Identification and Modulation of Microenvironment Is Crucial for Effective Mesenchymal Stromal Cell Therapy in Acute Lung Injury.

Rationale: There are controversial reports on applications of mesenchymal stromal cells (MSCs) in patients with acute respiratory distress syndrome (ARDS). Objectives: We hypothesized that lung microenvironment was the main determinant of beneficial versus detrimental effects of MSCs during ARDS. Methods: Lung proteome was profiled in three models of injury induced by acid instillation and/or mechanical ventilation in mice. Human gene of glutathione peroxidase-1 was delivered before MSC administration; or MSCs carrying human gene of IL-10 or hepatocyte growth factor were administered after lung injury. An inhibitory cocktail against IL-6, fibronectin, and oxidative stress was used in in vitro studies using human small airway epithelial cells and human MSCs after exposure to plasma of patients with ARDS. Measurements and Main Results: Distinct proteomic profiles were observed in three lung injury models. Administration of MSCs protected lung from ventilator-induced injury, whereas it worsened acid-primed lung injuries associated with fibrotic development in lung environment that had high levels of IL-6 and fibronectin along with low antioxidant capacity. Correction of microenvironment with glutathione peroxidase-1, or treatment with MSCs carrying human gene of IL-10 or hepatocyte growth factor after acid-primed injury, reversed the detrimental effects of native MSCs. Proteomic profiles obtained in the mouse models were also similarly observed in human ARDS. Treatment with the inhibitory cocktail in samples of patients with ARDS retained protective effects of MSCs in small airway epithelial cells. Conclusions: MSCs can be beneficial or detrimental depending on microenvironment at the time of administration. Identification of potential beneficiaries seems to be crucial to guide MSC therapy in ARDS.

T regulatory cells and dendritic cells protect against transfusion-related acute lung injury via IL-10.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities and is characterized by acute respiratory distress following blood transfusion. Donor antibodies are frequently involved; however, the pathogenesis and protective mechanisms in the recipient are poorly understood, and specific therapies are lacking. Using newly developed murine TRALI models based on injection of anti-major histocompatibility complex class I antibodies, we found CD4+CD25+FoxP3+ T regulatory cells (Tregs) and CD11c+ dendritic cells (DCs) to be critical effectors that protect against TRALI. Treg or DC depletion in vivo resulted in aggravated antibody-mediated acute lung injury within 90 minutes with 60% mortality upon DC depletion. In addition, resistance to antibody-mediated TRALI was associated with increased interleukin-10 (IL-10) levels, and IL-10 levels were found to be decreased in mice suffering from TRALI. Importantly, IL-10 injection completely prevented and rescued the development of TRALI in mice and may prove to be a promising new therapeutic approach for alleviating lung injury in this serious complication of transfusion.

Distinctive Roles and Mechanisms of Human Neutrophil Peptides in Experimental Sepsis and Acute Respiratory Distress Syndrome.

OBJECTIVES: To examine the effects and mechanisms of human neutrophil peptides in systemic infection and noninfectious inflammatory lung injury. DESIGN: Prospective experimental study. SETTING: University hospital-based research laboratory. SUBJECTS: In vitro human cells and in vivo mouse models. INTERVENTIONS: Wild-type (Friend virus B-type) and conditional leukocyte human neutrophil peptides transgenic mice were subjected to either sepsis induced by cecal ligation and puncture or acute lung injury by intratracheal instillation of hydrochloric acid followed by mechanical ventilation. Using human neutrophil peptides as bait, the basal cell adhesion molecule (CD239) and the purinergic P2Y purinoceptor 6 receptor were identified as the putative human neutrophil peptides receptor complex in human lung epithelial cells. MEASUREMENTS AND MAIN RESULTS: In the cecal ligation and puncture sepsis model, Friend virus B-type mice exhibited higher systemic bacterial load, cytokine production, and lung injury than human neutrophil peptides transgenic mice. Conversely, an increased lung cytokine production was seen in Friend virus B-type mice, which was further enhanced in human neutrophil peptides transgenic mice in response to two-hit lung injury induced by hydrochloric acid and mechanical ventilation. The human neutrophil peptides-mediated inflammatory response was mediated through the basal cell adhesion molecule-P2Y purinoceptor 6 receptor signal pathway in human lung epithelial cells. CONCLUSIONS: Human neutrophil peptides are critical in host defense against infectious sepsis by their cationic antimicrobial properties but may exacerbate tissue injury when neutrophil-mediated inflammatory responses are excessive in noninfectious lung injury. Targeting the basal cell adhesion molecule/P2Y purinoceptor 6 signaling pathway may serve as a novel approach to attenuate the neutrophil-mediated inflammatory responses and injury while maintaining the antimicrobial function of human neutrophil peptides in critical illness.

Dual effects of human neutrophil peptides in a mouse model of pneumonia and ventilator-induced lung injury.

BACKGROUND: Pneumonia is a major cause of high morbidity and mortality in critically illness, and frequently requires support with mechanical ventilation. The latter can lead to ventilator-induced lung injury characterized by neutrophil infiltration. The cationic human neutrophil peptides (HNP) stored in neutrophils can kill microorganisms, but excessive amount of HNP released during phagocytosis may contribute to inflammatory responses and worsen lung injury. Based on our previous work, we hypothesized that blocking the cell surface purinergic receptor P2Y6 will attenuate the HNP-induced inflammatory responses while maintaining their antimicrobial activity in pneumonia followed by mechanical ventilation. METHODS: Plasma HNP levels were measured in patients with pneumonia who received mechanical ventilation and in healthy volunteers. FVB littermate control and HNP transgenic (HNP+) mice were randomized to receive P. aeruginosa intranasally. The P2Y6 antagonist (MRS2578) or vehicle control was given after P. aeruginosa instillation. Additional mice underwent mechanical ventilation at either low pressure (LP) or high pressure (HP) ventilation 48 h after pneumonia, and were observed for 24 h. RESULTS: Plasma HNP concentration increased in patients with pneumonia as compared to healthy subjects. The bacterial counts in the bronchoalveolar lavage fluid (BALF) were lower in HNP+ mice than in FVB mice 72 h after P. aeruginosa instillation. However, upon receiving HP ventilation, HNP+ mice had higher levels of cytokines and chemokines in BALF than FVB mice. These inflammatory responses were attenuated by the treatment with MRS2578 that did not affect the microbial effects of HNP. CONCLUSIONS: HNP exerted dual effects by exhibiting antimicrobial activity in pneumonia alone condition while enhancing inflammatory responses in pneumonia followed by HP mechanical ventilation. Blocking P2Y6 can attenuate the inflammation without affecting the antibacterial property of HNP. The P2Y6 receptor may be a novel therapeutic target in attenuation of the leukocyte-mediated excessive host responses in inflammatory lung diseases.

Incidence of CNS tumors in Appalachian children.

Determine whether the risk of astrocytomas in Appalachian children is higher than the national average. We compared the incidence of pediatric brain tumors in Appalachia versus non-Appalachia regions, covering years 2000-2011. The North American Association of Central Cancer Registries (NAACCR) collects population-based data from 55 cancer registries throughout U.S. and Canada. All invasive primary (i.e. non-metastatic tumors), with age at diagnosis 0-19 years old, were included. Nearly 27,000 and 2200 central nervous system (CNS) tumors from non-Appalachia and Appalachia, respectively comprise the cohorts. Age-adjusted incidence rates of each main brain tumor subtype were compared. The incidence rate of pediatric CNS tumors was 8% higher in Appalachia, 3.31 [95% CI 3.17-3.45] versus non-Appalachia, 3.06, [95% CI 3.02-3.09] for the years 2001-2011, all rates are per 100,000 population. Astrocytomas accounted for the majority of this difference, with the rate being 16% higher in Appalachian children, 1.77, [95% CI 1.67-1.87] versus non-Appalachian children, 1.52, [95% CI 1.50-1.55]. Among astrocytomas, World Health Organization (WHO) grade I astrocytomas were 41% higher in Appalachia, 0.63 [95% CI 0.56-0.70] versus non-Appalachia 0.44 [95% CI 0.43-0.46] for the years 2004-2011. This is the first study to demonstrate that Appalachian children are at greater risk of CNS neoplasms, and that much of this difference is in WHO grade I astrocytomas, 41% more common. The cause of this increased incidence is unknown and we discuss the importance of this in relation to genetic and environmental findings in Appalachia.

Mechanical Stress and the Induction of Lung Fibrosis via the Midkine Signaling Pathway.

RATIONALE: Lung-protective ventilatory strategies have been widely used in patients with acute respiratory distress syndrome (ARDS), but the ARDS mortality rate remains unacceptably high and there is no proven pharmacologic therapy. OBJECTIVES: Mechanical ventilation can induce oxidative stress and lung fibrosis, which may contribute to high dependency on ventilator support and increased ARDS mortality. We hypothesized that the novel cytokine, midkine (MK), which can be up-regulated in oxidative stress, plays a key role in the pathogenesis of ARDS-associated lung fibrosis. METHODS: Blood samples were collected from 17 patients with ARDS and 10 healthy donors. Human lung epithelial cells were challenged with hydrogen chloride followed by mechanical stretch for 72 hours. Wild-type and MK gene-deficient (MK(-/-)) mice received two-hit injury of acid aspiration and mechanical ventilation, and were monitored for 14 days. MEASUREMENTS AND MAIN RESULTS: Plasma concentrations of MK were higher in patients with ARDS than in healthy volunteers. Exposure to mechanical stretch of lung epithelial cells led to an epithelial-mesenchymal transition profile associated with increased expression of angiotensin-converting enzyme, which was attenuated by silencing MK, its receptor Notch2, or NADP reduced oxidase 1. An increase in collagen deposition and hydroxyproline level and a decrease in lung tissue compliance seen in wild-type mice were largely attenuated in MK(-/-) mice. CONCLUSIONS: Mechanical stretch can induce an epithelial-mesenchymal transition phenotype mediated by the MK-Notch2-angiotensin-converting enzyme signaling pathway, contributing to lung remodeling. The MK pathway is a potential therapeutic target in the context of ARDS-associated lung fibrosis.

Monocyte interaction accelerates HCl-induced lung epithelial remodeling.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is characterized by overwhelming inflammatory responses and lung remodeling. We hypothesized that leukocyte infiltration during the inflammatory response modulates epithelial remodeling through a mechanism of epithelial-mesenchymal transition (EMT). METHODS: Human lung epithelial cells were treated for 30 min with hydrochloric acid (HCl). Human monocytes were then cocultured with the epithelial cells for up to 48 h, in the presence or absence of blocking peptides against lymphocyte function-associated antigen-1 (LFA-1), or tyrphostin A9, a specific inhibitor for platelet-derived growth factor (PDGF) receptor tyrosine kinase. RESULTS: Exposure of lung epithelial cells to HCl resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and production of interleukin (IL)-8 at 24 h. The expression of the epithelial markers E-cadherin decreased while the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) increased at 24 h and remained high at 48 h. The addition of monocytes augmented the profiles of lower expression of epithelial markers and higher mesenchymal markers accompanied by increased collagen deposition. This EMT profile was associated with an enhanced production of IL-8 and PDGF. Treatment of the lung epithelial cells with the LAF-1 blocking peptides CD11a237-246 or/and CD18112-122 suppressed monocyte adhesion, production of IL-8, PDGF and hydroxyproline as well as EMT markers. Treatment with tyrphostin A9 prevented the EMT profile shift induced by HCl stimulation. CONCLUSIONS: The interaction between epithelial cells and monocytes enhanced epithelial remodelling after initial injury through EMT signalling that is associated with the release of soluble mediators, including IL-8 and PDGF.

Human neutrophil peptides mediate endothelial-monocyte interaction, foam cell formation, and platelet activation.

OBJECTIVE: Neutrophils are involved in the inflammatory responses during atherosclerosis. Human neutrophil peptides (HNPs) released from activated neutrophils exert immune modulating properties. We hypothesized that HNPs play an important role in neutrophil-mediated inflammatory cardiovascular responses in atherosclerosis. METHODS AND RESULTS: We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor-related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation. CONCLUSIONS: HNPs exert proatherosclerotic properties that appear to be mediated through LRP8 signaling pathways, suggesting an important role for HNPs in the development of inflammatory cardiovascular diseases.

Bispectral Index Monitoring With Density Spectral Array for Delirium Detection.

BACKGROUND: Delirium in hospitalized patients often goes undetected. Cerebral state monitors, which measure limited-channel electroencephalography, have shown potential for improving delirium detection. OBJECTIVE: The aim of this study was to compare an FDA-approved cerebral state monitor, bispectral index monitoring with density spectral array (DSA), for delirium identification with clinical screening methods. METHODS: Hospitalized patients receiving psychiatric consultation were assessed for delirium using the 3-minute Diagnostic Interview for Confusion Assessment Method (3D-CAM) and underwent bispectral index monitor + DSA monitoring. Visual inspection of frequency band power of the DSA was performed by 2 trained independent raters. Average hue values were calculated for each frequency band using image analysis software as the device did not allow for extraction of raw electroencephalography data. Delirious versus nondelirious group averages, sensitivity, specificity, and area under the curve were calculated for significant DSA variables and the 3D-CAM. RESULTS: In an initial cohort of 43 patients, visual ratings of the DSA were not associated with delirium (P > 0.1). In a larger cohort of 123 subjects, multiple band hue ratios were associated with delirium, although none survived correction for multiple comparisons. In a subgroup of 74 non-neurological patients, low theta/low delta ratio was significantly associated with delirium (P = 0.001) (sensitivity/specificity/area under the curve: 83%/70%/0.757; 3D-CAM: 67%/77%/0.717; paired-sample area under the curve difference: -0.040, P = 0.68). In 21 patients with dementia, low theta power demonstrated significantly greater sensitivity/specificity/area under the curve of 83%/78%/0.824, whereas 3D-CAM achieved 50%/78%/0.639 (P = 0.04). CONCLUSION: Bispectral index monitor + DSA was similar to 3D-CAM for detecting delirium in hospitalized patients with and without neurological disorders, and was significantly more accurate in patients with dementia. More studies are needed to validate the use of cerebral state monitors for quantitative delirium detection.

Assessing adolescent tobacco use in a substance use treatment program with self-report and biological measures.

Tobacco use in adolescents can alter their lifetime health outcomes. Despite the importance of early identification and treatment, adolescent tobacco use, including that of electronic vapor products (e.g., e-cigarettes), is often missed. In a state-funded substance use treatment program, we added biological measures, including urinary cotinine and exhaled carbon monoxide to self-report measures to assess recent and lifetime tobacco use. We conducted a retrospective review of the de-identified charts to examine the feasibility of screening for self-report and biological measures of tobacco use. Self-report, urinary cotinine, and exhaled carbon monoxide samples were obtained at every visit, including intake and follow-up. There were 52 adolescents with a total of 400 clinic visits to the program. Of those 400 visits, 258 included self-reported tobacco use and 142 included a denial of using any form of tobacco. However, of those 142 visits with a negative self-report of tobacco, 31 tested positive for cotinine and 6 had positive exhaled carbon monoxide. Although 111 of the 142 had negative cotinine, 5 had positive carbon monoxide, but all of those self-reported recent cannabis use. Despite using a sensitive measure of self-report of tobacco use, almost 22% of visits had a discordant self-report with a biological measure that indicated tobacco use. Considering the lifelong impact of adolescent tobacco use, clinicians should consider augmenting self-report with biological measures of tobacco use. Identification of tobacco use in adolescents with substance use can assist clinicians in providing education about tobacco use, such as electronic vapor products, and individualizing treatments.

Circuit projection from suprachiasmatic nucleus to ventral tegmental area: a novel circadian output pathway.

The suprachiasmatic nucleus (SCN) is a circadian pacemaker that synchronizes a number of vital processes. Although a great deal of research has focused on input pathways to SCN and on the central clock itself, relatively little is known about SCN output signaling pathways. The ventral tegmental area (VTA) has been extensively studied for its influence in motivated learning and, recently, for a potential role in arousal and sleep-wake regulation. Here we present data that SCN indirectly projects to VTA via the medial preoptic nucleus (MPON). Microinjection of the retrograde, transynaptic tracer pseudorabies virus (PRV) in rat VTA consistently labeled SCN neurons at time points indicative of an indirect circuit projection. To specify intermediate relay nuclei between SCN and VTA, putative relays were lesioned 1 week prior to PRV injections in VTA. Unilateral lesions of MPON reduced PRV labeling in SCN by 81.6% in the ipsilateral hemisphere and 75.8% in the contralateral hemisphere. Bilateral lesions of the caudal-dorsal lateral septum, another putative relay nucleus and dorsal injection control, did not significantly reduce PRV labeling in the SCN. Single-unit extracellular recordings under halothane anesthesia revealed a novel population of VTA neurons that selectively fired during the active circadian phase. These results show that SCN provides an indirect circuit pathway to VTA via MPON, and that VTA neurons exhibit a circadian rhythm in their impulse activity. This pathway may function in the circadian regulation of numerous behavioral processes including arousal and motivation.

Novel neurons in ventral tegmental area fire selectively during the active phase of the diurnal cycle.

The ventral tegmental area (VTA) contains dopamine (DA) and gamma-aminobutyric acid (GABA) neurons involved in motivation and behavioral state. These phenomena are also influenced by circadian factors. The goal of our studies was to examine the impulse activity of neurochemically identified VTA neurons during dark (active) vs light (rest) phases of the circadian cycle. Using extracellular single-unit recordings with juxtacellular labeling in anesthetized rats, we found multiple neuronal subpopulations including 'novel neurons' that selectively fired during the dark phase. These novel neurons were electrophysiologically categorized into two groups, 'novel wide-spike' and 'novel thin-spike' neurons. Characterization of novel wide-spike neurons found they were consistently non-dopaminergic and non-GABAergic [tyrosine hydroxylase (TH)(-), glutamic acid decarboxylase (GAD)(-)]. However, they were inhibited by the D2 agonist quinpirole, an effect that could be reversed by the D2 antagonist eticlopride. Physiologically, they were fast firing (mean = 18.9 +/- 1.2 spikes/s), low bursting neurons (median = 6.2 +/- 3.0% of spikes in bursts) with spike durations > or = 2.0 ms, but slightly shorter than TH(+) neurons. They were also consistently non-responsive to footpad stimulation. The novel thin-spike neurons were neurochemically heterogeneous, and were located more ventrally than thin-spike neurons found during the light phase. These findings reveal previously unknown populations of VTA neurons whose activities are sensitive to diurnal phase, and whose functions may be in the temporal regulation of arousal and motivational processes.

Impaired olfactory behavior in mice deficient in the alpha subunit of G(o).

The ability to respond to chemical signals is essential for the survival and reproduction of most organisms. Olfactory signaling involves odorant receptor-mediated activation of G(olf), a homologue of G(s), on the dendrites of olfactory neurons. Olfactory receptor cells, however, also express Galpha(i2) and Galpha(o) on their axons, with all neurons expressing G(o) and a subset G(i2). Despite their abundance, possible contributions of G(o) and G(i2) to chemoreception remain unexplored. We investigated whether homologous recombinant mice deficient in the alpha subunit of G(o) are able to respond to odorants, whether possible olfactory impairments are dependent on genetic background, and whether formation of glomeruli in their olfactory bulbs is compromised. In an olfactory habituation/dishabituation test, G(o)-/- mice were unresponsive when exposed to odorants. Analysis of variance shows that performance of G(o)+/- mice crossed into the CD-1 background is also diminished in this test compared to their G(o)+/+ counterparts. Following food deprivation, G(o)-/- mice in the 129 Sv-ter/C57BL/6 genetic background were unable to locate a buried food pellet until they were approximately 10 weeks of age after which they performed as well as their litter mate controls. However, CD-1 G(o)-/- mice could locate a buried food pellet even when tested immediately after weaning. Despite their compromised olfactory responsiveness, histological examination did not reveal gross alterations in the olfactory bulbs of G(o)-/- mice. Thus, Galpha(o) is necessary for the expression of olfactory behavior under normal conditions and dependent on genetic background, but is not essential for the formation and maintenance of glomeruli.

Linking context with reward: a functional circuit from hippocampal CA3 to ventral tegmental area.

Reward-motivated behavior is strongly influenced by the learned significance of contextual stimuli in the environment. However, the neural pathways that mediate context-reward relations are not well understood. We have identified a circuit from area CA3 of dorsal hippocampus to ventral tegmental area (VTA) that uses lateral septum (LS) as a relay. Theta frequency stimulation of CA3 excited VTA dopamine (DA) neurons and inhibited non-DA neurons. DA neuron excitation was likely mediated by disinhibition because local antagonism of γ-aminobutyric acid receptors blocked responses to CA3 stimulation. Inactivating components of the CA3-LS-VTA pathway blocked evoked responses in VTA and also reinstatement of cocaine-seeking by contextual stimuli. This transsynaptic link between hippocampus and VTA appears to be an important substrate by which environmental context regulates goal-directed behavior.