Amy O'Brien-Ladner

Release of interleukin-1 by human alveolar macrophages after in vitro irradiation.

Therapeutic thoracic irradiation may induce two late pulmonary injury syndromes: radiation pneumonitis and subsequent pulmonary fibrosis. The alveolar macrophage has been considered a radioresistant cell and not a target cell involved in the pathogenesis of either type of radiation-induced lung injury. Alveolar macrophage-derived cytokines, including interleukin-1 (IL-1) and tumor necrosis factor (TNF), have been demonstrated to participate in inflammatory and fibrotic responses in the lung after various other types of lung injury. To evaluate whether the release of cytokines by alveolar macrophages is induced by radiation doses used clinically, alveolar macrophages recovered from nonsmoking volunteers were exposed in vitro to a single dose of 2 Gy and then maintained in culture for 18 h. Culture supernatants and cell lysates were then recovered and analyzed for IL-1 alpha and IL-1 beta by radioimmunoassay. Supernatants of irradiated alveolar macrophages contained significantly increased amounts of IL-1 alpha (P < 0.04) and IL-1 beta (P < 0.02) as well as total IL-1 (IL-1 alpha and IL-1 beta) (P < 0.02) compared to nonirradiated alveolar macrophages. Cell lysates of irradiated alveolar macrophages also contained increased amounts of IL-1 alpha and IL-1 beta, although differences from controls were not significant. The finding of increased release of IL-1 by alveolar macrophages after exposure to a single, clinically relevant dose of radiation suggests that the function of human alveolar macrophages is likely altered during therapeutic use of thoracic irradiation. Whether this release of IL-1 by alveolar macrophages contributes to early lung inflammation induced by thoracic irradiation is unclear.

Hypoxia-inducible factor-1 α/platelet derived growth factor axis in HIV-associated pulmonary vascular remodeling

BackgroundHuman immunodeficiency virus (HIV) infected patients are at increased risk for the development of pulmonary arterial hypertension (PAH). Recent reports have demonstrated that HIV associated viral proteins induce reactive oxygen species (ROS) with resultant endothelial cell dysfunction and related vascular injury. In this study, we explored the impact of HIV protein induced oxidative stress on production of hypoxia inducible factor (HIF)-1α and platelet-derived growth factor (PDGF), critical mediators implicated in the pathogenesis of HIV-PAH.MethodsThe lungs from 4-5 months old HIV-1 transgenic (Tg) rats were assessed for the presence of pulmonary vascular remodeling and HIF-1α/PDGF-BB expression in comparison with wild type controls. Human primary pulmonary arterial endothelial cells (HPAEC) were treated with HIV-associated proteins in the presence or absence of pretreatment with antioxidants, for 24 hrs followed by estimation of ROS levels and western blot analysis of HIF-1α or PDGF-BB.ResultsHIV-Tg rats, a model with marked viral protein induced vascular oxidative stress in the absence of active HIV-1 replication demonstrated significant medial thickening of pulmonary vessels and increased right ventricular mass compared to wild-type controls, with increased expression of HIF-1α and PDGF-BB in HIV-Tg rats. The up-regulation of both HIF-1α and PDGF-B chain mRNA in each HIV-Tg rat was directly correlated with an increase in right ventricular/left ventricular+septum ratio. Supporting our in-vivo findings, HPAECs treated with HIV-proteins: Tat and gp120, demonstrated increased ROS and parallel increase of PDGF-BB expression with the maximum induction observed on treatment with R5 type gp-120CM. Pre-treatment of endothelial cells with antioxidants or transfection of cells with HIF-1α small interfering RNA resulted in abrogation of gp-120CM mediated induction of PDGF-BB, therefore, confirming that ROS generation and activation of HIF-1α plays critical role in gp120 mediated up-regulation of PDGF-BB.ConclusionIn summary, these findings indicate that viral protein induced oxidative stress results in HIF-1α dependent up-regulation of PDGF-BB and suggests the possible involvement of this pathway in the development of HIV-PAH.

Bleomycin injury of the lung in a mast-cell-deficient model

Lung mast cell hyperplasia and fibrosis is induced by bleomycin lung injury. The role of the mast cell in this process of injury and resultant fibrosis is unclear. Mutant mi/mi mice, profoundly mast-cell-deficient, were treated with intraperitoneal bleomycin and demonstrated minimal acute inflammatory and chronic fibrotic responses. Lung histamine values determined at 14 and 42 days after bleomycin injury in mi/mi mice were not increased compared to untreated mi/mi animals. However, lung histamine levels in normal mice demonstrate a 300% increase over controls on Day 14 after bleomycin injury, and then returned to baseline by Day 42. The mi/mi BAL cell recovery at 2 weeks after injury and lung hydroxyproline levels at 4 weeks after injury were not altered from baseline. The normal litter mates, in contrast, demonstrated significant increases compared to controls in both of these parameters (p<0.01,p<0.04). Although the mi/mi mouse is also deficient in basophils, natural killer cells and functional osteoclasts, there is no evidence of lowered pulmonary defense mechanism and neutrophils and alveolar macrophages are present in normal numbers. This investigation supports the hypothesis that the mast cell contributes to bleomycin-induced lung injury and fibrosis.

Alveolar macrophages accumulate iron and ferritin after in vivo exposure to iron or tungsten dusts

Extracellular iron present in alveolar structures may contribute to oxidative lung injury induced by toxic mineral dusts by enhancing dust-induced generation of hydroxyl radicals. Alveolar macrophages (AMs) can sequester iron within ferritin and limit generation of hydroxyl radicals. In the current study we sought to assess whether AMs accumulate iron and ferritin after in vivo exposure to a dust with high iron content, to iron oxide, or to an inflammatory dust, calcium tungstate. We performed lung lavage 1, 7, 14, 28, 42, and 56 days after intratracheal instillation of mineral dust in saline solution or instillation of saline solution alone and quantitated cell recovery and AM content of iron and ferritin. Instillation of iron oxide increased neutrophil recovery only on a day 1 when compared with results in controls, whereas calcium tungstate increased neutrophil recovery through day 14. AMs recovered after instillation of iron oxide contained increased amounts of iron and ferritin, beginning on day 1 and progressing through day 56 after treatment (7.57 +/- 0.38 microgram iron per 10(6) AMs vs 1.54 +/- 0.28 microgram iron per 10(6) AMs for controls, p < 0.001; and 5908 +/- 768 ng ferritin per 10(6) AMs vs 395 +/- 20 ng ferritin per 10(6) AMs, p < 0.001). AMs recovered after calcium tungstate instillation also contained increased amounts of iron and ferritin beginning 14 days after treatment, with greatest content 42 days after treatment (4.85 +/- 0.68 microgram iron per 10(6) AMs, p < 0.001, and 2274 +/- 736 ng ferritin per 10(6) AMs, p < 0.001). Tumor necrosis factor, which can enhance iron accumulation by macrophages, was spontaneously released by AMs recovered from tungsten-treated rats. These studies indicate that AMs accumulate iron and ferritin in response to both iron loading of the lungs with iron oxide exposure and lung inflammation induced by calcium tungstate exposure.

Deferoxamine mimics the pattern of hypoxia-related injury at the microvasculature

Oxygen is essential for the maintenance of life, and when oxygen levels decline to critical levels, a program of complex mechanisms exists to i) sense hypoxia, ii) respond to minimize acute tissue injury, and iii) result in adaptations that offer protection against further hypoxia challenges. Alternative adaptation-related protection may also be inducible through the increased activity of hypoxia-inducible factors activated by hypoxia mimics such as iron chelation with deferoxamine (DFA). We have characterized a set of hypoxia-related responses at the microvasculature and postulated that microvascular injury in response to hypoxia could be reproduced by the reduction of bioavailable iron through chelation by DFA. We were able to induce a similar degree of leukocyte adherence and emigration and vascular leak with DFA infusion as compared with hypoxia exposure in an intact physiological rodent model. However, in contrast to hypoxia-exposed groups, we were unable to detect reactive oxygen species or alter the injury pattern with reactive oxygen species scavenger in the groups treated with DFA. Thus, we demonstrate that DFA mimics the pattern and intensity of hypoxia-related injury on the microvasculature; however, differences in the time course and mechanism of injury were identified. In addition, DFA saturated with iron did not completely reverse the effects of DFA, suggesting a mechanism(s) beyond a reduction in the bioavailability of iron. These findings may have importance in the targeting of iron for the development of hypoxia mimics that may offer protection against subsequent hypoxia exposure in clinical setting such as myocardial infarction and stroke.

Enhanced autophagy in pulmonary endothelial cells on exposure to HIV-Tat and morphine: Role in HIV-related pulmonary arterial hypertension

ABSTRACT Intravenous drug use is one of the major risk factors for HIV-infection in HIV-related pulmonary arterial hypertension patients. We previously demonstrated exaggerated pulmonary vascular remodeling with enhanced apoptosis followed by increased proliferation of pulmonary endothelial cells on simultaneous exposure to both opioids and HIV protein(s). Here we hypothesize that the exacerbation of autophagy may be involved in the switching of endothelial cells from an early apoptotic state to later hyper-proliferative state. Treatment of human pulmonary microvascular endothelial cells (HPMECs) with both the HIV-protein Tat and morphine resulted in an oxidative stress-dependent increase in the expression of various markers of autophagy and formation of autophagosomes when compared to either Tat or morphine monotreatments as demonstrated by western blot, transmission electron microscopy and immunofluorescence. Autophagy flux experiments suggested increased formation rather than decreased clearance of autolysosomes. Inhibition of autophagy resulted in a significant increase in apoptosis and reduction in proliferation of HPMECs with combined morphine and Tat (M+T) treatment compared to monotreatments whereas stimulation of autophagy resulted in opposite effects. Significant increases in the expression of autophagy markers as well as the number of autophagosomes and autolysosomes was observed in the lungs of SIV-infected macaques and HIV-infected humans exposed to opioids. Overall our findings indicate that morphine in combination with viral protein(s) results in the induction of autophagy in pulmonary endothelial cells that may lead to an increase in severity of angio-proliferative remodeling of the pulmonary vasculature on simian and human immunodeficiency virus infection in the presence of opioids.

Frontiers: Integration of a Research Participant Registry with Medical Clinic Registration and Electronic Health Records.

VOLUME 8 • ISSUE 5 WWW.CTSJOURNAL.COM Introduction P articipant recruitment is a signifi cant challenge to the success of clinical research. Without sufficient study participants, investigators cannot meet accrual goals and studies either must be abandoned without reaching recruitment targets or prolonged. 1,2 Recruitment of study participants can be facilitated by maintaining registries of people who agree to be contacted for future studies. Registries range from simple databases that include only contact information, to more sophisticated regional and national registries that include individual health information and that can be searched for individuals who meet specifi c study criteria. 3–7 Such registries clearly facilitate recruitment, but they can be costly to develop, advertise, and maintain. 3,5,6,8–11 Frontiers: Th e Heartland Institute for Clinical and Translational Research is an academic home for clinical and translational research, headquartered at the University of Kansas Medical Center (KUMC) and partly funded by a Clinical and Translational Science Award (CTSA) from the National Institutes of Health since 2011. Th e vision of Frontiers is to improve the health of the region through a novel and transformative translational research enterprise from the bench to bedside to community. In this paper, we report our experience in establishing the Frontiers Research Participant Registry and highlight the unique combination of features that diff erentiate it from other registries: (1) Registry enrollment is integrated into outpatient clinic registration, as all clinic patients have the opportunity to provide permission to be contacted for studies for which they are eligible; (2) Th e registry is aligned with the complete electronic health record (EHR) that can be searched to provide the total numbers of clinic patients who meet inclusion/exclusion criteria; (3) Investigators with IRB-approved studies can receive contact information for those patients who meet criteria and who have given permission to be contacted; (4) Th e registry provides a HIPAA-compliant means for investigators to contact only those potential participants who meet inclusion/exclusion criteria based on their EHR. Together, these features have yielded a highly successful program with a growing database of over 31,000 individuals who have agreed to be contacted for research recruitment.