Search

Stress correlates with reduced tumor size and burden, VEGF and CCL2 levels, and infiltrating macrophages compared to unstressed mice.

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.

Neither norepinephrine nor recombinant CCL2 have proliferative effects on B16F10 cells.

Restraint stress increases tumor CD3+/CD4+ T cell infiltration

Neither norepinephrine nor recombinant CCL2 have toxic effects on B16F10 cells.

Stress confers survival in a murine model of melanoma.

Supplementary Table for qPCR sequences

Supplementary Figure 2 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Supplementary Figure 4 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Supplementary Figure 3 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma. [Cancer Res 2007;67(19):9398–406]

Supplementary Figure 1B from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Supplementary Figure 5 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

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.

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., A post‐mortem examination of COVID‐19 infected lung tissues and an observational study were performed in order to evaluate whether expression of cell fitness marker hFwe‐Lose in patient's nasopharyngeal swabs could predict hospitalization or death from COVID‐19.

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.

COVID-19 pandemic has devastated large urban areas across the country. Most rural areas have so far been able to avoid the initial surge in cases due to the low population density. However, as the pandemic advances, rural areas are at an increased risk for the second wave of the epidemic. Rural areas are especially vulnerable due to the older population, higher comorbidities, and lack of access to healthcare. This field report discusses the experiences and issues faced by the rural Appalachian community during the ongoing COVID-19 pandemic.

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-1αfl/fl/LysMcre, or HIF-2αfl/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-1αfl/fl/LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2αfl/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.

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-Lc3a(Lc3a), and MAP1-Lc3b(Lc3b), and increased p62/STSQM1 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, Lc3a, and Lc3b 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.

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.

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 21,000xg, 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.

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.

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.

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.

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.

Autophagy is a biological process characterized by self-digestion and involves induction of autophagosome formation, leading to degradation of autophagic cargo. Aging is associated with the reduction of autophagy activity leading to neurodegenerative disorders, chronic inflammation, and susceptibility to infection; however, the underlying mechanism is unclear. DNA methylation by DNA methyltransferases reduces the expression of corresponding genes. Since macrophages are major players in inflammation and defense against infection we determined the differences in methylation of autophagy genes in macrophages derived from young and aged mice. We found that promoter regions of Atg5 and LC3B are hypermethylated in macrophages from aged mice and this is accompanied by low gene expression. Treatment of aged mice and their derived macrophages with methyltransferase inhibitor (2)-epigallocatechin-3-gallate (EGCG) or specific DNA methyltransferase 2 (DNMT2) siRNA restored the expression of Atg5 and LC3 in vivo and in vitro. Our study builds a foundation for the development of novel therapeutics aimed to improve autophagy in the elderly population and suggests a role for DNMT2 in DNA methylation activities.

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.

We describe the development and implementation of a randomized controlled trial to investigate the impact of genomic counseling on a cohort of patients with heart failure (HF) or hypertension (HTN), managed at a large academic medical center, the Ohio State University Wexner Medical Center (OSUWMC). Our study is built upon the existing Coriell Personalized Medicine Collaborative (CPMC®). OSUWMC patient participants with chronic disease (CD) receive eight actionable complex disease and one pharmacogenomic test report through the CPMC® web portal. Participants are randomized to either the in-person post-test genomic counseling—active arm, versus web-based only return of results—control arm. Study-specific surveys measure: (1) change in risk perception; (2) knowledge retention; (3) perceived personal control; (4) health behavior change; and, for the active arm (5), overall satisfaction with genomic counseling. This ongoing partnership has spurred creation of both infrastructure and procedures necessary for the implementation of genomics and genomic counseling in clinical care and clinical research. This included creation of a comprehensive informed consent document and processes for prospective return of actionable results for multiple complex diseases and pharmacogenomics (PGx) through a web portal, and integration of genomic data files and clinical decision support into an EPIC-based electronic medical record. We present this partnership, the infrastructure, genomic counseling approach, and the challenges that arose in the design and conduct of this ongoing trial to inform subsequent collaborative efforts and best genomic counseling practices.

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 21,000xg, 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.

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...

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.

Established in 2002, the Ohio State University Medical Center Program in Pharmacogenomics, lead by Wolfgang Sadee, is comprised of nearly 50 members dedicated to the discovery, investigation and translation of genetic biomarkers with the primary goal of advancing personalized healthcare. This article describes the research teams, bioinformatics infrastructure, supporting laboratories and Centers for Personalized Healthcare and for Clinical and Translational Science, current molecular genetic studies, translational and clinical pharmacogenomic studies, examples of biomarkers under development, and the future directions of the program.

To determine the association between excess weight and processes of care and outcomes for critically ill adults.Prospective cohort study.Three medical intensive care units at two hospitals.Five hundred eighty mechanically ventilated adult patients admitted between February 1, 2006 and January 31, 2008.None.After adjusting weight based on the recorded fluid balance before enrollment, 21.9% of subjects were categorized into different body mass index categories than without this adjustment. We used a competing risk analysis with events of interest considered death during hospitalization and successful liberation from mechanical ventilation. We found no statistically significant difference between body mass index categories (25 kg/m² vs. 25 to30 kg/m² vs. ≥30 kg/m²) in the competing risks analyses when the results were unadjusted or adjusted for severity of illness and comorbidities. When the analyses were adjusted for the use of continuous infusions of opioids and/or sedatives and ventilator parameters (tidal volume per ideal body weight, positive end-expiratory pressure, and airway pressure), subjects with an overweight fluid-balance-adjusted body mass index had significantly lower hazard ratios for dying while hospitalized (adjusted hazard ratio 0.68 [95% confidence interval 0.47-0.99], p=.044), and those with an obese fluid-adjusted body mass index had significantly higher hazard ratios for successful extubation (adjusted hazard ratio 1.53 [95% confidence interval 1.14-2.06], p=.005). An analysis of longer-term mortality found lower adjusted hazard ratios for subjects with overweight (adjusted hazard ratio 0.74 [95% confidence interval 0.56-0.96]) and obese (adjusted hazard ratio 0.74 [95% confidence interval 0.59-0.94]) fluid-balance-adjusted body mass indices.Processes of provided care may affect the observed association between excess weight and outcomes for critically ill adults and should be considered when making inferences about observed results. It is unknown if disparities in processes of care are due to clinically justified reasons for variation, bias against heavier patients, or other reasons.

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.

Previously, we reported that M-CSF induced monocyte survival through the activation of Akt, p38MAPK and Erk1/2 kinases. Here, we found that Src family kinases were upstream of these kinases and played a central role in regulating M-CSF-induced monocyte survival. We observed that M-CSF promoted c-Src activation in monocytes and MDMs in a time-dependent manner. Src inhibitors reduced M-CSF-mediated phosphorylation of the M-CSF receptor (M-CSFR), Akt, Erk1/2, and p38 MAPK. We also observed that Src directly phosphorylated the M-CSFR. Notably, the inhibitors blocked phosphorylation of specific tyrosine residues within the M-CSFR. We further demonstrated that the Src inhibitor, PP2, attenuated M-CSF-induced NF-κB activation and M-CSF-induced monocyte survival. These findings indicated that Src family kinases mediate monocyte survival through the regulation of receptor phosphorylation and modulation of downstream signaling events. Thus, we predict that targeting Src family kinases may have therapeutic implication in inflammatory diseases.

Cardiovascular disease (CVD) is the leading cause of death worldwide. According to the World Health Organization (WHO), an estimate of 17.3 million people died from CVDs in 2008 and by 2030, the number of deaths is estimated to reach almost 23.6 million. Despite the development of a variety of treatment options, heart failure management has failed to inhibit myocardial scar formation and replace the lost cardiomyocyte mass with new functional contractile cells. This shortage is complicated by the limited ability of the heart for self-regeneration. Accordingly, novel management approaches have been introduced into the field of cardiovascular research, leading to the evolution of gene- and cell-based therapies. Stem cell-based therapy (aka, cardiomyoplasty) is a rapidly growing alternative for regenerating the damaged myocardium and attenuating ischemic heart disease. However, the optimal cell type to achieve this goal has not been established yet, even after a decade of cardiovascular stem cell research. Mesenchymal stem cells (MSCs) in particular have been extensively investigated as a potential therapeutic approach for cardiac regeneration, due to their distinctive characteristics. In this paper, we focus on the therapeutic applications of MSCs and their transition from the experimental benchside to the clinical bedside.