developed significantly more tumors with a diameter larger than 2 mm, which is in line with higher angiogenic capacity. In conclusion, we demonstrated that individual differences in a stable behavioral trait are linked to individual differences in angiogenic capacity and tumor progression.

Individual differences in behavior of inbred Lewis rats are associated with severity of joint destruction in adjuvant-induced arthritis.

The aim of our study was to test the hypothesis that differences in behavioral characteristics are linked to severity of arthritis in association with neuro-endocrine and immune reactivity in an inbred strain of rats. Lewis rats were selected as high-active (HA) and low-active (LA) animals based on their exploratory activity in the open field. Subsequently, adjuvant-arthritis (AA) was induced in both groups. We observed no differences in the severity of inflammation as determined by paw swelling and redness. However, LA and HA animals differed in the severity of bone destruction as determined on radiographs taken on day 30 after induction of AA. LA rats had more osteoporosis, periostal new bone formation, and bone destruction than HA rats. There were no differences between HA and LA rats in corticosterone response after acute or chronic immune challenge. Splenocytes of LA rats had a lower mitogen-induced IL-10 and IFNgamma production during AA. Histological examination revealed more intense factor VIII staining in arthritic joints of LA animals, indicating more pronounced synovial angiogenesis. In addition, LA rats had higher plasma VEGF, an important angiogenic factor. Expression of RANKL, a crucial factor promoting bone resorption, was also higher in joints of LA animals. Our data demonstrate that activity in the open field, a behavioral trait, is associated with the severity of bone destruction in AA. Lower production of bone-protective cytokines and a higher rate of angiogenesis leading to more synovial proliferation may be responsible for the more severe joint destruction in LA animals.