Alcohol abuse is associated with enhanced pulmonary and systemic xanthine oxidoreductase activity.

Acute respiratory distress syndrome (ARDS) is a common and devastating disorder. Alcohol use disorders (AUDs) increase ARDS risk and worsen outcomes through mechanisms that may include enhancement of pulmonary oxidative stress. Alcohol consumption increases activity of the enzyme xanthine oxidoreductase (XOR) that contributes to production of both reactive oxygen species (ROS) and uric acid, a damage-associated molecular pattern. These by-products have the potential to modulate proinflammatory pathways, such as those involving cyclooxygenase (COX)-2, and to activate the nucleotide-binding domain, leucine-rich-containing family, pyrin-domain containing-3 (NLRP3) inflammasome. We sought to determine if pulmonary and systemic XOR activity was altered by AUDs. Bronchoscopy with bronchoalveolar lavage (BAL) and blood sampling was performed in otherwise healthy human subjects with AUDs and controls. Uric acid in epithelial-lining fluid, derived from BAL, was substantially higher among individuals with AUDs and did not normalize after 7 days of abstinence; serum uric acid did not differ across groups. XOR enzyme activity in fresh BAL cells and serum was significantly increased in subjects with AUDs. XOR protein in BAL cells from AUD subjects was increased in parallel with COX-2 expression, and furthermore, mRNA expression of NLRP3 inflammasome components was sustained in LPS-stimulated BAL cells from AUD subjects in conjunction with increased IL-1ß. Our data suggest that AUDs augment pulmonary and systemic XOR activity that may contribute to ROS and uric acid generation, promoting inflammation. Further investigations will be necessary to determine if XOR inhibition can mitigate alcohol-associated pulmonary oxidative stress, diminish inflammation, and improve ARDS outcomes.

Alcohol abuse and smoking alter inflammatory mediator production by pulmonary and systemic immune cells.

Alcohol use disorders (AUDs) and tobacco smoking are associated with an increased predisposition for community-acquired pneumonia and the acute respiratory distress syndrome. Mechanisms are incompletely established but may include alterations in response to pathogens by immune cells, including alveolar macrophages (AMs) and peripheral blood mononuclear cells (PBMCs). We sought to determine the relationship of AUDs and smoking to expression of IFNγ, IL-1ß, IL-6, and TNFα by AMs and PBMCs from human subjects after stimulation with lipopolysaccharide (LPS) or lipoteichoic acid (LTA). AMs and PBMCs from healthy subjects with AUDs and controls, matched on smoking, were cultured with LPS (1 µg/ml) or LTA (5 µg/ml) in the presence and absence of the antioxidant precursor N-acetylcysteine (10 mM). Cytokines were measured in cell culture supernatants. Expression of IFNγ, IL-1ß, IL-6, and TNFα in AMs and PBMCs was significantly increased in response to stimulation with LPS and LTA. AUDs were associated with augmented production of proinflammatory cytokines, particularly IFNγ and IL-1ß, by AMs and PBMCs in response to LPS. Smoking diminished the impact of AUDs on AM cytokine expression. Expression of basal AM and PBMC Toll-like receptors-2 and -4 was not clearly related to differences in cytokine expression; however, addition of N-acetylcysteine with LPS or LTA led to diminished AM and PBMC cytokine secretion, especially among current smokers. Our findings suggest that AM and PBMC immune cell responses to LPS and LTA are influenced by AUDs and smoking through mechanisms that may include alterations in cellular oxidative stress.

The Relationship Between Airway Antioxidant Levels, Alcohol Use Disorders, and Cigarette Smoking.

BACKGROUND: Alcohol use disorders (AUDs) and cigarette smoking are associated with pulmonary oxidative stress, likely related to antioxidant depletion. Pulmonary oxidative stress may adversely affect innate immunity, leading to increased pneumonia susceptibility and severity, including development of the acute respiratory distress syndrome. In people with AUDs, most of whom smoke, antioxidant therapy can potentially restore immune cell function and attenuate pneumonia development. Challenges to human investigations of antioxidant therapies include an inability to identify pulmonary oxidative stress noninvasively and the optimal route to deliver pulmonary antioxidants. We sought to determine whether bronchoalveolar lavage (BAL) measures of thiol antioxidants from a 50-ml upper airway aliquot approximated those in the alveolar space and to determine whether AUDs and/or smoking affected these relationships. METHODS: Healthy human subjects with and without AUDs, including smokers and nonsmokers, underwent BAL. Samples obtained after the first 50-ml normal saline aliquot were analyzed as representing bronchial airways; subsequent 50-ml aliquots were analyzed as representative of the alveolar space. Reduced and oxidized (GSSG) glutathione, cysteine (Cys), and its oxidized species, cystine, along with mixed disulfides (MDs) were quantified using high-performance liquid chromatography. The percent of total thiols present in their oxidized forms, and thiol redox potentials, were calculated. RESULTS: Positive correlations between upper and lower BAL fluid thiol species were observed that were most robust for GSSG (ρ = 0.85), Cys (ρ = 0.83), and MDs (ρ = 0.69), but poor for thiol redox potential measures. In contrast to nonsmokers (either with or without AUDs), in subjects with AUDs who smoked, upper BAL fluid %GSSG, Cys, and MD measures were relatively increased compared to lower. CONCLUSIONS: A small volume BAL procedure may be suitable to assess intrapulmonary oxidative stress related to thiol depletion. Factors including AUDs and smoking may disproportionately increase upper airways oxidative stress that could be relevant for therapeutic interventions.

Neuroendocrine signaling via the serotonin transporter regulates clearance of apoptotic cells.

Serotonin (5-hydroxytryptamine; 5-HT) is a CNS neurotransmitter increasingly recognized to exert immunomodulatory effects outside the CNS that contribute to the pathogenesis of autoimmune and chronic inflammatory diseases. 5-HT signals to activate the RhoA/Rho kinase (ROCK) pathway, a pathway known for its ability to regulate phagocytosis. The clearance of apoptotic cells (i.e. efferocytosis) is a key modulator of the immune response that is inhibited by the RhoA/ROCK pathway. Because efferocytosis is defective in many of the same illnesses where 5-HT has been implicated in disease pathogenesis, we hypothesized that 5-HT would suppress efferocytosis via activation of RhoA/ROCK. The effect of 5-HT on efferocytosis was examined in murine peritoneal and human alveolar macrophages, and its mechanisms were investigated using pharmacologic blockade and genetic deletion. 5-HT impaired efferocytosis by murine peritoneal macrophages and human alveolar macrophages. 5-HT increased phosphorylation of myosin phosphatase subunit 1 (Mypt-1), a known ROCK target, and inhibitors of RhoA and ROCK reversed the suppressive effect of 5-HT on efferocytosis. Peritoneal macrophages expressed the 5-HT transporter and 5-HT receptors (R) 2a, 2b, but not 2c. Inhibition of 5-HTR2a and 5-HTR2b had no effect on efferocytosis, but blockade of the 5-HT transporter prevented 5-HT-impaired efferocytosis. Genetic deletion of the 5-HT transporter inhibited 5-HT uptake into peritoneal macrophages, prevented 5-HT-induced phosphorylation of Mypt-1, reversed the inhibitory effect of 5-HT on efferocytosis, and decreased cellular peritoneal inflammation. These results suggest a novel mechanism by which 5-HT might disrupt efferocytosis and contribute to the pathogenesis of autoimmune and chronic inflammatory diseases.

Growth differentiation factor-15 and prognosis in acute respiratory distress syndrome: a retrospective cohort study.

INTRODUCTION: We sought to determine whether higher levels of the novel biomarker growth differentiation factor-15 (GDF-15) are associated with poor outcomes and the presence of pulmonary vascular dysfunction (PVD) in patients with acute respiratory distress syndrome (ARDS). METHODS: We conducted a retrospective cohort study in patients enrolled in the Acute Respiratory Distress Syndrome Network Fluid and Catheter Treatment (FACT) Trial. Patients enrolled in the FACT Trial who received a pulmonary artery catheter (PAC), had plasma available from the same study day and sufficient hemodynamic data to determine the presence of PVD were included. Logistic regression was used to determine the association between GDF-15 level and 60-day mortality. RESULTS: Of the 513 patients enrolled in the FACT Trial assigned to receive a PAC, 400 were included in this analysis. Mortality at 60 days was significantly higher in patients whose GDF-15 levels were in the third (28%) or fourth (49%) quartile when compared to patients with GDF-15 levels in the first quartile (12%) (P <0.001). Adjusting for severity of illness measured by APACHE III score, the odds of death for patients with GDF-15 levels in the fourth quartile when compared to the first quartile was 4.26 (95% CI 2.18, 10.92, P <0.001). When added to APACHE III alone for prediction of 60-day mortality, GDF-15 levels increased the area under the receiver operating characteristic curve from 0.72 to 0.77. At an optimal cutoff of 8,103 pg/mL, the sensitivity and specificity of GDF-15 for predicting 60-day mortality were 62% (95% CI 53%, 71%) and 76% (95% CI 71%, 81%), respectively. Levels of GDF-15 were not useful in identifying the presence of PVD, as defined by hemodynamic measurements obtained by a PAC. CONCLUSIONS: In patients with ARDS, higher levels of GDF-15 are significantly associated with poor outcome but not PVD.

Protandim does not influence alveolar epithelial permeability or intrapulmonary oxidative stress in human subjects with alcohol use disorders.

Alcohol use disorders (AUDs), including alcohol abuse and dependence, have been linked to the development of acute lung injury (ALI). Prior clinical investigations suggested an association between AUDs and abnormal alveolar epithelial permeability mediated through pulmonary oxidative stress that may partially explain this relationship. We sought to determine if correcting pulmonary oxidative stress in the setting of AUDs would normalize alveolar epithelial permeability in a double-blinded, randomized, placebo-controlled trial of Protandim, a nutraceutical reported to enhance antioxidant activity. We randomized 30 otherwise healthy AUD subjects to receive directly observed inpatient oral therapy with either Protandim (1,350 mg/day) or placebo. Subjects underwent bronchoalveolar lavage (BAL) and blood sampling before study drug administration and after 7 days of therapy; all AUD subjects completed the study protocol without adverse events. BAL total protein was measured at each timepoint as an indicator of alveolar epithelial permeability. In subjects with AUDs, before study drug initiation, BAL total protein values were not significantly higher than in 11 concurrently enrolled controls (P = 0.07). Over the 7-day study period, AUD subjects did not exhibit a significant change in BAL total protein, regardless of their randomization to Protandim {n = 14, -2% [intraquartile range (IQR), -56-146%]} or to placebo [n = 16, 77% (IQR -20-290%); P = 0.19]. Additionally, among those with AUDs, no significant changes in BAL oxidative stress indexes, epithelial growth factor, fibroblast growth factor, interleukin-1ß, or interleukin-10 were observed regardless of drug type received. Plasma thiobarbituric acid reactive substances, a marker of lipid peroxidation, decreased significantly over time among AUD subjects randomized to placebo (P < 0.01). These results suggest that Protandim for 7 days in individuals with AUDs who are newly abstinent does not alter alveolar epithelial permeability. However, our work demonstrates the feasibility of safely conducting clinical trials that include serial bronchoscopies in a vulnerable population at risk for acute lung injury.

Alveolar macrophage gene expression is altered in the setting of alcohol use disorders.

BACKGROUND: Alcohol use disorders (AUDs) are associated with an increased susceptibility to a variety of common and devastating pulmonary diseases including community- and hospital-acquired pneumonias, as well as the acute respiratory distress syndrome (ARDS). Alveolar macrophages play an important role in preventing the development of these disorders through maintaining lung sterility and resolving lung inflammation. Although alcohol exposure has been associated with aberrant alveolar macrophage function in animal models, the clinical relevance of these observations in humans is not established. Therefore, we sought to determine the effects of AUDs on human alveolar macrophage gene expression. METHODS: Whole genome microarray analysis was performed on alveolar macrophages obtained by bronchoalveolar lavage from a test cohort of subjects with AUDs (n = 7), and controls (n = 7) who were pair-matched on age, gender, and smoking. Probe set expression differences in this cohort were validated by real-time reverse transcription-polymerase chain reaction (RT RT-PCR). Functional analysis with web-based bioinformatics tools was utilized with microarray data to assess differentially expressed candidate genes (p < 0.01) based on alcohol consumption. Alveolar macrophage mRNA samples from a second cohort of subjects with AUDs (n = 7) and controls (n = 7) were used to confirm gene expression differences related to AUDs. RESULTS: In both the test and the confirmatory cohorts, AUDs were associated with upregulation of alveolar macrophage gene expression related to apoptosis, including perforin-1, granzyme A, and CXCR4 (fusin). Pathways governing the regulation of progression through cell cycle and immune response were also affected, as was upregulation of gene expression for mitochondrial superoxide dismutase. Overall, 12 genes' expression was affected by AUDs independent of smoking. CONCLUSIONS: Alcohol use disorders are associated with unique changes in human alveolar macrophage gene expression. Novel therapies targeting alveolar macrophage gene expression in the setting of AUDs may prove to be clinically useful in limiting susceptibility for pulmonary disorders in these individuals.

Acute lung injury but not sepsis is associated with increased colony formation by peripheral blood mononuclear cells.

Acute lung injury (ALI) and severe sepsis are common critical illnesses associated with the mobilization of bone marrow-derived cells into the circulation. By identifying and determining these cells' functional characteristics, unique prognostic biomarkers can be developed to help investigators understand the mechanisms underlying the pathophysiology of these disorders. We previously demonstrated an increased colony-forming unit (CFU) ability of circulating peripheral blood mononuclear cells (PBMCs) in patients with ALI, compared with healthy control subjects, that also correlated with improved survival. Here we hypothesized that the increased CFUs in ALI are associated with lung injury, and therefore ALI will result in an increased number of CFUs compared with patients exhibiting severe sepsis. To test this, blood was collected from 80 patients (63 with ALI, and 17 with severe sepsis) within 72 hours of diagnosis, and from 5 healthy control subjects. A CFU assay was performed on isolated PBMCs. Lung injury scores and the need for mechanical ventilation were greater in patients with ALI than in patients with severe sepsis (P < 0.0001 for each). CFU numbers were highest in patients with ALI compared with patients manifesting severe sepsis or control subjects (median CFU number [25-75% quartiles] of 61 [13-104] versus 17 [3-34] versus 5 [2-13], P < 0.0005). A trend toward improved survival was demonstrated in patients with high (> or = 48) CFUs (P = 0.06). No relationship between CFUs and mechanical ventilation was evident. Our findings suggest that increased colony-forming ability by PBMCs in ALI results from lung injury, independent of sepsis and mechanical ventilation. Factors contributing to colony formation by PBMCs in ALI, and the role PBMCs play in its pathogenesis remain to be fully established.

Alcohol use disorders affect antimicrobial proteins and anti-pneumococcal activity in epithelial lining fluid obtained via bronchoalveolar lavage.

AIMS: Our overall objective was to examine whether characteristics of epithelial lining fluid (ELF) from subjects with alcohol use disorders (AUDs) obtained via bronchoalveolar lavage (BAL) contribute to their predisposition to pneumococcal pneumonia. We sought to compare the anti-pneumococcal activity of acellular human BAL from subjects with AUDs to matched controls. Further, differences in BAL lysozyme activity and lactoferrin concentrations between these two groups were examined to determine the effect of AUDs on these antimicrobial proteins. METHODS: BAL was performed in subjects with AUDs and matched controls. Acellular BAL was used at varying concentrations in an in vitro killing assay of Streptococcus pneumoniae, type 2, and the percent kill of organisms per microgram per milliliter total BAL protein was ascertained. Lysozyme activity and lactoferrin concentrations were measured in BAL from subjects and controls at measured concentrations of BAL protein. RESULTS: AUD subjects (n = 15) and controls (n = 10) were enrolled in these investigations who were balanced in terms of smoking history. Using a mixed effect model, across the range of BAL protein concentrations, killing of pneumococcus tended to be less potent with BAL fluid from AUD subjects. Additionally, lysozyme activity and lactoferrin concentrations were significantly lower in the AUD group. CONCLUSIONS: The predisposition for pneumococcal pneumonia among those with AUDs may be in part mediated through effects of alcohol on substances within ELF that include antimicrobial proteins. Clarifying the composition and activity of ELF antimicrobial proteins in the setting of AUDs via investigations with human BAL fluid can help establish their contribution to the susceptibility for pulmonary infections in these individuals.

The impact of alcohol use disorders on pulmonary immune cell inflammatory responses to Streptococcus pneumoniae.

Community-acquired pneumonia due to Streptococcus pneumoniae occurs commonly in alcohol use disorders (AUDs). Pneumonia in the AUD patient is associated with poorer outcomes, and specific therapies to mitigate disease severity in these patients do not exist. Numerous investigations have attributed increased severity of pneumonia in AUDs to aberrant function of the alveolar macrophage (AM), a lung immune cell critical in host defense initiation. No studies have examined the response of human AMs to S. pneumoniae in AUDs. We hypothesized that the inflammatory mediators released by AMs after S. pneumoniae stimulation would differ quantitatively in individuals with AUDs compared to non-AUD participants. We further postulated that AM inflammatory mediators would be diminished after exposure to the antioxidant, N-acetylcysteine (NAC). For comparison, responses of peripheral blood mononuclear cells (PBMCs) to pneumococcal protein were also examined. Otherwise healthy participants with AUDs and smoking-matched controls underwent bronchoalveolar lavage and peripheral blood sampling to obtain AMs and PBMCs, respectively. Freshly collected cells were cultured with increasing doses of heat-killed S. pneumoniae protein, with and without exposure to N-acetylcysteine. Cell culture supernatants were collected, and inflammatory mediators were measured, including interferon (IFN)-γ, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α. IFN-γ and IL-6 were significantly higher in unstimulated AM cell culture supernatants from subjects with AUDs. After stimulation with pneumococcal protein, a dose-response and time-dependent increase in pro-inflammatory cytokine production by both AMs and PBMCs was also observed; differences were not observed between AUD and non-AUD subjects. Addition of NAC to pneumococcal-stimulated AMs and PBMCs was generally associated with diminished cytokine production, with the exception of IL-1ß that was elevated in AM culture supernatants from subjects with AUDs. Our observations suggest that AUDs contribute to basal alterations in AM pro-inflammatory cytokine elaboration, but did not support consistent differences in pneumococcal-stimulated AM or PBMC inflammatory mediator secretion that were referable to AUDs.

The PhenoGen informatics website: tools for analyses of complex traits.

BACKGROUND: With the advent of "omics" (e.g. genomics, transcriptomics, proteomics and phenomics), studies can produce enormous amounts of data. Managing this diverse data and integrating with other biological data are major challenges for the bioinformatics community. Comprehensive new tools are needed to store, integrate and analyze the data efficiently. DESCRIPTION: The PhenoGen Informatics website http://phenogen.uchsc.edu is a comprehensive toolbox for storing, analyzing and integrating microarray data and related genotype and phenotype data. The site is particularly suited for combining QTL and microarray data to search for "candidate" genes contributing to complex traits. In addition, the site allows, if desired by the investigators, sharing of the data. Investigators can conduct "in-silico" microarray experiments using their own and/or "shared" data. CONCLUSION: The PhenoGen website provides access to tools that can be used for high-throughput data storage, analyses and interpretation of the results. Some of the advantages of the architecture of the website are that, in the future, the present set of tools can be adapted for the analyses of any type of high-throughput "omics" data, and that access to new tools, available in the public domain or developed at PhenoGen, can be easily provided.

Impact of Regular Cannabis Use on Biomarkers of Lower Urinary Tract Function.

OBJECTIVE: To evaluate the differences in the composition and quantities of urine peptides in regular cannabis users and nonusers by liquid chromatography tandem mass spectrometry analysis. MATERIALS AND METHODS: Urine specimens from healthy control subjects and cannabis users were utilized to identify the differences in the number and quantity of urine proteins by liquid chromatography tandem mass spectrometry analysis. Significantly altered proteins were determined by a permutation testing statistical method. Heat map, dendrogram, pathway, and network analyses were performed to assess the degree of expression and the potential relationships between proteins in both groups. RESULTS: A total of 1337 proteins were detected in both groups with 19 proteins being significantly altered in cannabis users. Innate immunity and carbohydrate metabolic pathways were highly linked with upregulated proteins in the cannabis group. Additionally, 91 proteins were present and 46 proteins were absent only in cannabis users in comparison with the control cohort. Our results suggest that regular use of cannabis is associated with significant alterations in a number of urinary peptides, with a large number of proteins present or absent only in cannabis users. Pathway analyses demonstrated an increased immune response in cannabis users compared with controls. CONCLUSION: Our observations potentially indicate activation (or inhibition) of specific signaling pathways in the lower urinary tract during chronic exposure to exogenous cannabinoids. Our study provides initial proteomic knowledge for future investigations on the potential role of exocannabinoids in the development of intravesical therapies to treat lower urinary tract disorders.

Streamlining microarray technology in a prototype core laboratory.

The overall scheme of a microarray experiment is summarized in Figure 3. The real benefit of using microarrays in research is gaining knowledge from the abundant data provided from the series of related microarray experiments. The core laboratory has produced three cDNA arrays, a mouse 15K array, a human 13K array, and a human apoptosis array with 350 plus apoptotic elements. Recently, the 15K array is being used in building a database for gene expression in several areas of the mouse brain and for studying transgenic and knockout mice. The apoptosis array has been used by cancer researchers to further elucidate changes and interactions between genes leading to cell death and cancer. The UCHSC Gene Expression Array Core is supported by the National Institute on Aging and the National Cancer Institute (Bethesda, MD) to serve all academic users and has a special interest in providing a solid technological base for genomic researchers interested in alcohol and cancer research.