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Objective: To qualify the use of patient clinical records as non-human-subject for research purpose, electronic medical record data must be de-identified so there is minimum risk to protected health information exposure. This study demonstrated a robust framework for structured data de-identification that can be applied to any relational data source that needs to be de-identified.Methods: Using a real world clinical data warehouse, a pilot implementation of limited subject areas were used to demonstrate and evaluate this new de-identification process. Query results and performances are compared between source and target system to validate data accuracy and usability.Results: The combination of hashing, pseudonyms, and session dependent randomizer provides a rigorous de-identification framework to guard against 1) source identifier exposure; 2) internal data analyst manually linking to source identifiers; and 3) identifier cross-link among different researchers or multiple query sessions by the same researcher. In addition, a query rejection option is provided to refuse queries resulting in less than preset numbers of subjects and total records to prevent users from accidental subject identification due to low volume of data. This framework does not prevent subject re-identification based on prior knowledge and sequence of events. Also, it does not deal with medical free text de-identification, although text de-identification using natural language processing can be included due its modular design.Conclusion: We demonstrated a framework resulting in HIPAA Compliant databases that can be directly queried by researchers. This technique can be augmented to facilitate inter-institutional research data sharing through existing middleware such as caGrid. [ABSTRACT FROM AUTHOR]

Over the last 15 years, investigators have identified small noncoding RNAs as regulators of gene expression. One type of noncoding RNAs are termed microRNAs (miRNAs). miRNAs are evolutionary conserved, approximately 22-nucleotide single-stranded RNAs that target genes by inducing mRNA degradation or by inhibiting translation. miRNAs are implicated in many critical cellular processes, including apoptosis, proliferation, and differentiation. Furthermore, it is estimated that miRNAs may be responsible for regulating the expression of nearly one-third of the human genome. Despite the identification of greater than 500 mature miRNAs, very little is known about their biological functions and functional targets. In the last 5 years, researchers have increasingly focused on the functional relevance and role that miRNAs play in the pathogenesis of human disease. miRNAs are known to be important in solid organ and hematological malignancies, heart disease, as potential modulators of the immune response, and organ development. It is anticipated that miRNA analysis will emerge as an important complement to proteomic and genomic studies to further our understanding of disease pathogenesis. Despite the application of genomics and proteomics to the study of human lung disease, few studies have examined miRNA expression. This perspective is not meant to be an exhaustive review of miRNA biology but will provide an overview of both miRNA biogenesis and our current understanding of the role of miRNAs in lung disease as well as a perspective on the importance of integrating this analysis as a tool for identifying and understanding the biological pathways in lung-disease pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Identifying predictors of drug use recurrence (DUR) is critical to combat the addiction epidemic. Wearable devices and phone-based applications for obtaining self-reported assessments in the patient's natural environment (e.g., ecological momentary assessment; EMA) have been used in various healthcare settings. However, the utility of combining these technologies to predict DUR in substance use disorder (SUD) has not yet been explored. This study investigates the combined use of wearable technologies and EMA as a potential mechanism for identifying physiological/behavioral biomarkers of DUR., Methods: Participants, recruited from an SUD treatment program, were provided with a commercially available wearable device that continuously monitors biometric signals (e.g., heart rate/variability [HR/HRV], sleep characteristics). They were also prompted daily to complete an EMA via phone-based application (EMA-APP) that included questionnaires regarding mood, pain, and craving., Results: Seventy-seven participants are included in this pilot study (34 participants experienced a DUR during enrollment). Wearable technologies revealed that physiological markers were significantly elevated in the week prior to DUR relative to periods of sustained abstinence (p<0.001). Results from the EMA-APP revealed that those who experienced a DUR reported greater difficulty concentrating, exposure to triggers associated with substance use, and increased isolation the day prior to DUR (p<0.001). Compliance with study procedures during the DUR week was lower than any other period of measurement (p<0.001)., Conclusions: These results suggest that data acquired via wearable technologies and the EMA-APP may serve as a method of predicting near-term DUR, thereby potentially prompting intervention before drug use occurs., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to disclose/declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Risk stratification of COVID-19 patients is essential for pandemic management. Changes in the cell fitness marker, hFwe-Lose, can precede the host immune response to infection, potentially making such a biomarker an earlier triage tool. Here, we evaluate whether hFwe-Lose gene expression can outperform conventional methods in predicting outcomes (e.g., death and hospitalization) in COVID-19 patients. We performed a post-mortem examination of infected lung tissue in deceased COVID-19 patients to determine hFwe-Lose's biological role in acute lung injury. We then performed an observational study (n = 283) to evaluate whether hFwe-Lose expression (in nasopharyngeal samples) could accurately predict hospitalization or death in COVID-19 patients. In COVID-19 patients with acute lung injury, hFwe-Lose is highly expressed in the lower respiratory tract and is co-localized to areas of cell death. In patients presenting in the early phase of COVID-19 illness, hFwe-Lose expression accurately predicts subsequent hospitalization or death with positive predictive values of 87.8-100% and a negative predictive value of 64.1-93.2%. hFwe-Lose outperforms conventional inflammatory biomarkers and patient age and comorbidities, with an area under the receiver operating characteristic curve (AUROC) 0.93-0.97 in predicting hospitalization/death. Specifically, this is significantly higher than the prognostic value of combining biomarkers (serum ferritin, D-dimer, C-reactive protein, and neutrophil-lymphocyte ratio), patient age and comorbidities (AUROC of 0.67-0.92). The cell fitness marker, hFwe-Lose, accurately predicts outcomes in COVID-19 patients. This finding demonstrates how tissue fitness pathways dictate the response to infection and disease and their utility in managing the current COVID-19 pandemic., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Our previous study found that zinc finger protein 71 (ZNF71) mRNA expression was associated with chemosensitivity and its protein expression was prognostic of non-small-cell lung cancer (NSCLC). The Krüppel associated box (KRAB) transcriptional repression domain is commonly present in human zinc finger proteins, which are linked to imprinting, silencing of repetitive elements, proliferation, apoptosis, and cancer. This study revealed that ZNF71 KRAB had a significantly higher expression than the ZNF71 KRAB -less isoform in NSCLC tumors ( n = 197) and cell lines ( n = 117). Patients with higher ZNF71 KRAB expression had a significantly worse survival outcome than patients with lower ZNF71 KRAB expression (log-rank p = 0.04; hazard ratio (HR): 1.686 [1.026, 2.771]), whereas ZNF71 overall and KRAB -less expression levels were not prognostic in the same patient cohort. ZNF71 KRAB expression was associated with epithelial-to-mesenchymal transition (EMT) in both patient tumors and cell lines. ZNF71 KRAB was overexpressed in NSCLC cell lines resistant to docetaxel and paclitaxel treatment compared to chemo-sensitive cell lines, consistent with its association with poor prognosis in patients. Therefore, ZNF71 KRAB isoform is a more effective prognostic factor than ZNF71 overall and KRAB -less expression for NSCLC. Functional analysis using CRISPR-Cas9 and RNA interference (RNAi) screening data indicated that a knockdown/knockout of ZNF71 did not significantly affect NSCLC cell proliferation in vitro.

Globally, stroke is a leading cause of death and disability. Traditional risk factors like hypertension, diabetes, and obesity do not fully account for all stroke cases. Recent infection is regarded as changes in systemic immune signaling, which can increase thrombosis formation and other stroke risk factors. We have previously shown that administration of lipopolysaccharide (LPS) 30-minutes prior to stroke increases in infarct volume. In the current study, we found that animals intermittently exposed to LPS have larger cortical infarcts when compared to saline controls. To elucidate the mechanism behind this phenomenon, several avenues were investigated. We observed significant upregulation of tumor necrosis factor-alpha (TNF-α) mRNA, especially in the ipsilateral hemisphere of both saline and LPS exposed groups compared to sham surgery animals. We also observed significant reductions in expression of genes involved in autophagy in the ipsilateral hemisphere of LPS stroke animals. In addition, we assessed DNA methylation of autophagy genes and observed a significant increase in the ipsilateral hemisphere of LPS stroke animals. Intermittent exposure to LPS increases cortical infarct volume, downregulates autophagy genes, and induces hypermethylation of the corresponding CpG islands. These data suggest that intermittent immune activation may deregulate epigenetic mechanisms and promote neuropathological outcomes after stroke.

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31 + microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Psychological stressors have been implicated in the progression of various tumor types. We investigated a role for stress in tumor immune cell chemotaxis in the B16F10 mouse model of malignant melanoma. We exposed female mice to 6-hour periods of restraint stress (RST) for 7 days, then implanted B16F10 malignant melanoma tumor cells and continued the RST paradigm for 14 additional days. We determined serum corticosterone and liver catecholamine concentrations in these mice. To evaluate the tumor microenvironment, we performed IHC and examined cytokine expression profiles using ELISA-based analysis of tumor homogenates. We found that tumors in mice subjected to RST grew significantly slower, had reduced tumor C-C motif ligand 2 (CCL2), and contained fewer F4/80-positive macrophages than tumors from unstressed mice. We observed a concomitant increase in norepinephrine among the RST mice. An in vitro assay confirmed that norepinephrine downregulates CCL2 production in both mouse and human macrophages, and that pretreatment with the pan-β-adrenergic receptor inhibitor nadolol rescues this activity. Furthermore, RST had no effect on tumor growth in transgenic CCL2-deficient mice. This study suggests that stress reduces malignant melanoma by reducing recruitment of tumor-promoting macrophages by CCL2., (©2020 American Association for Cancer Research.)

The etiology and pathogenesis of pulmonary fibrosis is poorly understood. We and others reported that M-CSF/CSF-1, M-CSF-R and downstream AKT activation plays an important role in lung fibrosis in mice models and in IPF patients. To understand potential molecular pathways used by M-CSF-R activation to direct lung fibrosis, we used a novel transgenic mouse model that expresses a constitutively-active form of AKT, myristoylated AKT (Myr-Akt), driven by the c-fms (M-CSF-R) promoter. We were particularly interested in the basal immune state of the lungs of these Myr-Akt mice to assess M-CSF-R-related priming for lung fibrosis. In support of a priming effect, macrophages isolated from the lungs of unchallenged Myr-Akt mice displayed an M2-tropism, enhanced co-expression of M-CSF-R and α -SMA, reduced autophagy reflected by reduced expression of the key autophagy genes Beclin -1, MAP1- Lc 3 a ( Lc 3 a ), and MAP1- Lc 3 b ( Lc 3 b ), and increased p 62/ STSQM 1 expression compared with littermate WT mice. Furthermore, Myr-Akt mice had more basal circulating fibrocytes than WT mice. Lastly, upon bleomycin challenge, Myr-Akt mice showed enhanced collagen deposition, increased F4/80+ and CD45+ cells, reduced autophagy genes Beclin -1, Lc 3 a , and Lc 3 b expression, and a shorter life-span than WT littermates. These data provide support that M-CSF-R/AKT activation may have a priming effect which can predispose lung tissue to pulmonary fibrosis., Competing Interests: Conflicts of Interest The authors report no conflicts of interest in this study.

Extracellular vesicles (EVs) are mRNA-containing cell fragments shed into circulation during pathophysiological events. DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) regulate gene expression by modifying DNA methylation and altering transcription. Sepsis is a systemic insult resulting in vascular dysfunction, which can lead to shock and death. We analysed plasma from ICU patients for circulating EV numbers, defined as particles isolated from 1 mL plasma at 21,000x g , and DNMTs mRNA content as prognostic markers of septic shock. Compared to plasma from critically ill patients with or without sepsis, plasma from septic shock patients contained more EVs per mL, expressed as total DNMTs mRNAs over 5 days, and more individual DNMT mRNAs at each day. A comparison of EV- DNMT1 (maintenance methylation) with EV- DNMT3A + DNMT3B ( de novo methylation) expression correlated highly with severity, and EVs from septic shock patients carried more total DNMT mRNAs and more DNMT3A + DNMT3B mRNAs than control or sepsis EVs. Total plasma EVs also correlated with sepsis severity. EV-DNMT mRNAs load, when coupled with total plasma EV number, may be a novel method to diagnose septic shock upon ICU admittance and offer opportunities to more precisely intervene with standard therapy or other targeted interventions to regulate EV release and/or specific DNMT activity., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.)

We previously demonstrated that decreased miR-17∼92 cluster expression was 1) present in lungs from human infants who died with bronchopulmonary dysplasia (BPD); 2) inversely correlated with DNA methyltransferase (DNMT) expression and promoter methylation; and 3) correlated with a subsequent diagnosis of BPD at 36 wk gestational age. We tested the hypothesis that plasma miR-17 levels would be lowest in infants who ultimately develop severe BPD. Secondly, we utilized our well-characterized murine model of severe BPD that combines perinatal inflammation with postnatal hyperoxia to test the hypothesis that alterations in lung miR-17∼92, DNMT, and promoter methylation in our model would mirror our findings in tissues from premature human infants. Plasma was obtained during the first 5 days of life from premature infants born ≤32 wk gestation. Lung tissues were harvested from mice exposed to maternal inflammation and neonatal hyperoxia for 14 days after birth. miR-17∼92 cluster expression and DNA methyltransferase expression were measured by qRT-PCR, and promoter methylation was assessed by Methyl-Profiler assay. Plasma miR-17 levels are significantly lower in the first week of life in human infants who develop severe BPD compared with mild or moderate BPD. Data from our severe BPD murine model reveal that lung miR-17∼92 cluster expression is significantly attenuated, and levels inversely correlated with DNMT expression and miR-17∼92 cluster promoter methylation. Collectively, our data support a plausible role for epigenetically altered miR-17∼92 cluster in the pathogenesis of severe BPD., (Copyright © 2016 the American Physiological Society.)

Phosphatase and tensin homolog (Pten) is a negative regulator of cell proliferation and growth. Using a Cre-recombinase approach with Lox sequences flanking the fibroblast-specific protein 1 (Fsp1 aka S100A4; a mesenchymal marker), we probed sites of expression using a β-galactosidase Rosa26(LoxP) reporter allele; the transgene driving deletion of Pten (exons 4-5) was found throughout the brain parenchyma and pituitary, suggesting that deletion of Pten in Fsp1-positive cells may influence behavior. Because CNS-specific deletion of Pten influences social and anxiety-like behaviors and S100A4 is expressed in astrocytes, we predicted that loss of Pten in Fsp1-expressing cells would result in deficits in social interaction and increased anxiety. We further predicted that environmental enrichment would compensate for genetic deficits in these behaviors. We conducted a battery of behavioral assays on Fsp1-Cre;Pten(LoxP/LoxP) male and female homozygous knockouts (Pten(-/-)) and compared their behavior to Pten(LoxP/LoxP) (Pten(+/+)) conspecifics. Despite extensive physical differences (including reduced hippocampal size) and deficits in sensorimotor function, Pten(-/-) mice behaved remarkably similar to control mice on nearly all behavioral tasks. These results suggest that the social and anxiety-like phenotypes observed in CNS-specific Pten(-/-) mice may depend on neuronal Pten, as lack of Pten in Fsp1-expressing cells of the CNS had little effect on these behaviors., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mobile sensor data-to-knowledge (MD2K) was chosen as one of 11 Big Data Centers of Excellence by the National Institutes of Health, as part of its Big Data-to-Knowledge initiative. MD2K is developing innovative tools to streamline the collection, integration, management, visualization, analysis, and interpretation of health data generated by mobile and wearable sensors. The goal of the big data solutions being developed by MD2K is to reliably quantify physical, biological, behavioral, social, and environmental factors that contribute to health and disease risk. The research conducted by MD2K is targeted at improving health through early detection of adverse health events and by facilitating prevention. MD2K will make its tools, software, and training materials widely available and will also organize workshops and seminars to encourage their use by researchers and clinicians., (© The Author 2015. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Rationale: Bronchopulmonary dysplasia remains a significant cause of neonatal morbidity; however, the identification of novel targets to predict or prevent the development of bronchopulmonary dysplasia remains elusive. Proper microRNA (miR)-17∼92 cluster is necessary for normal lung development, and alterations in expression are reported in other pulmonary diseases. The overall hypothesis for our work is that altered miR-17∼92 cluster expression contributes to the molecular pathogenesis of bronchopulmonary dysplasia., Objectives: The current studies tested the hypothesis that alterations in miR-17∼92 cluster and DNA methyltransferase expression are present in bronchopulmonary dysplasia., Methods: miR-17∼92 cluster expression, promoter methylation, and DNA methyltransferase expression were determined in autopsy lung samples obtained from premature infants who died with bronchopulmonary dysplasia, or from term/near-term infants who died from nonrespiratory causes. Expression of miR-17∼92 cluster members miR-17 and -19b was measured in plasma samples collected in the first week of life from a separate cohort of preterm infants at a second institution in whom bronchopulmonary dysplasia was diagnosed subsequently., Measurements and Main Results: Autopsy tissue data indicated that miR-17∼92 expression is significantly lower in bronchopulmonary dysplasia lungs and is inversely correlated with promoter methylation and DNA methyltransferase expression when compared with that of control subjects without bronchopulmonary dysplasia. Plasma sample analyses indicated that miR-17 and -19b expression was decreased in infants who subsequently developed bronchopulmonary dysplasia., Conclusions: Our data are the first to demonstrate altered expression of the miR-17∼92 cluster in bronchopulmonary dysplasia. The consistency between our autopsy and plasma findings further support our working hypothesis that the miR-17∼92 cluster contributes to the molecular pathogenesis of bronchopulmonary dysplasia.

Background: We have previously shown that critically ill children transfused with red blood cells (RBCs) of longer storage durations have more suppressed monocyte function after transfusion compared to children transfused with fresher RBCs and that older stored RBCs directly suppress monocyte function in vitro, through unknown mechanisms. We hypothesized that RBC-derived microvesicles (MVs) were responsible for monocyte suppression., Study Design and Methods: To determine the role of stored RBC unit-derived MVs, we cocultured monocytes with supernatants, isolated MVs, or supernatants that had been depleted of MVs from prestorage leukoreduced RBCs that had been stored for either 7 or 30 days. Isolated MVs were characterized by electron microscopy and flow cytometry. Monocyte function after coculture experiments was measured by cytokine production after stimulation with lipopolysaccharide (LPS)., Results: Monocyte function was suppressed after exposure to supernatants from 30-day RBC units compared to monocytes cultured in medium alone (LPS-induced tumor necrosis factor-α production, 17,611 ± 3,426 vs. 37,486 ± 5,598 pg/mL; p = 0.02). Monocyte function was not suppressed after exposure to MV fractions. RBC supernatants that had been depleted of MVs remained immunosuppressive. Treating RBC supernatants with heat followed by RNase (to degrade protein-bound RNA) prevented RBC supernatant-induced monocyte suppression., Conclusion: Our findings implicate soluble mediators of stored RBC-induced monocyte suppression outside of MV fractions and suggest that extracellular protein-bound RNAs (such as microRNA) may play a role in transfusion-related immunomodulation., (© 2015 AABB.)

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a progressive decline in lung function which can be attributed to excessive scarring, inflammation and airway remodelling. Mannose-6-phosphate (M6P) is a strong inhibitor of fibrosis and its administration has been associated with beneficial effects in tendon repair surgery as well as nerve repair after injury. Given this promising therapeutic approach we developed an improved analogue of M6P, namely PXS64, and explored its anti-fibrotic effects in vitro. Normal human lung fibroblasts (NHLF) and human lung fibroblast 19 cells (HF19) were exposed to active recombinant human TGF-β1 to induce increases in fibrotic markers. rhTGF-β1 increased constitutive protein levels of fibronectin and collagen in the NHLF cells, whereas HF19 cells showed increased levels of fibronectin, collagen as well as αSMA (alpha smooth muscle actin). PXS64 demonstrated a robust inhibitory effect on all proteins analysed. IPF patient fibroblasts treated with PXS64 presented an improved phenotype in terms of their morphological appearance, as well as a decrease in fibrotic markers (collagen, CTGF, TGF-β3, tenascin C, αSMA and THBS1). To explore the cell signalling pathways involved in the anti-fibrotic effects of PXS64, proteomics analysis with iTRAQ labelling was performed and the data demonstrated a specific antagonistic effect on the TGF-β1 pathway. This study shows that PXS64 effectively inhibits the production of extracellular matrix, as well as myofibroblast differentiation during fibrosis. These results suggest that PXS64 influences tissue remodelling by inhibiting TGF-β1 signalling in NHLF and HF19 cell lines, as well as in IPF patient fibroblasts. Thus PXS64 is a potential candidate for preclinical application in pulmonary fibrosis., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

: Cardiovascular disease is the number 1 cause of morbidity and mortality in the United States. The most common manifestation of cardiovascular disease is myocardial infarction (MI), which can ultimately lead to congestive heart failure. Cell therapy (cardiomyoplasty) is a new potential therapeutic treatment alternative for the damaged heart. Recent preclinical and clinical studies have shown that mesenchymal stem cells (MSCs) are a promising cell type for cardiomyoplasty applications. However, a major limitation is the poor survival rate of transplanted stem cells in the infarcted heart. miR-133a is an abundantly expressed microRNA (miRNA) in the cardiac muscle and is downregulated in patients with MI. We hypothesized that reprogramming MSCs using miRNA mimics (double-stranded oligonucleotides) will improve survival of stem cells in the damaged heart. MSCs were transfected with miR-133a mimic and antagomirs, and the levels of miR-133a were measured by quantitative real-time polymerase chain reaction. Rat hearts were subjected to MI and MSCs transfected with miR-133a mimic or antagomir were implanted in the ischemic hearts. Four weeks after MI, cardiac function, cardiac fibrosis, miR-133a levels, and apoptosis-related genes (Apaf-1, Caspase-9, and Caspase-3) were measured in the heart. We found that transfecting MSCs with miR-133a mimic improves survival of MSCs as determined by the MTT assay. Similarly, transplantation of miR-133a mimic transfected MSCs in rat hearts subjected to MI led to a significant increase in cell engraftment, cardiac function, and decreased fibrosis when compared with MSCs only or MI groups. At the molecular level, quantitative real-time polymerase chain reaction data demonstrated a significant decrease in expression of the proapoptotic genes; Apaf-1, caspase-9, and caspase-3 in the miR-133a mimic transplanted group. Furthermore, luciferase reporter assay confirmed that miR-133a is a direct target for Apaf-1. Overall, bioengineering of stem cells through miRNAs manipulation could potentially improve the therapeutic outcome of patients undergoing stem cell transplantation for MI.

The four variables, hypoxia, acidity, high glutathione (GSH) concentration and fast reducing rate (redox) are distinct and varied characteristics of solid tumors compared to normal tissue. These parameters are among the most significant factors underlying the metabolism and physiology of solid tumors, regardless of their type or origin. Low oxygen tension contributes to both inhibition of cancer cell proliferation and therapeutic resistance of tumors; low extracellular pH, the reverse of normal cells, mainly enhances tumor invasion; and dysregulated GSH and redox potential within cancer cells favor their proliferation. In fact, cancer cells under these microenvironmental conditions appreciably alter tumor response to cytotoxic anti-cancer treatments. Recent experiments measured the in vivo longitudinal data of these four parameters with tumor development and the corresponding presence and absence of tumor macrophage HIF-1α or HIF-2α in a mouse model of breast cancer. In the current paper, we present a mathematical model-based system of (ordinary and partial) differential equations to monitor tumor growth and susceptibility to standard chemotherapy with oxygen level, pH, and intracellular GSH concentration. We first show that our model simulations agree with the corresponding experiments, and then we use our model to suggest treatments of tumors by altering these four parameters in tumor microenvironment. For example, the model qualitatively predicts that GSH depletion can raise the level of reactive oxygen species (ROS) above a toxic threshold and result in inhibition of tumor growth.

The ETS-family transcription factors Ets1 and Ets2 are evolutionarily conserved effectors of the RAS/ERK signaling pathway, but their function in Ras cellular transformation and biology remains unclear. Taking advantage of Ets1 and Ets2 mouse models to generate Ets1/Ets2 double knockout mouse embryonic fibroblasts, we demonstrate that deletion of both Ets1 and Ets2 was necessary to inhibit HrasG12V induced transformation both in vitro and in vivo. HrasG12V expression in mouse embryonic fibroblasts increased ETS1 and ETS2 expression and binding to cis-regulatory elements on the c-Myc proximal promoter, and consequently induced a robust increase in MYC expression. The expression of the oncogenic microRNA 17-92 cluster was increased in HrasG12V transformed cells, but was significantly reduced when ETS1 and ETS2 were absent. MYC and ETS1 or ETS2 collaborated to increase expression of the oncogenic microRNA 17-92 cluster in HrasG12V transformed cells. Enforced expression of exogenous MYC or microRNA 17-92 rescued HrasG12V transformation in Ets1/Ets2-null cells, revealing a direct function for MYC and microRNA 17-92 in ETS1/ETS2-dependent HrasG12V transformation.

Reports demonstrate the role of M-CSF (CSF1) in tumor progression in mouse models as well as the prognostic value of macrophage numbers in breast cancer patients. Recently, a subset of CD14+ monocytes expressing the Tie2 receptor, once thought to be predominantly expressed on endothelial cells, has been characterized. We hypothesized that increased levels of CSF1 in breast tumors can regulate differentiation of Tie2- monocytes to a Tie2+ phenotype. We treated CD14+ human monocytes with CSF1 and found a significant increase in CD14+/Tie2+ positivity. To understand if CSF1-induced Tie2 expression on these cells improved their migratory ability, we pre-treated CD14+ monocytes with CSF1 and used Boyden chemotaxis chambers to observe enhanced response to angiopoietin-2 (ANG2), the chemotactic ligand for the Tie2 receptor. We found that CSF1 pre-treatment significantly augmented chemotaxis and that Tie2 receptor upregulation was responsible as siRNA targeting Tie2 receptor abrogated this effect. To understand any augmented angiogenic effect produced by treating these cells with CSF1, we cultured human umbilical vein endothelial cells (HUVECs) with conditioned supernatants from CSF1-pre-treated CD14+ monocytes for a tube formation assay. While supernatants from CSF1-pre-treated TEMs increased HUVEC branching, a neutralizing antibody against the CSF1R abrogated this activity, as did siRNA against the Tie2 receptor. To test our hypothesis in vivo, we treated PyMT tumor-bearing mice with CSF1 and observed an expansion in the TEM population relative to total F4/80+ cells, which resulted in increased angiogenesis. Investigation into the mechanism of Tie2 receptor upregulation on CD14+ monocytes by CSF1 revealed a synergistic contribution from the PI3 kinase and HIF pathways as the PI3 kinase inhibitor LY294002, as well as HIF-1α-deficient macrophages differentiated from the bone marrow of HIF-1αfl/fl/LysMcre mice, diminished CSF1-stimulated Tie2 receptor expression.

MicroRNAs (miRNAs) have emerged as important regulators in the post-transcriptional control of gene expression. The discovery of their presence not only in tissues but also in extratissular fluids, including blood, urine and cerebro-spinal fluid, together with their changes in expression in various pathological conditions, has implicated these extracellular miRNAs as informative biomarkers of disease. However, exploiting miRNAs in this capacity requires methodological rigour. Here, we report several key procedural aspects of miRNA isolation from plasma and serum, as exemplified by research in cardiovascular and pulmonary diseases. We also highlight the advantages and disadvantages of various profiling methods to determine the expression levels of plasma- and serum-derived miRNAs. Attention to such methodological details is critical, as circulating miRNAs become diagnostic tools for various human diseases., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The median survival of patients with idiopathic pulmonary fibrosis (IPF) continues to be approximately 3 years from the time of diagnosis, underscoring the lack of effective medical therapies for this disease. In the United States alone, approximately 40,000 patients die of this disease annually. In November 2012, the NHLBI held a workshop aimed at coordinating research efforts and accelerating the development of IPF therapies. Basic, translational, and clinical researchers gathered with representatives from the NHLBI, patient advocacy groups, pharmaceutical companies, and the U.S. Food and Drug Administration to review the current state of IPF research and identify priority areas, opportunities for collaborations, and directions for future research. The workshop was organized into groups that were tasked with assessing and making recommendations to promote progress in one of the following six critical areas of research: (1) biology of alveolar epithelial injury and aberrant repair; (2) role of extracellular matrix; (3) preclinical modeling; (4) role of inflammation and immunity; (5) genetic, epigenetic, and environmental determinants; (6) translation of discoveries into diagnostics and therapeutics. The workshop recommendations provide a basis for directing future research and strategic planning by scientific, professional, and patient communities and the NHLBI.

The mechanisms of poultry particulate matter (PM)-induced agricultural respiratory disorders are not thoroughly understood. Hence, it is hypothesized in this article that poultry PM induces the release of interleukin-8 (IL-8) by lung epithelial cells that is regulated upstream by the concerted action of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK). To test this hypothesis, the widely used cultured human lung epithelial cells (A549) were chosen as the model system. Poultry PM caused a significant activation of PLA2 in A549 cells, which was attenuated by AACOCF3 (cPLA2 inhibitor) and PD98059 (ERK-1/2 upstream inhibitor). Poultry PM induced upstream ERK-1/2 phosphorylation and downstream cPLA2 serine phosphorylation, in a concerted fashion, in cells with enhanced association of ERK-1/2 and cPLA2. The poultry PM-induced cPLA2 serine phosphorylation and IL-8 release were attenuated by AACOCF3, PD98059, and by transfection with dominant-negative ERK-1/2 DNA in cells. The poultry PM-induced IL-8 release by the bone marrow-derived macrophages of cPLA2 knockout mice was significantly lower. For the first time, this study demonstrated that the poultry PM-induced IL-8 secretion by human lung epithelial cells was regulated by cPLA2 activation through ERK-mediated serine phosphorylation, suggesting a mechanism of airway inflammation among poultry farm workers.

In response to elevated glucocorticoid levels, erythroid progenitors rapidly expand to produce large numbers of young erythrocytes. Previous work demonstrates hematopoietic changes in rodents exposed to various physical and psychological stressors, however, the effects of chronic psychological stress on erythropoiesis has not be delineated. We employed laboratory, clinical and genomic analyses of a murine model of chronic restraint stress (RST) to examine the influence of psychological stress on erythropoiesis. Mice exposed to RST demonstrated markers of early erythroid expansion involving the glucocorticoid receptor. In addition, these RST-exposed mice had increased numbers of circulating reticulocytes and increased erythropoiesis in primary and secondary erythroid tissues. Mice also showed increases in erythroid progenitor populations and elevated expression of the erythroid transcription factor KLF1 in these cells. Together this work reports some of the first evidence of psychological stress affecting erythroid homeostasis through glucocorticoid stimulation.

The purpose of this study was to elucidate the mechanism of the airborne poultry dust (particulate matter, PM)-induced respiratory tract inflammation, a common symptom in agricultural respiratory diseases. The study was based on the hypothesis that poultry PM would induce the release of inflammatory cytokine interleukin-8 (IL-8) by respiratory epithelial cells under the upstream regulation by cytosolic phospholipase A2 (cPLA2) activation and subsequent formation of cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites (eicosanoids). Human lung epithelial cells (A549) in culture were treated with the poultry PM (0.1-1.0 mg) for different lengths of time, following which PLA2 activity, release of eicosanoids and secretion of IL-8 in cells were determined. Poultry PM (1.0 mg/ml) caused a significant activation of PLA2 in a time-dependent manner (15-60 min), which was significantly attenuated by the calcium-chelating agents, cPLA2-specific inhibitor (AACOCF3) and antioxidant (vitamin C) in A549 cells. Poultry PM also significantly induced the release of COX- and LOX-catalyzed eicosanoids (prostaglandins, thromboxane A2 and leukotrienes B4 and C4) and upstream activation of AA LOX in the cells. Poultry PM also significantly induced release of IL-8 by the cells in a dose- and time-dependent manner, which was significantly attenuated by the calcium chelating agents, antioxidants and COX- and LOX-specific inhibitors. The current study for the first time revealed that the poultry PM-induced IL-8 release from the respiratory epithelial cells was regulated upstream by reactive oxygen species, cPLA2-, COX- and LOX-derived eicosanoid lipid signal mediators.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a disease of progressive lung fibrosis with a high mortality rate. In organ repair and remodeling, epigenetic events are important. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and can target epigenetic molecules important in DNA methylation. The miR-17~92 miRNA cluster is critical for lung development and lung epithelial cell homeostasis and is predicted to target fibrotic genes and DNA methyltransferase (DNMT)-1 expression., Objectives: We investigated the miR-17~92 cluster expression and its role in regulating DNA methylation events in IPF lung tissue., Methods: Expression and DNA methylation patterns of miR-17~92 were determined in human IPF lung tissue and fibroblasts and fibrotic mouse lung tissue. The relationship between the miR-17~92 cluster and DNMT-1 expression was examined in vitro. Using a murine model of pulmonary fibrosis, we examined the therapeutic potential of the demethylating agent, 5'-aza-2'-deoxycytidine., Measurements and Main Results: Compared with control samples, miR-17~92 expression was reduced in lung biopsies and lung fibroblasts from patients with IPF, whereas DNMT-1 expression and methylation of the miR-17~92 promoter was increased. Several miRNAs from the miR-17~92 cluster targeted DNMT-1 expression resulting in a negative feedback loop. Similarly, miR-17~92 expression was reduced in the lungs of bleomycin-treated mice. Treatment with 5'-aza-2'-deoxycytidine in a murine bleomycin-induced pulmonary fibrosis model reduced fibrotic gene and DNMT-1 expression, enhanced miR-17~92 cluster expression, and attenuated pulmonary fibrosis., Conclusions: This study provides insight into the pathobiology of IPF and identifies a novel epigenetic feedback loop between miR-17~92 and DNMT-1 in lung fibrosis.

Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation.

Altered inflammatory cytokine profiles are often observed in individuals suffering from major depression. Recent clinical work reports on elevated IL-6 and decreased IL-10 in depression. Elevated IL-6 has served as a consistent biomarker of depression and IL-10 is proposed to influence depressive behavior through its ability to counterbalance pro-inflammatory cytokine expression. Clinical and animal studies suggest a role for IL-10 in modifying depressive behavior. Murine restraint stress (RST) is regularly employed in the study of behavioral and biological symptoms associated with depressive disorders. While responses to acute RST exposure have been widely characterized, few studies have examined the ongoing and longitudinal effects of extended RST and fewer still have examined the lasting impact during the post-stress period. Consistent with clinical data, we report that a protocol of prolonged murine RST produced altered cytokine profiles similar to those observed in major depressive disorder. Parallel to these changes in circulating cytokines, IL-10 mRNA expression was diminished in the cortex and hippocampus throughout the stress period and following cessation of RST. Moreover, chronic RST promoted depressive-like behavior throughout the 28-day stress period and these depressive-like complications were maintained weeks after cessation of RST. Because of the correlation between IL-10 suppression and depressive behavior and because many successful antidepressant therapies yield increases in IL-10, we examined the effects of IL-10 treatment on RST-induced behavioral changes. Behavioral deficits induced by RST were reversed by exogenous administration of recombinant IL-10. This work provides one of the first reports describing the biological and behavioral impact following prolonged RST and, taken together, this study provides details on the correlation between responses to chronic RST and those seen in depressive disorders.

Under hypoxia, tumor cells, and tumor-associated macrophages produce VEGF (vascular endothelial growth factor), a signaling molecule that induces angiogenesis. The same macrophages, when treated with GM-CSF (granulocyte/macrophage colony-stimulating factor), produce sVEGFR-1 (soluble VEGF receptor-1), a soluble protein that binds with VEGF and inactivates its function. The production of VEGF by macrophages is regulated by HIF-1α (hypoxia inducible factor-1α), and the production of sVEGFR-1 is mediated by HIF-2α. Recent experiments measured the effect of inhibiting tumor growth by GM-CSF treatment in mice with HIF-1α-deficient or HIF-2α-deficient macrophages. In the present paper, we represent these experiments by a mathematical model based on a system of partial differential equations. We show that the model simulations agree with the above experiments. The model can then be used to suggest strategies for inhibiting tumor growth. For example, the model qualitatively predicts the extent to which GM-CSF treatment in combination with a small molecule inhibitor that stabilizes HIF-2α will reduce tumor volume and angiogenesis.

Macrophage secretion of vascular endothelial growth factor (VEGF) in response to hypoxia contributes to tumor growth and angiogenesis. In addition to VEGF, hypoxic macrophages stimulated with GM-CSF secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using mice with a monocyte/macrophage-selective deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α, we recently demonstrated that the antitumor response to GM-CSF was dependent on HIF-2α-driven sVEGFR-1 production by tumor-associated macrophages, whereas HIF-1α specifically regulated VEGF production. We therefore hypothesized that chemical stabilization of HIF-2α using an inhibitor of prolyl hydroxylase domain 3 (an upstream inhibitor of HIF-2α activation) would increase sVEGFR-1 production from GM-CSF-stimulated macrophages. Treatment of macrophages with the prolyl hydroxylase domain 3 inhibitor AKB-6899 stabilized HIF-2α and increased sVEGFR-1 production from GM-CSF-treated macrophages, with no effect on HIF-1α accumulation or VEGF production. Treatment of B16F10 melanoma-bearing mice with GM-CSF and AKB-6899 significantly reduced tumor growth compared with either drug alone. Increased levels of sVEGFR-1 mRNA, but not VEGF mRNA, were detected within the tumors of GM-CSF- and AKB-6899-treated mice, correlating with decreased tumor vascularity. Finally, the antitumor and antiangiogenic effects of AKB-6899 were abrogated when mice were simultaneously treated with a sVEGFR-1 neutralizing Ab. These results demonstrate that AKB-6899 decreases tumor growth and angiogenesis in response to GM-CSF by increasing sVEGFR-1 production from tumor-associated macrophages. Specific activation of HIF-2α can therefore decrease tumor growth and angiogenesis.

The field of pharmacogenomics aims to incorporate individual patient genomic information into treatment selection. This is a rapidly evolving field with significant clinical promise. Implementation into clinical practice has several challenges that must be overcome. Genomics-based information encompasses large databases and requires expert knowledge for interpretation. Existing research suggests there are already several areas where pharmacogenomics-based decision-making is ripe for adoption into clinical practice. Impediments exist that must be overcome prior to large-scale implementation of existing pharmacogenomics-based therapies. There are several institutions and corporations at the forefront of implementation that are leading the development; however, larger systems-based approaches will be necessary. This article will discuss some of the present successes and future challenges that are necessary to overcome in order to implement a more patient-centered healthcare system.

M-CSF is overexpressed in breast cancer and is known to stimulate macrophages to produce VEGF resulting in angiogenesis. It has recently been shown that the growth factor GM-CSF injected into murine breast tumors slowed tumor growth by secreting soluble VEGF receptor-1 (sVEGFR-1) that binds and inactivates VEGF. This study presents a mathematical model that includes all the components above, as well as MCP-1, tumor cells, and oxygen. The model simulations are representative of the in vivo data through predictions of tumor growth using different protocol strategies for GM-CSF for the purpose of predicting higher degrees of treatment success. For example, our model predicts that once a week dosing of GM-CSF would be less effective than daily, twice a week, or three times a week treatment because of the presence of essential factors required for the anti-tumor effect of GM-CSF., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lung vascular alterations and pulmonary hypertension associated with oxidative stress have been reported to be involved in idiopathic lung fibrosis (ILF). Therefore, here, we hypothesize that the widely used lung fibrosis inducer, bleomycin, would cause cytoskeletal rearrangement through thiol-redox alterations in the cultured lung vascular endothelial cell (EC) monolayers. We exposed the monolayers of primary bovine pulmonary artery ECs to bleomycin (10 µg) and studied the cytotoxicity, cytoskeletal rearrangements, and the macromolecule (fluorescein isothiocyanate-dextran, 70,000 mol. wt.) paracellular transport in the absence and presence of two thiol-redox protectants, the classic water-soluble N-acetyl-L-cysteine (NAC) and the novel hydrophobic N,N'-bis-2-mercaptoethyl isophthalamide (NBMI). Our results revealed that bleomycin induced cytotoxicity (lactate dehydrogenase leak), morphological alterations (rounding of cells and filipodia formation), and cytoskeletal rearrangement (actin stress fiber formation and alterations of tight junction proteins, ZO-1 and occludin) in a dose-dependent fashion. Furthermore, our study demonstrated the formation of reactive oxygen species, loss of thiols (glutathione, GSH), EC barrier dysfunction (decrease of transendothelial electrical resistance), and enhanced paracellular transport (leak) of macromolecules. The observed bleomycin-induced EC alterations were attenuated by both NAC and NBMI, revealing that the novel hydrophobic thiol-protectant, NBMI, was more effective at µM concentrations as compared to the water-soluble NAC that was effective at mM concentrations in offering protection against the bleomycin-induced EC alterations. Overall, the results of the current study suggested the central role of thiol-redox in vascular EC dysfunction associated with ILF.

Approach for in vivo real-time assessment of tumor tissue extracellular pH (pH(e)), redox, and intracellular glutathione based on L-band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony-stimulating factor. It was observed that tumor pH(e) is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony-stimulating factor decreased the value of pH(e) by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony-stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pH(e) mapping was performed using recently proposed variable frequency proton-electron double-resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pH(e) and a difference of about 0.4 pH units between average pH(e) values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pH(e), extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors., (Copyright © 2011 Wiley Periodicals, Inc.)

We have characterized the immune system involvement in the disease processes of idiopathic pulmonary fibrosis in novel ways. To do so, we analyzed lung tissue from 21 cases of idiopathic pulmonary fibrosis and 21 (non-fibrotic, non-cancerous) controls for immune cell and inflammation-related markers. The immunohistochemical analysis of the tissue was grouped by patterns of severity in disease pathology. There were significantly greater numbers of CD68(+) and CD80(+) cells and significantly fewer CD3(+), CD4(+), and CD45RO(+) cells in areas of relatively (histologically) normal lung in biopsy samples from idiopathic pulmonary fibrosis patients compared with controls. In zones of active disease, characterized by epithelial cell regeneration and fibrosis, there were significantly more cells expressing CD4, CD8, CD20, CD68, CD80, chemokine receptor 6 (CCR6), S100, IL-17, tumor necrosis factor-α, and retinoic acid-related orphan receptors compared with histologically normal lung areas from idiopathic pulmonary fibrosis patients. Inflammation was implicated in these active regions by the cells that expressed retinoid orphan receptor-α, -β, and -γ, CCR6, and IL-17. The regenerating epithelial cells predominantly expressed these pro-inflammatory molecules, as evidenced by co-expression analyses with epithelial cytokeratins. Macrophages in pseudo-alveoli and CD3(+) T cells in the fibrotic interstitium also expressed IL-17. Co-expression of IL-17 with retinoid orphan receptors and epithelial cytoskeletal proteins, CD68, and CD3 in epithelial cells, macrophages, and T-cells, respectively, confirmed the production of IL-17 by these cell types. There was little staining for forkhead box p3, CD56, or CD34 in any idiopathic pulmonary fibrosis lung regions. The fibrotic regions had fewer immune cells overall. In summary, our study shows participation of innate and adaptive mononuclear cells in active-disease regions of idiopathic pulmonary fibrosis lung, where the regenerating epithelial cells appear to propagate inflammation. The regenerative mechanisms become skewed to ultimately result in lethal, fibrotic restriction of lung function.