Gut microbiome dynamics and associations with mortality in critically ill patients.

BACKGROUND: Critical illness and care within the intensive care unit (ICU) leads to profound changes in the composition of the gut microbiome. The impact of such changes on the patients and their subsequent disease course remains uncertain. We hypothesized that specific changes in the gut microbiome would be more harmful than others, leading to increased mortality in critically ill patients. METHODS: This was a prospective cohort study of critically ill adults in the ICU. We obtained rectal swabs from 52 patients and assessed the composition the gut microbiome using 16 S rRNA gene sequencing. We followed patients throughout their ICU course and evaluated their mortality rate at 28 days following admission to the ICU. We used selbal, a machine learning method, to identify the balance of microbial taxa most closely associated with 28-day mortality. RESULTS: We found that a proportional ratio of four taxa could be used to distinguish patients with a higher risk of mortality from patients with a lower risk of mortality (p = .02). We named this binarized ratio our microbiome mortality index (MMI). Patients with a high MMI had a higher 28-day mortality compared to those with a low MMI (hazard ratio, 2.2, 95% confidence interval 1.1-4.3), and remained significant after adjustment for other ICU mortality predictors, including the presence of the acute respiratory distress syndrome (ARDS) and the Acute Physiology and Chronic Health Evaluation (APACHE II) score (hazard ratio, 2.5, 95% confidence interval 1.4-4.7). High mortality was driven by taxa from the Anaerococcus (genus) and Enterobacteriaceae (family), while lower mortality was driven by Parasutterella and Campylobacter (genera). CONCLUSIONS: Dysbiosis in the gut of critically ill patients is an independent risk factor for increased mortality at 28 days after adjustment for clinically significant confounders. Gut dysbiosis may represent a potential therapeutic target for future ICU interventions.

Comparing Long-Term Prognosis in Chronic Critically Ill Patients: A Case Series Study of Medical versus Surgical Sepsis.

Background and Objectives: Chronic critical illness (CCI) is a syndrome characterized by persistent organ dysfunction that requires critical care therapy for ≥14 days. Sepsis and respiratory failure constitute the two primary causes of CCI. A better understanding of this patient population and their clinical course may help to risk-stratify them early during hospitalization. Our objective was to identify whether the source of sepsis (medical versus surgical) affected clinical trajectory and prognosis in patients developing CCI. Materials and Methods: We describe a cohort of patients having acute respiratory failure and sepsis and requiring critical care therapy in the medical (MICU) or surgical (SICU) critical care units for ≥14 days. Given the relative infrequency of CCI, we use a case series design to examine mortality, functional status, and place of residence (home versus non-home) at one year following their index hospitalization. Results: In medical patients developing CCI (n = 31), the severity of initial organ dysfunction, by SOFA score, was significantly associated with the development of CCI (p = 0.002). Surgical patients with CCI (n = 7) experienced significantly more ventilator-free days within the first 30 days following sepsis onset (p = 0.004), as well as less organ dysfunction at day 14 post-sepsis (p < 0.0001). However, one-year mortality, one-year functional status, and residency at home were not statistically different between cohorts. Moreover, 57% of surgical patients and 26% of medical patients who developed CCI were living at home for one year following their index hospitalization (p = 0.11). Conclusions: While surgical patients who develop sepsis-related CCI experience more favorable 30-day outcomes as compared with medical patients, long-term outcomes do not differ significantly between groups. This suggests that reversing established organ dysfunction and functional disability, regardless of etiology, is more challenging compared to preventing these complications at an earlier stage.

Analysis of CDR3 Sequences from T-Cell Receptor ß in Acute Respiratory Distress Syndrome.

Acute Respiratory Distress Syndrome (ARDS) is an illness that typically develops in people who are significantly ill or have serious injuries. ARDS is characterized by fluid build-up that occurs in the alveoli. T-cells are implicated as playing a role in the modulation of the aberrant response leading to excessive tissue damage and, eventually, ARDS. Complementarity Determining Region 3 (CDR3) sequences derived from T-cells are key players in the adaptive immune response. This response is governed by an elaborate specificity for distinct molecules and the ability to recognize and vigorously respond to repeated exposures to the same molecules. Most of the diversity in T-cell receptors (TCRs) is contained in the CDR3 regions of the heterodimeric cell-surface receptors. For this study, we employed the novel technology of immune sequencing to assess lung edema fluid. Our goal was to explore the landscape of CDR3 clonal sequences found within these samples. We obtained more than 3615 CDR3 sequences across samples in the study. Our data demonstrate that: (1) CDR3 sequences from lung edema fluid exhibit distinct clonal populations, and (2) CDR3 sequences can be further characterized based on biochemical features. Analysis of these CDR3 sequences offers insight into the CDR3-driven T-cell repertoire of ARDS. These findings represent the first step towards applications of this technology with these types of biological samples in the context of ARDS.

Surfactant Protein D Influences Mortality During Abdominal Sepsis by Facilitating Escherichia coli Colonization in the Gut.

Determine the role of surfactant protein D (SPD) in sepsis. DESIGN: Murine in vivo study. SETTING: Research laboratory at an academic medical center. PATIENTS: SPD knockout (SPD-/-) and wild-type (SPD+/+) mice. INTERVENTIONS: SPD-/- and SPD+/+ mice were subjected to cecal ligation and puncture (CLP). After CLP, Escherichia coli bacteremia was assessed in both groups. Cecal contents from both groups were cultured to assess for colonization by E. coli. To control for parental effects on the microbiome, SPD-/- and SPD+/+ mice were bred from heterozygous parents, and levels of E. coli in their ceca were measured. Gut segments were harvested from mice, and SPD protein expression was measured by Western blot. SPD-/- mice were gavaged with green fluorescent protein, expressing E. coli and recombinant SPD (rSPD). MEASUREMENTS AND MAIN RESULTS: SPD-/- mice had decreased mortality and decreased E. coli bacteremia compared with SPD+/+ mice following CLP. At baseline, SPD-/- mice had decreased E. coli in their cecal flora. When SPD-/- and SPD+/+ mice were bred from heterozygous parents and then separated after weaning, less E. coli was cultured from the ceca of SPD-/- mice. E. coli gut colonization was increased by gavage of rSPD in SPD-/- mice. The source of enteric SPD in SPD+/+ mice was the gallbladder. CONCLUSIONS: Enteral SPD exacerbates mortality after CLP by facilitating colonization of the mouse gut with E. coli.

Tracheal aspirate transcriptomic and miRNA signatures of extreme premature birth with bronchopulmonary dysplasia.

OBJECTIVE: Extreme preterm infants are a growing population in neonatal intensive care units who carry a high mortality and morbidity. Multiple factors play a role in preterm birth, resulting in major impact on organogenesis leading to complications including bronchopulmonary dysplasia (BPD). The goal of this study was to identify biomarker signatures associated with prematurity and BPD. STUDY DESIGN: We analyzed miRNA and mRNA profiles in tracheal aspirates (TAs) from 55 infants receiving invasive mechanical ventilation. Twenty-eight infants were extremely preterm and diagnosed with BPD, and 27 were term babies receiving invasive mechanical ventilation for elective procedures. RESULT: We found 22 miRNAs and 33 genes differentially expressed (FDR < 0.05) in TAs of extreme preterm infants with BPD vs. term babies without BPD. Pathway analysis showed associations with inflammatory response, cellular growth/proliferation, and tissue development. CONCLUSIONS: Specific mRNA-miRNA signatures in TAs may serve as biomarkers for BPD pathogenesis, a consequence of extreme prematurity.

Learning gene networks underlying clinical phenotypes using SNP perturbation.

Availability of genome sequence, molecular, and clinical phenotype data for large patient cohorts generated by recent technological advances provides an opportunity to dissect the genetic architecture of complex diseases at system level. However, previous analyses of such data have largely focused on the co-localization of SNPs associated with clinical and expression traits, each identified from genome-wide association studies and expression quantitative trait locus mapping. Thus, their description of the molecular mechanisms behind the SNPs influencing clinical phenotypes was limited to the single gene linked to the co-localized SNP. Here we introduce PerturbNet, a statistical framework for learning gene networks that modulate the influence of genetic variants on phenotypes, using genetic variants as naturally occurring perturbation of a biological system. PerturbNet uses a probabilistic graphical model to directly model the cascade of perturbation from genetic variants to the gene network to the phenotype network along with the networks at each layer of the biological system. PerturbNet learns the entire model by solving a single optimization problem with an efficient algorithm that can analyze human genome-wide data within a few hours. PerturbNet inference procedures extract a detailed description of how the gene network modulates the genetic effects on phenotypes. Using simulated and asthma data, we demonstrate that PerturbNet improves statistical power for detecting disease-linked SNPs and identifies gene networks and network modules mediating the SNP effects on traits, providing deeper insights into the underlying molecular mechanisms.

Whole blood RNA sequencing reveals a unique transcriptomic profile in patients with ARDS following hematopoietic stem cell transplantation.

BACKGROUND: The acute respiratory distress syndrome (ARDS) is characterized by the acute onset of hypoxemia and bilateral lung infiltrates in response to an inciting event, and is associated with high morbidity and mortality. Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) are at increased risk for ARDS. We hypothesized that HSCT patients with ARDS would have a unique transcriptomic profile identifiable in peripheral blood compared to those that did not undergo HSCT. METHODS: We isolated RNA from banked peripheral blood samples from a biorepository of critically ill ICU patients. RNA-Seq was performed on 11 patients with ARDS (5 that had undergone HSCT and 6 that had not) and 12 patients with sepsis without ARDS (5 that that had undergone HCST and 7 that had not). RESULTS: We identified 687 differentially expressed genes between ARDS and ARDS-HSCT (adjusted p-value < 0.01), including IFI44L, OAS3, LY6E, and SPATS2L that had increased expression in ARDS vs. ARDS-HSCT; these genes were not differentially expressed in sepsis vs sepsis-HSCT. Gene ontology enrichment analysis revealed that many differentially expressed genes were related to response to type I interferon. CONCLUSIONS: Our findings reveal significant differences in whole blood transcriptomic profiles of patients with non-HSCT ARDS compared to ARDS-HSCT patients and point toward different immune responses underlying ARDS and ARDS-HSCT that contribute to lung injury.

Circulating RIPK3 levels are associated with mortality and organ failure during critical illness.

BACKGROUND: Necroptosis is a form of programmed necrotic cell death that is rapidly emerging as an important pathophysiological pathway in numerous disease states. Necroptosis is dependent on receptor-interacting protein kinase 3 (RIPK3), a protein shown to play an important role in experimental models of critical illness. However, there is limited clinical evidence regarding the role of extracellular RIPK3 in human critical illness. METHODS: Plasma RIPK3 levels were measured in 953 patients prospectively enrolled in 5 ongoing intensive care unit (ICU) cohorts in both the USA and Korea. RIPK3 concentrations among groups were compared using prospectively collected phenotypic and outcomes data. RESULTS: In all 5 cohorts, extracellular RIPK3 levels in the plasma were higher in patients who died in the hospital compared with those who survived to discharge. In a combined analysis, increasing RIPK3 levels were associated with elevated odds of in-hospital mortality (odds ratio [OR] 1.7 for each log10-unit increase in RIPK3 level, P < 0.0001). When adjusted for baseline severity of illness, the OR for in-hospital mortality remained statistically significant (OR 1.33, P = 0.007). Higher RIPK3 levels were also associated with more severe organ failure. CONCLUSIONS: Our findings suggest that elevated levels of RIPK3 in the plasma of patients admitted to the ICU are associated with in-hospital mortality and organ failure. FUNDING: Supported by NIH grants P01 HL108801, R01 HL079904, R01 HL055330, R01 HL060234, K99 HL125899, and KL2TR000458-10. Supported by Samsung Medical Center grant SMX1161431.

Phospholipase D isoforms differentially regulate leukocyte responses to acute lung injury.

Phospholipase D (PLD) plays important roles in cellular responses to tissue injury that are critical to acute inflammatory diseases, such as the acute respiratory distress syndrome (ARDS). We investigated the expression of PLD isoforms and related phospholipid phosphatases in patients with ARDS, and their roles in a murine model of self-limited acute lung injury (ALI). Gene expression microarray analysis on whole blood obtained from patients that met clinical criteria for ARDS and clinically matched controls (non-ARDS) demonstrated that PLD1 gene expression was increased in patients with ARDS relative to non-ARDS and correlated with survival. In contrast, PLD2 expression was associated with mortality. In a murine model of self-resolving ALI, lung Pld1 expression increased and Pld2 expression decreased 24 h after intrabronchial acid. Total lung PLD activity was increased 24 h after injury. Pld1-/- mice demonstrated impaired alveolar barrier function and increased tissue injury relative to WT and Pld2-/- , whereas Pld2-/- mice demonstrated increased recruitment of neutrophils and macrophages, and decreased tissue injury. Isoform-specific PLD inhibitors mirrored the results with isoform-specific Pld-KO mice. PLD1 gene expression knockdown in human leukocytes was associated with decreased phagocytosis by neutrophils, whereas reactive oxygen species production and phagocytosis decreased in M2-macrophages. PLD2 gene expression knockdown increased neutrophil and M2-macrophage transmigration, and increased M2-macrophage phagocytosis. These results uncovered selective regulation of PLD isoforms after ALI, and opposing effects of selective isoform knockdown on host responses and tissue injury. These findings support therapeutic strategies targeting specific PLD isoforms for the treatment of ARDS.

Multicohort Analysis of Whole-Blood Gene Expression Data Does Not Form a Robust Diagnostic for Acute Respiratory Distress Syndrome.

OBJECTIVES: To identify a novel, generalizable diagnostic for acute respiratory distress syndrome using whole-blood gene expression arrays from multiple acute respiratory distress syndrome cohorts of varying etiologies. DATA SOURCES: We performed a systematic search for human whole-blood gene expression arrays of acute respiratory distress syndrome in National Institutes of Health Gene Expression Omnibus and ArrayExpress. We also included the Glue Grant gene expression cohorts. STUDY SELECTION: We included investigator-defined acute respiratory distress syndrome within 48 hours of diagnosis and compared these with relevant critically ill controls. DATA EXTRACTION: We used multicohort analysis of gene expression to identify genes significantly associated with acute respiratory distress syndrome, both with and without adjustment for clinical severity score. We performed gene ontology enrichment using Database for Annotation, Visualization and Integrated Discovery and cell type enrichment tests for both immune cells and pneumocyte gene expression. Finally, we selected a gene set optimized for diagnostic power across the datasets and used leave-one-dataset-out cross validation to assess robustness of the model. DATA SYNTHESIS: We identified datasets from three adult cohorts with sepsis, one pediatric cohort with acute respiratory failure, and two datasets of adult patients with trauma and burns, for a total of 148 acute respiratory distress syndrome cases and 268 critically ill controls. We identified 30 genes that were significantly associated with acute respiratory distress syndrome (false discovery rate < 20% and effect size >1.3), many of which had been previously associated with sepsis. When metaregression was used to adjust for clinical severity scores, none of these genes remained significant. Cell type enrichment was notable for bands and neutrophils, suggesting that the gene expression signature is one of acute inflammation rather than lung injury per se. Finally, an attempt to develop a generalizable diagnostic gene set for acute respiratory distress syndrome showed a mean area under the receiver-operating characteristic curve of only 0.63 on leave-one-dataset-out cross validation. CONCLUSIONS: The whole-blood gene expression signature across a wide clinical spectrum of acute respiratory distress syndrome is likely confounded by systemic inflammation, limiting the utility of whole-blood gene expression studies for uncovering a generalizable diagnostic gene signature.

NLRP3 Inflammasome Deficiency Protects against Microbial Sepsis via Increased Lipoxin B4 Synthesis.

RATIONALE: Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection, is a major public health concern with high mortality and morbidity. Although inflammatory responses triggered by infection are crucial for host defense against invading microbes, the excessive inflammation often causes tissue damage leading to organ dysfunction. Resolution of inflammation, an active immune process mediated by endogenous lipid mediators (LMs), is important to maintain host homeostasis. OBJECTIVES: We sought to determine the role of the nucleotide-binding domain, leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3) inflammasome in polymicrobial sepsis and regulation of LM biosynthesis. METHODS: We performed cecal ligation and puncture (CLP) using mice lacking NLRP3 inflammasome-associated molecules to assess mortality. Inflammation was evaluated by using biologic fluids including plasma, bronchoalveolar, and peritoneal lavage fluid. Local acting LMs in peritoneal lavage fluid from polymicrobacterial septic mice were assessed by mass spectrometry-based metabololipidomics. MEASUREMENTS AND MAIN RESULTS: Genetic deficiency of NLRP3 inhibited inflammatory responses and enhanced survival of CLP-induced septic mice. NLRP3 deficiency reduced proinflammatory LMs and increased proresolving LM, lipoxin B4 (LXB4) in septic mice, and in macrophages stimulated with LPS and ATP. Activation of the NLRP3 inflammasome induced caspase-7 cleavage and pyroptosis. Caspase-7 deficiency similarly reduced inflammation and mortality in CLP-induced sepsis, and increased LXB4 production in vivo and in vitro. Exogenous application of LXB4 reduced inflammation, pyroptosis, and mortality of mice after CLP. CONCLUSIONS: Genetic deficiency of NLRP3 promoted resolution of inflammation in polymicrobial sepsis by relieving caspase-7-dependent repression of LXB4 biosynthesis, and increased survival potentially via LXB4 production and inhibition of proinflammatory cytokines.

Inflammasomes: Key Mediators of Lung Immunity.

Inflammasomes are key inflammatory signaling platforms that detect microbial substances, sterile environmental insults, and molecules derived from host cells. Activation of the inflammasome promotes caspase-1-mediated secretion of proinflammatory cytokines interleukin (IL)-1ß and IL-18 and pyroptosis. Recent developments in this field demonstrate the crucial role of the inflammasome in a wide range of disease models. Although inflammasomes are a crucial part of host defense mechanisms against pathogens, the exuberant immune response resulting from inflammasome activation also contributes to the development of various diseases. As ongoing studies further elucidate the regulation and function of the inflammasome, more evidence has emerged that the inflammasome appears to play a pivotal role in the development of multiple inflammatory diseases. Here, we discuss recent insights into how inflammasomes are regulated to activate caspase-1 and implicated in human diseases. We also review the contributions of the inflammasome to pulmonary diseases.

A time-varying group sparse additive model for genome-wide association studies of dynamic complex traits.

MOTIVATION: Despite the widespread popularity of genome-wide association studies (GWAS) for genetic mapping of complex traits, most existing GWAS methodologies are still limited to the use of static phenotypes measured at a single time point. In this work, we propose a new method for association mapping that considers dynamic phenotypes measured at a sequence of time points. Our approach relies on the use of Time-Varying Group Sparse Additive Models (TV-GroupSpAM) for high-dimensional, functional regression. RESULTS: This new model detects a sparse set of genomic loci that are associated with trait dynamics, and demonstrates increased statistical power over existing methods. We evaluate our method via experiments on synthetic data and perform a proof-of-concept analysis for detecting single nucleotide polymorphisms associated with two phenotypes used to assess asthma severity: forced vital capacity, a sensitive measure of airway obstruction and bronchodilator response, which measures lung response to bronchodilator drugs. AVAILABILITY AND IMPLEMENTATION: Source code for TV-GroupSpAM freely available for download at http://www.cs.cmu.edu/~mmarchet/projects/tv_group_spam, implemented in MATLAB. CONTACT: epxing@cs.cmu.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Low quality of dying and death in patients with septic shock as perceived by nurses and resident physicians.

Septic shock is a disease with both high prevalence and mortality. Few studies have evaluated the quality of dying and death (QODD) in patients with septic shock. The authors compared the QODD of patients who died of septic shock versus other causes. They prospectively collected QODD surveys from nurses and residents caring for 196 patients who died in the medical intensive care unit (ICU) at an urban, university hospital. Patients were included in the analysis if either a nurse or resident returned a survey. Chart review established cause of death. The authors compared total QODD scores (on a scale of 0-100) and a single-item score (QODD-1; on a scale of 0-10) of patients who died of septic shock versus other causes. Survey response rates were 59% (n = 155) for residents and 49% (n = 129) for nurses. Nurses rated patients as having lower total QODD and QODD-1 scores for septic (Δ 7.5 points, p = 0.03, and 0.9 points, p = 0.05, respectively). Residents rated septic patients with lower QODD-1 scores than nonseptic patients (Δ 0.8 points, p = 0.03). This study shows that nurses rate patients with septic shock as having lower QODD than patients dying of other causes. These findings are important for clinicians who counsel families of patients dying of septic shock.

Circulating microRNAs as biomarkers in patients with allergic rhinitis and asthma.

BACKGROUND: MicroRNAs (miRNAs) are emerging as important regulatory molecules that might be involved in the pathogenesis of various diseases. Circulating miRNAs might be noninvasive biomarkers to diagnose and characterize asthma and allergic rhinitis (AR). OBJECTIVE: We sought to determine whether miRNAs are differentially expressed in the blood of asthmatic patients compared with those in the blood of nonasthmatic patients with AR and nonallergic nonasthmatic subjects. Furthermore, we sought to establish whether miRNAs could be used to characterize or subtype asthmatic patients. METHODS: Expression of plasma miRNAs was measured by using real-time quantitative PCR in 35 asthmatic patients, 25 nonasthmatic patients with AR, and 19 nonallergic nonasthmatic subjects. Differentially expressed miRNAs were identified by using Kruskal-Wallis 1-way ANOVA with Bonferroni P value adjustment to correct for multiple comparisons. A random forest classification algorithm combined with a leave-one-out cross-validation approach was implemented to assess the predictive capacities of the profiled miRNAs. RESULTS: We identified 30 miRNAs that were differentially expressed among healthy, allergic, and asthmatic subjects. These miRNAs fit into 5 different expression pattern groups. Among asthmatic patients, miRNA expression profiles identified 2 subtypes that differed by high or low peripheral eosinophil levels. Circulating miR-125b, miR-16, miR-299-5p, miR-126, miR-206, and miR-133b levels were most predictive of allergic and asthmatic status. CONCLUSIONS: Subsets of circulating miRNAs are uniquely expressed in patients with AR and asthmatic patients and have potential for use as noninvasive biomarkers to diagnose and characterize these diseases.

Gene expression profiling of asthma phenotypes demonstrates molecular signatures of atopy and asthma control.

BACKGROUND: Recent studies have used cluster analysis to identify phenotypic clusters of asthma with differences in clinical traits, as well as differences in response to therapy with anti-inflammatory medications. However, the correspondence between different phenotypic clusters and differences in the underlying molecular mechanisms of asthma pathogenesis remains unclear. OBJECTIVE: We sought to determine whether clinical differences among children with asthma in different phenotypic clusters corresponded to differences in levels of gene expression. METHODS: We explored differences in gene expression profiles of CD4(+) lymphocytes isolated from the peripheral blood of 299 young adult participants in the Childhood Asthma Management Program study. We obtained gene expression profiles from study subjects between 9 and 14 years of age after they participated in a randomized, controlled longitudinal study examining the effects of inhaled anti-inflammatory medications over a 48-month study period, and we evaluated the correspondence between our earlier phenotypic cluster analysis and subsequent follow-up clinical and molecular profiles. RESULTS: We found that differences in clinical characteristics observed between subjects assigned to different phenotypic clusters persisted into young adulthood and that these clinical differences were associated with differences in gene expression patterns between subjects in different clusters. We identified a subset of genes associated with atopic status, validated the presence of an atopic signature among these genes in an independent cohort of asthmatic subjects, and identified the presence of common transcription factor binding sites corresponding to glucocorticoid receptor binding. CONCLUSION: These findings suggest that phenotypic clusters are associated with differences in the underlying pathobiology of asthma. Further experiments are necessary to confirm these findings.

UCP2-induced fatty acid synthase promotes NLRP3 inflammasome activation during sepsis.

Cellular lipid metabolism has been linked to immune responses; however, the precise mechanisms by which de novo fatty acid synthesis can regulate inflammatory responses remain unclear. The NLRP3 inflammasome serves as a platform for caspase-1-dependent maturation and secretion of proinflammatory cytokines. Here, we demonstrated that the mitochondrial uncoupling protein-2 (UCP2) regulates NLRP3-mediated caspase-1 activation through the stimulation of lipid synthesis in macrophages. UCP2-deficient mice displayed improved survival in a mouse model of polymicrobial sepsis. Moreover, UCP2 expression was increased in human sepsis. Consistently, UCP2-deficient mice displayed impaired lipid synthesis and decreased production of IL-1ß and IL-18 in response to LPS challenge. In macrophages, UCP2 deficiency suppressed NLRP3-mediated caspase-1 activation and NLRP3 expression associated with inhibition of lipid synthesis. In UCP2-deficient macrophages, inhibition of lipid synthesis resulted from the downregulation of fatty acid synthase (FASN), a key regulator of fatty acid synthesis. FASN inhibition by shRNA and treatment with the chemical inhibitors C75 and cerulenin suppressed NLRP3-mediated caspase-1 activation and inhibited NLRP3 and pro-IL-1ß gene expression in macrophages. In conclusion, our results suggest that UCP2 regulates the NLRP3 inflammasome by inducing the lipid synthesis pathway in macrophages. These results identify UCP2 as a potential therapeutic target in inflammatory diseases such as sepsis.

Knockdown of Drosha in human alveolar type II cells alters expression of SP-A in culture: a pilot study.

Human surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. SP-A is synthesized and secreted by alveolar type II (ATII) cells, one of the two cell types of the distal lung epithelium (ATII and ATI). We have shown that miRNA interactions with sequence polymorphisms on the SP-A mRNA 3'UTRs mediate differential expression of SP-A1 and SP-A2 gene variants in vitro. In the present study, we describe a physiologically relevant model to study miRNA regulation of SP-A in human ATII. For these studies, we purified and cultured human ATII on an air-liquid interface matrix (A/L) or plastic wells without matrix (P). Gene expression analyses confirmed that cells cultured in A/L maintained the ATII phenotype for over 5 days, whereas P-cultured cells differentiated to ATI. When we transfected ATII with siRNAs to inhibit the expression of Drosha, a critical effector of miRNA maturation, the levels of SP-A mRNA and protein increased in a time dependent manner. We next characterized cultured ATII and ATI by studying expression of 1,066 human miRNAs using miRNA PCR arrays. We detected expression of >300 miRNAs with 24 miRNAs differentially expressed in ATII versus ATI, 12 of which predicted to bind SP-A 3'UTRs, indicating that these may be implicated in SP-A downregulation in ATI. Thus, miRNAs not only affect SP-A expression, but also may contribute to the maintenance of the ATII cell phenotype and/or the trans-differentiation of ATII to ATI cells, and may represent new molecular markers that distinguish ATII and ATI.

Family structure, experiences with end-of-life decision making, and who asked about advance directives impacts advance directive completion rates.

BACKGROUND: Advance directives are an important but underutilized resource. Reasons for this underutilization need to be determined. OBJECTIVE: We investigated factors associated with completion of advance directives among inpatients. DESIGN: We conducted prospective, structured interviews on family structure, health care, disease, and end-of-life experiences. We compared those with completed advance directives and those without. SETTING/SUBJECTS: We interviewed 130 inpatients in an urban university hospital. MEASUREMENTS: We used bivariate analysis and logistic regression to identify characteristics of patients with living wills and health care proxies versus patients without them. RESULTS: Twenty-one percent of patients had a living will and 35% had a health care proxy. Patients with completed living wills were older (p≤0.0046), had more comorbidities (p=0.018), were widowed (p=0.02), and were more often admitted with chronic disease (p=0.009) compared to those without living wills. Patients with health care proxies were older (p<0.001), had religious affiliations (p=0.04), more children (p=0.03), and more often widowed (p≤0.001) than those without health care proxies. Patients were 10.8 times (95% confidence interval [CI] 4.59-25.3), 46.5 times (95% CI 15.1-139.4), and 68.6 times (95% CI 13.0-361.3) more likely to complete a living will when asked by medical staff, legal staff, or family and friends, respectively, than those not asked. Patients with health care proxies were 1.68 times (95% CI 0.81-3.47), 4.34 times (95% CI 1.50-12.6), and 18.0 times (95% CI 2.03-158.8) more likely to have been asked by the same groups. Patients with experience in end-of-life decision-making were 2.54 times more likely to possess a living will (95%CI 1.01-6.42) and 3.53 times more likely to possess a health care proxy (95% CI 1.51-8.25) than those without experiences. CONCLUSIONS: Having been asked about advance directives by medical staff, legal staff, or family and friends increases the likelihood that patients will possess an advance directive. Those with prior experience with end-of-life decision-making are more likely to possess an advance directive. Family structure and health care utilization also impacts possession of advance directives.