Kristina L. Bailey

Muramic Acid, Endotoxin, 3-Hydroxy Fatty Acids, and Ergosterol Content Explain Monocyte and Epithelial Cell Inflammatory Responses to Agricultural Dusts

In agricultural and other environments, inhalation of airborne microorganisms is linked to respiratory disease development. Bacterial endotoxins, peptidoglycans, and fungi are potential causative agents, but relative microbial characterization and inflammatory comparisons amongst agricultural dusts are not well described. The aim of this study was to determine the distribution of microbial endotoxin, 3-hydroxy fatty acids (3-OHFA), muramic acid, and ergosterol and evaluate inflammatory responses in human monocytes and bronchial epithelial cells with various dust samples. Settled surface dust was obtained from five environments: swine facility, dairy barn, grain elevator, domestic home (no pets), and domestic home with dog. Endotoxin concentration was determined by recombinant factor C (rFC). 3-OHFA, muramic acid, and ergosterol were measured using gas chromatography–mass spectrometry. Dust-induced inflammatory cytokine secretion in human monocytes and bronchial epithelial cells was evaluated. Endotoxin-independent dust-induced inflammatory responses were evaluated. Endotoxin and 3-OHFA levels were highest in agricultural dusts. Muramic acid, endotoxin, 3-OHFA, and ergosterol were detected in dusts samples. Muramic acid was highest in animal farming dusts. Ergosterol was most significant in grain elevator dust. Agricultural dusts induced monocyte tumor necrosis factor (TNF) α, interleukin (IL)-6, IL-8, and epithelial cell IL-6 and IL-8 secretion. Monocyte and epithelial IL-6 and IL-8 secretion was not dependent on endotoxin. House dust(s) induced monocyte TNFα, IL-6, and IL-8 secretion. Swine facility dust generally produced elevated responses compared to other dusts. Agricultural dusts are complex with significant microbial component contribution. Large animal farming dust(s)-induced inflammation is not entirely dependent on endotoxin. Addition of muramic acid to endotoxin in large animal farming environment monitoring is warranted.

Lessons learned: critical care management of patients with Ebola in the United States.

Objective:This report will describe the preparations for and the provision of care of two patients with Ebola virus disease in the biocontainment unit at the University of Nebraska Medical Center. Data Sources:Patient medical records. Study Selection:Not applicable. Data Extraction:Not applicable. Data Synthesis:Not applicable. Conclusions:Safe and effective care of patients with Ebola virus disease requires significant communication and planning. Adherence to a predetermined isolation protocol is essential, including proper donning and doffing of personal protective equipment. Location of the patient care area and the logistics of laboratory testing, diagnostic imaging, and the removal of waste must be considered. Patients with Ebola virus disease are often dehydrated and need adequate vascular access for fluid resuscitation, nutrition, and phlebotomy for laboratory sampling. Advanced planning for acute life-threatening events and code status must be considered. Intensivist scheduling should account for the significant amount of time required for the care of patients with Ebola virus disease. With appropriate precautions and resources, designated hospitals in the United States can safely provide care for patients with Ebola virus disease.

Co-Exposure to Cigarette Smoke and Alcohol Decreases Airway Epithelial Cell Cilia Beating in a Protein Kinase Cε-Dependent Manner

Alcohol use disorders are associated with increased lung infections and exacerbations of chronic lung diseases. Whereas the effects of cigarette smoke are well recognized, the interplay of smoke and alcohol in modulating lung diseases is not clear. Because innate lung defense is mechanically maintained by airway cilia action and protein kinase C (PKC)-activating agents slow ciliary beat frequency (CBF), we hypothesized that the combination of smoke and alcohol would decrease CBF in a PKC-dependent manner. Primary ciliated bronchial epithelial cells were exposed to 5% cigarette smoke extract plus100 mmol/L ethanol for up to 24 hours and assayed for CBF and PKCε. Smoke and alcohol co-exposure activated PKCε by 1 hour and decreased both CBF and total number of beating cilia by 6 hours. A specific activator of PKCε, DCP-LA, slowed CBF after maximal PKCε activation. Interestingly, activation of PKCε by smoke and alcohol was only observed in ciliated cells, not basal bronchial epithelium. In precision-cut mouse lung slices treated with smoke and alcohol, PKCε activation preceded CBF slowing. Correspondingly, increased PKCε activity and cilia slowing were only observed in mice co-exposed to smoke and alcohol, regardless of the sequence of the combination exposure. No decreases in CBF were observed in PKCε knockout mice co-exposed to smoke and alcohol. These data identify PKCε as a key regulator of cilia slowing in response to combined smoke and alcohol-induced lung injury.

Agricultural exposures in patients with COPD in health systems serving rural areas

ABSTRACT Background: Agricultural exposure is a risk factor for the development of chronic obstructive pulmonary disease (COPD). However, there are no good estimates of the number of COPD patients with a history of agricultural exposure. Methods: We conducted a telephone interview of subjects with COPD identified by reviewing all pulmonary function tests at the Omaha Veterans Administration Hospital between November 2004 and March 2005. Obstructive lung disease was defined as a FEV1/FVC ratio of less than 70%. The survey detailed demographic data, smoking history, pulmonary symptoms, and history of agricultural exposures. Results: Participants included 150 veterans (mean age 68.2 years ±10.8). A history of agricultural exposure was elicited in 68% of subjects. Of those who had worked in agriculture, the types of exposures varied, with 14% in hog confinement barns, 20% on dairy farms, 8% on poultry farms, and 87% exposed to grain dust. There was a trend of diminishing FEV1 with increasing years of agricultural exposure. Conclusions: In health systems that serve rural areas, patients with COPD commonly have a history of agricultural exposures that may contribute to the development of COPD. Health care workers in these areas should include agricultural exposures as an important part of the social/occupational history in these patients.

Ethanol blocks adenosine uptake via inhibiting the nucleoside transport system in bronchial epithelial cells.

BACKGROUND Adenosine uptake into cells by nucleoside transporters plays a significant role in governing extracellular adenosine concentration. Extracellular adenosine is an important signaling molecule that modulates many cellular functions via 4 G-protein-coupled receptor subtypes (A(1), A(2A), A(2B), and A(3)). Previously, we demonstrated that adenosine is critical in maintaining airway homeostasis and airway repair and that airway host defenses are impaired by alcohol. Taken together, we hypothesized that ethanol impairs adenosine uptake via the nucleoside transport system. METHODS To examine ethanol-induced alteration on adenosine transport, we used a human bronchial epithelial cell line (BEAS-2B). Cells were preincubated for 10 minutes in the presence and absence of varying concentrations of ethanol (EtOH). In addition, some cells were pretreated with S-(4-Nitrobenzyl)-6-thioinosine (100 microM: NBT), a potent adenosine uptake inhibitor. Uptake was then determined by addition of [(3)H]-adenosine at various time intervals. RESULTS Increasing EtOH concentrations resulted in increasing inhibition of adenosine uptake when measured at 1 minute. Cells pretreated with NBT effectively blocked adenosine uptake. In addition, short-term EtOH revealed increased extracellular adenosine concentration. Conversely, adenosine transport became desensitized in cells exposed to EtOH (100 mM) for 24 hours. To determine the mechanism of EtOH-induced desensitization of adenosine transport, cAMP activity was assessed in response to EtOH. Short-term EtOH exposure (10 minutes) had little or no effect on adenosine-mediated cAMP activation, whereas long-term EtOH exposure (24 hours) blocked adenosine-mediated cAMP activation. Western blot analysis of lysates from unstimulated BEAS-2B cells detected a single 55 kDa band indicating the presence of hENT1 and hENT2, respectively. Real-time RT-PCR of RNA from BEAS-2B revealed transcriptional expression of ENT1 and ENT2. CONCLUSIONS Collectively, these data reveal that acute exposure of cells to EtOH inhibits adenosine uptake via a nucleoside transporter, and chronic exposure of cells to EtOH desensitizes the adenosine transporter to these inhibitory effects of ethanol. Furthermore, our data suggest that inhibition of adenosine uptake by EtOH leads to an increased extracellular adenosine accumulation, influencing the effect of adenosine at the epithelial cell surface, which may alter airway homeostasis.

Aging causes a slowing in ciliary beat frequency, mediated by PKCε

The elderly are at much higher risk for developing pneumonia than younger individuals. Pneumonia is a leading cause of death and is the third most common reason for hospitalization in the elderly. One reason that elderly people may be more susceptible to pneumonia is a breakdown in the lungs first line of defense, mucociliary clearance. Cilia beat in a coordinated manner to propel out invading microorganisms and particles. Ciliary beat frequency (CBF) is known to slow with aging, however, little is known about the mechanism(s) involved. We compared the CBF in BALB/c and C57BL/6 mice aged 2, 12, and 24 mo and found that CBF diminishes with age. Cilia in the mice at age 12 and 24 mo retained their ability to be stimulated by the β2 agonist procaterol. To help determine the mechanism of ciliary slowing, we measured protein kinase C alpha and epsilon (PKCα and PKCε) activity. There were no activity differences in PKCα between the mice aged 2, 12, or 24 mo. However, we demonstrated a significantly higher PKCε activity in the mice at 12 and 24 mo than the in the mice 2 mo of age. The increase in activity is likely due to a nearly threefold increase in PKCε protein in the lung during aging. To strengthen the connection between activation of PKCε and ciliary slowing, we treated tracheas of mice at 2 mo with the PKCε agonist 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA). We noted a similar decrease in baseline CBF, and the cilia remained sensitive to stimulation with β2 agonists. The mechanisms for the slowing of baseline CBF have not been previously determined. In this mouse model of aging we were able to show that decreases in CBF are related to an increase in PKCε activity.

Alcohol up-regulates TLR2 through a NO/cGMP dependent pathway.

BACKGROUND Heavy alcohol consumption is associated with severe bronchitis. This is likely related to increased inflammation in the airways of alcohol abusers. Toll-like receptor 2 (TLR2) is an important mediator of inflammation in the airway epithelium. TLR2 initiates an inflammatory cascade in response to gram-positive bacteria. We have previously shown that alcohol up-regulates TLR2 in the airway epithelium. However, the mechanism of alcohol-mediated up-regulation of TLR2 has not been identified. METHODS A human airway epithelial cell line, 16HBE14o-, was exposed to biologically relevant concentrations of alcohol (100 mM) in the presence and absence of N(omega)-Nitro-l-arginine methyl ester hydrochloride, a nitric oxide (NO) synthase inhibitor; and Rp-8-Br-cGMP-S, an antagonist analogue of cGMP. TLR2 was measured using real-time PCR and Western blots. In addition, 16HBE14o- cells were incubated with sodium nitroprusside (SNP), an NO donor, and 8-Br-cGMP, a cGMP analogue. TLR2 was measured using real-time PCR. RESULTS N(omega)-Nitro-l-arginine methyl ester hydrochloride blocked the alcohol-mediated up-regulation of TLR2. This indicates that NO plays a key role in alcohols up-regulation of TLR2. SNP, a NO donor, up-regulated TLR2. Rp-8-Br-CGMP-S attenuated alcohols up-regulation of TLR2, suggesting that NO was working through cGMP/PKG. 8-Br-cGMP up-regulated TLR2, also demonstrating the importance of cGMP/PKG. CONCLUSIONS Alcohol up-regulates TLR2 through a NO/cGMP/PKG dependent pathway in the airway epithelium. This is an important observation in the understanding how alcohol modulates airway inflammation. In addition, this is the first time that cyclic nucleotides have been shown to play a role in the regulation of TLR2.

Pulmonary health effects of agriculture.

Purpose of review Occupational exposures in the agricultural industry are associated with numerous lung diseases, including chronic obstructive pulmonary disease, asthma, hypersensitivity pneumonitis, lung cancer, and interstitial lung diseases. Efforts are ongoing to ascertain contributing factors to these negative respiratory outcomes and improve monitoring of environmental factors leading to disease. In this review, recently published studies investigating the deleterious effects of occupational exposures in the agricultural industry are discussed. Recent findings Occupational exposures to numerous agricultural environment aerosols, including pesticides, fungi, and bacteria are associated with impaired respiratory function and disease. Increases in certain farming practices, including mushroom and greenhouse farming, present new occupational exposure concerns. Improved detection methods may provide opportunities to better monitor safe exposure levels to known lung irritants. Summary In the agricultural industry, occupational exposures to organic and inorganic aerosols lead to increased risk for lung disease among workers. Increased awareness of respiratory risks and improved monitoring of agricultural environments are necessary to limit pulmonary health risks to exposed populations.

Alcohol Exposure Alters Mouse Lung Inflammation in Response to Inhaled Dust

Alcohol exposure is associated with increased lung infections and decreased mucociliary clearance. Occupational workers exposed to dusts from concentrated animal feeding operations (CAFOs) are at risk for developing chronic inflammatory lung diseases. Agricultural worker co-exposure to alcohol and organic dust has been established, although little research has been conducted on the combination effects of alcohol and organic dusts on the lung. Previously, we have shown in a mouse model that exposure to hog dust extract (HDE) collected from a CAFO results in the activation of protein kinase C (PKC), elevated lavage fluid cytokines/chemokines including interleukin-6 (IL-6), and the development of significant lung pathology. Because alcohol blocks airway epithelial cell release of IL-6 in vitro, we hypothesized that alcohol exposure would alter mouse lung inflammatory responses to HDE. To test this hypothesis, C57BL/6 mice were fed 20% alcohol or water ad libitum for 6 weeks and treated with 12.5% HDE by intranasal inhalation method daily during the final three weeks. Bronchoalveolar lavage fluid (BALF), tracheas and lungs were collected. HDE stimulated a 2–4 fold increase in lung and tracheal PKCε (epsilon) activity in mice, but no such increase in PKCε activity was observed in dust-exposed mice fed alcohol. Similarly, alcohol-fed mice demonstrated significantly less IL-6 in lung lavage in response to dust than that observed in control mice instilled with HDE. TNFα levels were also inhibited in the alcohol and HDE-exposed mouse lung tissue as compared to the HDE only exposed group. HDE-induced lung inflammatory aggregates clearly present in the tissue from HDE only exposed animals were not visually detectable in the HDE/alcohol co-exposure group. Statistically significant weight reductions and 20% mortality were also observed in the mice co-exposed to HDE and alcohol. These data suggest that alcohol exposure depresses the ability of the lung to activate PKCε-dependent inflammatory pathways to environmental dust exposure. These data also define alcohol as an important co-exposure agent to consider in the study of inhalation injury responses.

Alcohol Decreases RhoA Activity Through a Nitric Oxide (NO)/Cyclic GMP(cGMP)/Protein Kinase G (PKG)-Dependent Pathway in the Airway Epithelium

BACKGROUND Alcohol has been shown to have a number of harmful effects on the lung, including increasing the risk of pneumonia and bronchitis. How alcohol increases the risk of these diseases is poorly defined. RhoA is a small guanosine triphosphate (GTP)ase that plays an integral role in many basic functions of airway epithelial cells. It is not known how alcohol affects RhoA activity in the airway epithelium. We hypothesized that brief alcohol exposure modulates RhoA activity in the airway epithelium through a nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G (PKG)-dependent pathway. METHODS Primary airway epithelial cells were cultured and exposed to ethanol at various concentrations and times. The cell layers were harvested and RhoA activity was measured. RESULTS Alcohol induced a time- and concentration-dependent decrease in RhoA activity in airway epithelial cells. We were able to block this decrease in activity using Nω-nitro-l-arginine methyl ester (L-NAME) hydrochloride, a nitric oxide synthase (NOS) inhibitor. Likewise, we were able to demonstrate the same decrease in RhoA activation using 0.1 μM sodium nitroprusside, an NO donor. To determine the role of cGMP/PKG, we pretreated the cells with a cGMP antagonist analog, Rp-8Br-cGMPS. This blocked the decrease in RhoA activity caused by alcohol, suggesting that alcohol exerts its effect on RhoA activity through cGMP/PKG. CONCLUSIONS Alcohol decreases airway epithelial RhoA activity through an NO/cGMP/PKG-dependent pathway. RhoA activity controls many aspects of basic cellular function, including cell morphology, tight junction formation, and cell cycle progression and gene regulation. Dysregulation of RhoA activity can potentially have several consequences, including dysregulation of inflammation. This may partially explain how alcohol increases the risk of pneumonia and bronchitis.