Background

Despite the high prevalence of respiratory symptoms and obstructive lung disease in HIV-infected persons, the prevalence of bronchodilator reversibility (BDR) and asthma has not been systematically studied during the era of combination antiretroviral therapy (ART).

Objective

To determine the prevalence of asthma diagnosis and related pulmonary function abnormalities in an HIV-infected cohort and to identify potential mechanisms.

Methods

A cross-sectional analysis of 223 HIV-infected individuals with data on respiratory symptoms and diagnoses, pulmonary function, sputum cell counts, and asthma-related cytokines and chemokines in serum/sputum.

Results

Doctor-diagnosed asthma was present in 46 (20.6%) and BDR (≥200ml and ≥12% increase in FEV1 or FVC) in 20 participants (9.0%). Pulmonary symptoms and function were worse in those with doctor-diagnosed asthma. Doctor-diagnosed asthma was independently associated with female sex (p=0.04), body mass index >29.6kg/m2 (vs.<29.6kg/m2) (p=0.03), history of bacterial or Pneumocystis pneumonia (p=0.01), and with not currently taking ART (p=0.04), and in univariate analysis with parental history of asthma (n=180; p=0.004). High sputum eosinophil percentages (>2.3% based on the highest decile) were more likely in those with doctor-diagnosed asthma (p=0.02) or BDR (p=0.02). Doctor-diagnosed asthma tended to be more common with high sputum IL-4 (p=0.02) and RANTES (p=0.02), while BDR was associated with high plasma macrophage inflammatory protein (MIP)-1α (p=0.002), and sputum MIP-1β levels (p=0.001).

Conclusion

Asthma diagnosis and BDR are prevalent in an HIV-infected outpatient cohort, and associations with family history, obesity, allergic inflammation, prior infection, the absence of ART, and elevated HIV-stimulated cytokines suggest possible mechanisms of HIV-associated asthma.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disorder caused by mutation of the NF1 tumor suppressor gene. Spinal deformities are common skeletal manifestations in patients with NF1. To date, the mechanism of vertebral abnormalities remains unclear because of the lack of appropriate animal models for the skeletal manifestations of NF1. In the present study, we report a novel murine NF1 model, Nf1flox/−;Col2.3Cre+ mice. These mice display short vertebral segments. In addition, a significant reduction in cortical and trabecular bone mass of the vertebrae was observed in Nf1flox/−;Col2.3Cre+ mice as measured by dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). Peak stress and peak load were also significantly reduced in Nf1flox/−;Col2.3Cre+ mice as compared to controls. Furthermore, the lumbar vertebrae showed enlargement of the inter-vertebral canal, a characteristic feature of lumbar vertebrae in NF1 patients. Finally, histologic analysis demonstrated increased numbers of osteoclasts and decreased numbers of osteoblasts in the vertebrae of Nf1flox/−;Col2.3Cre+ mice in comparison to controls. In summary, Nf1flox/−;Col2.3Cre+ mice demonstrate multiple structural and functional abnormalities in the lumbar vertebrae which recapitulate the dystrophic vertebral changes in NF1 patients. This novel murine model provides a platform to understand the cellular and molecular mechanisms underlying the pathogenesis of spinal deficits in NF1 patients.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology with a variable and unpredictable course.

Objectives: The aim of this study was to identify and validate plasma proteins that are predictive of outcome in IPF.

Methods: Plasma samples were available for 241 patients with IPF (140 derivation and 101 validation). In the derivation cohort, concentrations of 92 proteins were analyzed using a multiplex bead-based immunoassay and concentrations of matrix metalloproteinase (MMP)-7, MMP-1, and surfactant protein D were assessed by ELISA. In the validation cohort concentrations of intercellular adhesion molecule (ICAM)-1, IL-8, and vascular cell adhesion molecule (VCAM)-1 were assessed by bead-based multiplex assay, and S100A12 and MMP-7 by ELISA. Associations of biomarkers with mortality, transplant-free survival, and disease progression were tested in the derivation and validation cohorts using nonparametric methods of survival analysis and the Cox proportional hazards model, and an integrated risk prediction score was derived and tested.

Measurements and Main Results: High concentrations of MMP-7, ICAM-1, IL-8, VCAM-1, and S100A12 predicted poor overall survival, poor transplant-free survival, and poor progression-free survival in the derivation cohort. In the independent validation cohort high concentrations of all five were predictive of poor transplant-free survival; MMP-7, ICAM-1, and IL-8 of overall survival; and ICAM-1 of poor progression-free survival. The personal clinical and molecular mortality prediction index derived in the derivation cohort was highly predictive of mortality in the validation cohort.

Conclusions: Our results suggest that plasma proteins should be evaluated as a tool for prognosis determination in prioritization of patients for lung transplantation and stratification in drug studies.

Pneumocystis jirovecii has been detected in lung tissue from patients with chronic obstructive pulmonary disease (COPD) and is associated with disease severity. The regional distribution of the organism in lungs is unknown, but differences in distribution of Pneumocystis could affect estimates of colonization prevalence. We examined the distribution of Pneumocystis in the lungs of 19 non-HIV-infected patients with COPD who were undergoing lung transplantation. DNA was extracted from explanted lungs. We found Pneumocystis colonization in lung tissue of 42.1% of patients with advanced COPD; however, there was significant regional variation in colonization between lung segments of individual patients. Colonization was detected more commonly in the lower and middle lobes than the upper lobes. These findings suggest that single samples from an individual may underestimate the prevalence of Pneumocystis colonization and future studies may obtain a higher yield of Pneumocystis colonization detection when sampling the lower lobes.

Prostaglandin E2 (PGE2), interleukin (IL)-23, and IL-1beta (β) propagate inflammatory bowel disease (IBD) by enhancing the development and function of IL-17 producing CD4+ T helper (Th17) cells. CD4+ T cells that express the C-type lectin-like receptor CD161 have been proposed to be the physiologic pool of circulating Th17 cells implicated in IBD. We sought to understand how PGE2, alone and in combination with IL-23 and IL-1β, modulate human peripheral CD161+ CD4+ memory T cells. We found that CD161+ cells comprise a significant proportion of human peripheral CD4+ memory T cells. PGE2 and IL-23 plus IL-1β synergistically induced early IL-17A secretion from CD161+CD4+ memory T cells and the selective enrichment of IL-17A+CD161+CD4+ memory T cells in culture. Conversely, IL-23 plus IL-1β partially opposed the PGE2-mediated repression of early IFN-γ secretion from CD161+ cells, as well as the PGE2-mediated depletion of IFN-γ+CD161+ cells. Our results suggest that PGE2 and IL-23 plus IL-1β induce the Th17 immune response preferentially in CD161+CD4+ memory T cells, while divergently regulating their ability to express IFN-γ. We hypothesize that Th17-mediated chronic inflammation in IBD depends on the net response of CD161+CD4+ memory T cells to both PGE2 and IL-23 plus IL-1β.

Background:

Pelvic and acetabular fractures have been known as one of the high risk factors for developing deep vein thrombosis (DVT), but thromboprophylaxis for patients with such fractures remains underused despite its widely accepted benefits. Current guidelines have not been universally adopted in clinical practice. The purpose of this study is to introduce a Thrombotic Risk Assessment Questionary (assessment table) according to evidence-based guidelines and evaluate its impact on the use of thromboprophylaxis for patients with pelvic and acetabular fractures.

Materials and Methods:

We retrospectively reviewed 305 consecutive patients with pelvic and acetabular fractures from August 1, 2008 through September 30, 2010. The control group without using the assessment table included 153 patients admitted during the first 13 months, and the assessment group using the assessment table included 152 patients admitted during the following months. Data on clinical outcomes of DVT, the number of patients receiving prophylaxis, and the time of the first dose of anticoagulant were collected.

Results:

Compared with the control group, Patients using the assessment table were more likely to be given DVT prophylaxis (84.2% vs. 37.3%, P < 0.05) and the time of the first dose of anticoagulant was reduced (4.32 days ± 4.78 days vs. 6.6 days ± 5.96 days, P < 0.05). Patients in the assessment group had lower risk of developing DVT (8.6% vs. 20.3%, P < 0.05).

Conclusion:

The assessment table can significantly improve the use of thromboprophylaxis after pelvic and acetabular fractures, which will likely reduce the incidence of DVT. Developing individual hospital prophylaxis strategy is an effective way to determine whether hospitalized patients should receive pharmacologic and/or mechanical prophylaxis or not.

Rationale: Studies demonstrating an association between chronic obstructive pulmonary disease and low bone mineral density (BMD) implicate factors distinct from treatments and severity of lung disease in the pathogenesis of osteoporosis. Whereas emphysema has been independently associated with vascular disease and other comorbidities, its association with BMD has not been well studied.

Objectives: We explored the associations of BMD with computed tomography (CT) measures of emphysema and other risk factors in current and former smokers.

Methods: One hundred ninety subjects completed a CT scan, pulmonary function testing, questionnaires, and dual x-ray absorptiometry measurements of hip and lumbar spine BMD. Subjects were classified as having normal BMD, osteopenia, or osteoporosis. Demographic, physiologic, and radiographic characteristics were compared and the association of BMD with radiographic emphysema, airflow obstruction, and osteoporosis risk factors was assessed.

Measurements and Main Results: No difference existed in age, tobacco exposure, oral steroid use, or physical activity across BMD categories. Both osteopenia and osteoporosis were associated with the presence of airflow obstruction, inhaled corticosteroid use, and female sex, and demonstrated a significant relationship with the presence of visual emphysema (P = 0.0003). Quantitative emphysema, but not CT-measured indices of airway wall thickness, was inversely associated with BMD. Visual emphysema alone was a significant predictor of osteopenia/osteoporosis (odds ratio = 2.55; 95% confidence interval, 1.24–5.25) in a model including obstruction severity, age, sex, and inhaled and oral steroid use.

Conclusions: Radiographic emphysema is a strong, independent predictor of low BMD in current and former smokers. This relationship suggests a common mechanistic link between emphysema and osteopenia/osteoporosis.

Background

Tobacco use is associated with an increased prevalence of cardiovascular disease. N-terminal pro-brain natiuretic peptide (NT-proBNP), a widely available biomarker that is associated with cardiovascular outcomes in other conditions, has not been investigated as a predictor of mortality in tobacco smokers. We hypothesized that NT-proBNP would be an independent prognostic marker in a cohort of well-characterized tobacco smokers without known cardiovascular disease.

Methods

Clinical data from 796 subjects enrolled in two prospective tobacco exposed cohorts was assessed to determine factors associated with elevated NT-proBNP and the relationship of these factors and NT-proBNP with mortality.

Results

Subjects were followed for a median of 562 (IQR 252 – 826) days. Characteristics associated with a NT-proBNP above the median (≥49 pg/mL) were increased age, female gender, and decreased body mass index. By time-to-event analysis, an NT-proBNP above the median (≥49 pg/mL) was a significant predictor of mortality (log rank p = 0.02). By proportional hazard analysis controlling for age, gender, cohort, and severity of airflow obstruction, an elevated NT-proBNP level (≥49 pg/mL) remained an independent predictor of mortality (HR = 2.19, 95% CI 1.07–4.46, p = 0.031).

Conclusions

Elevated NT-proBNP is an independent predictor of mortality in tobacco smokers without known cardiovascular disease, conferring a 2.2 fold increased risk of death. Future studies should assess the ability of this biomarker to guide further diagnostic testing and to direct specific cardiovascular risk reduction inventions that may positively impact quality of life and survival.

Background

Transforming growth factor beta 1 (TGFβ1) plays a major role in many lung diseases including lung cancer, pulmonary hypertension, and pulmonary fibrosis. TGFβ1 activates a signal transduction cascade that results in the transcriptional regulation of genes in the nucleus, primarily through the DNA-binding transcription factor SMAD3. The objective of this study is to identify genome-wide scale map of SMAD3 binding targets and the molecular pathways and networks affected by the TGFβ1/SMAD3 signaling in lung epithelial cells.

Methodology

We combined chromatin immunoprecipitation with human promoter region microarrays (ChIP-on-chip) along with gene expression microarrays to study global transcriptional regulation of the TGFβ1/SMAD3 pathway in human A549 alveolar epithelial cells. The molecular pathways and networks associated with TGFβ1/SMAD3 signaling were identified using computational approaches. Validation of selected target gene expression and direct binding of SMAD3 to promoters were performed by quantitative real time RT-PCR and electrophoretic mobility shift assay on A549 and human primary lung epithelial cells.

Results and Conclusions

Known TGFβ1 target genes such as SERPINE1, SMAD6, SMAD7, TGFB1 and LTBP3, were found in both ChIP-on-chip and gene expression analyses as well as some previously unrecognized targets such as FOXA2. SMAD3 binding of FOXA2 promoter and changed expression were confirmed. Computational approaches combining ChIP-on-chip and gene expression microarray revealed multiple target molecular pathways affected by the TGFβ1/SMAD3 signaling. Identification of global targets and molecular pathways and networks associated with TGFβ1/SMAD3 signaling allow for a better understanding of the mechanisms that determine epithelial cell phenotypes in fibrogenesis and carcinogenesis as does the discovery of the direct effect of TGFβ1 on FOXA2.

An investigation of the electrochemical activity of human white blood cells (WBC) for biofuel cell (BFC) applications is described. WBCs isolated from whole human blood were suspended in PBS and introduced into the anode compartment of a proton exchange membrane (PEM) fuel cell. The cathode compartment contained a 50 mM potassium ferricyanide solution. Average current densities between 0.9 and 1.6 μA cm-2 and open circuit potentials (Voc) between 83 and 102 mV were obtained, which were both higher than control values. Cyclic voltammetry was used to investigate the electrochemical activity of the activated WBCs in an attempt to elucidate the mechanism of electron transfer between the cells and electrode. Voltammograms were obtained for the WBCs, including peripheral blood mononuclear cells (PBMCs - a lymphocyte-monocyte mixture isolated on a Ficoll gradient), a B lymphoblastoid cell line (BLCL), and two leukemia cell lines, namely K562 and Jurkat. An oxidation peak at about 363 mV vs. SCE for the PMA (phorbol ester) activated primary cells, with a notable absence of a reduction peak was observed. Oxidation peaks were not observed for the BLCL, K562 or Jurkat cell lines. HPLC confirmed the release of serotonin (5-HT) from the PMA activated primary cells. It is believed that serotonin, among other biochemical species released by the activated cells, contributes to the observed BFC currents.

Frizzleds are receptors for Wnt signaling and are involved in skeletal morphogenesis. Little is known about the transcriptional regulation of frizzleds in bone cells. In the current study, we determined if two common and potentially functional genetic variants (rs2232157, rs2232158) in the frizzled-1 (FZD1) promoter region and their haplotypes influence FZD1 promoter activity in human osteoblast-like cells. We also determined if these variants are associated with femoral neck bone mineral density (BMD) and geometry in 1319 African ancestry men aged ≥40 years. Real-time quantitative PCR and western blot analysis demonstrated FZD1 mRNA and protein expression in the human osteoblast-like cell lines, MG63 and SaOS-2. Promoter activity was next assessed by transient expression of haplotype specific FZD1 promoter reporter plasmids in these cells. In comparison to the common GG haplotype, promoter activity was 3-fold higher for the TC haplotype in both cell lines (p<0.05). We previously demonstrated that rs2232158 is associated with differential FZD1 promoter activity and Egr1 binding and thus focused further functional analyses on the rs2232157 G-to-T polymorphism. Electrophoretic mobility shift assay demonstrated that distinct nuclear protein complexes were associated with rs2232157 in an allele specific manner. Bioinformatics analysis predicted that the G to T transversion creates an E2F1 binding site, further supporting the functional significance of rs2232157 in FZD1 promoter regulation. Individual SNPs and haplotypes were not associated with femoral neck BMD. The TC haplotype was associated with larger subperiosteal width and greater CSMI (p<0.05). These results suggest that FZD1 expression is regulated in a haplotype dependent manner in osteoblasts and that these same haplotypes may be associated with biomechanical indices of bone strength.

Background

Idiopathic pulmonary fibrosis (IPF) is a progressive and medically refractory lung disease with a grim prognosis. Although the etiology of IPF remains perplexing, abnormal adaptive immune responses are evident in many afflicted patients. We hypothesized that perturbations of human leukocyte antigen (HLA) allele frequencies, which are often seen among patients with immunologic diseases, may also be present in IPF patients.

Methods/Principal Findings

HLA alleles were determined in subpopulations of IPF and normal subjects using molecular typing methods. HLA-DRB1*15 was over-represented in a discovery cohort of 79 Caucasian IPF subjects who had lung transplantations at the University of Pittsburgh (36.7%) compared to normal reference populations. These findings were prospectively replicated in a validation cohort of 196 additional IPF subjects from four other U.S. medical centers that included both ambulatory patients and lung transplantation recipients. High-resolution typing was used to further define specific HLA-DRB1*15 alleles. DRB1*1501 prevalence in IPF subjects was similar among the 143 ambulatory patients and 132 transplant recipients (31.5% and 34.8%, respectively, p = 0.55). The aggregate prevalence of DRB1*1501 in IPF patients was significantly greater than among 285 healthy controls (33.1% vs. 20.0%, respectively, OR 2.0; 95%CI 1.3–2.9, p = 0.0004). IPF patients with DRB1*1501 (n = 91) tended to have decreased diffusing capacities for carbon monoxide (DLCO) compared to the 184 disease subjects who lacked this allele (37.8±1.7% vs. 42.8±1.4%, p = 0.036).

Conclusions/Significance

DRB1*1501 is more prevalent among IPF patients than normal subjects, and may be associated with greater impairment of gas exchange. These data are novel evidence that immunogenetic processes can play a role in the susceptibility to and/or manifestations of IPF. Findings here of a disease association at the HLA-DR locus have broad pathogenic implications, illustrate a specific chromosomal area for incremental, targeted genomic study, and may identify a distinct clinical phenotype among patients with this enigmatic, morbid lung disease.

High mobility group (HMG) proteins regulate chromatin architecture and gene expression. Constitutional rearrangement of an HMG family member, HMGA2, in an 8-year old boy resulted in extreme overgrowth and advanced bone development. Moreover, a recent genome-wide association study documented an association between a variant in the 3′ untranslated region of HMGA2 (rs1042725) and height in otherwise healthy individuals. We attempted to extend these findings by testing if this HMGA2 polymorphism is associated with other skeletal measures in two large population cohorts of diverse race/ethnicity. Genotyping was completed in 1,680 Afro-Caribbean men aged ≥40 years and 1,548 Caucasian American men aged ≥69 years. Bone mineral density (BMD) was assessed with peripheral quantitative computed tomography. The minor allele frequency of rs1042725 was 32% among Afro-Caribbeans and 48% among Caucasians (p<0.0001). No association was observed with height in either study cohort. However, presence of the minor allele of this SNP was associated with decreased tibia trabecular volumetric BMD in both populations (p=0.007 Afro-Caribbean; p=0.0007 Caucasian). Real time quantitative RT-PCR and Western blot analysis demonstrated HMGA2 mRNA and protein expression in the human fetal osteoblast cell line, hFOB. Our analyses suggest a novel association between a common genetic variant in HMGA2 and trabecular BMD in ethnically diverse older men. Additional research is needed to better understand the role of HMGA2 in the regulation of bone metabolism.

Rationale: The molecular mechanisms underlying acute exacerbations of idiopathic pulmonary fibrosis (IPF) are poorly understood. We studied the global gene expression signature of acute exacerbations of IPF.

Objectives: To understand the gene expression patterns of acute exacerbations of IPF.

Methods: RNA was extracted from 23 stable IPF lungs, 8 IPF lungs with acute exacerbation (IPF-AEx), and 15 control lungs and used for hybridization on Agilent gene expression microarrays. Functional analysis of genes was performed with Spotfire and Genomica. Gene validations for MMP1, MMP7, AGER, DEFA1–3, COL1A2, and CCNA2 were performed by real-time quantitative reverse transcription-polymerase chain reaction. Immunohistochemistry and in situ terminal deoxynucleotidyltransferase dUTP nick end-labeling assays were performed on the same tissues used for the microarray. ELISA for α-defensins was performed on plasma from control subjects, patients with stable IPF, and patients with IPF-AEx.

Measurements and Main Results: Gene expression patterns in IPF-AEx and IPF samples were similar for the genes that distinguish IPF from control lungs. Five hundred and seventy-nine genes were differentially expressed (false discovery rate < 5%) between stable IPF and IPF-AEx. Functional analysis of these genes did not indicate any evidence of an infectious or overwhelming inflammatory etiology. CCNA2 and α-defensins were among the most up-regulated genes. CCNA2 and α-defensin protein levels were also higher and localized to the epithelium of IPF-AEx, where widespread apoptosis was also detected. α-Defensin protein levels were increased in the peripheral blood of patients with IPF-AEx.

Conclusions: Our results indicate that IPF-AEx is characterized by enhanced epithelial injury and proliferation, as reflected by increases in CCNA2 and α-defensins and apoptosis of epithelium. The concomitant increase in α-defensins in the peripheral blood and lungs may suggest their use as biomarkers for this disorder.

Sphingosine 1-phospate (S1P) has been demonstrated to protect against the formation of lipopolysaccharide (LPS)-induced lung edema when administered concomitantly with LPS. In the present study, we sought to determine the effectiveness of S1P to attenuate lung injury in a translationally relevant canine model of acute lung injury (ALI) when administered as rescue therapy. Secondarily, we examined whether the attenuation of LPS-induced physiological lung injury following administration of S1P was, at least in part, due to an alteration in local and/or systemic inflammatory cytokine expression. We prospectively examined 18 one-year old male beagles in which we instilled bacterial LPS (2–4 mg/kg) intratracheally followed in one hour with intravenous S1P (85 μg/kg) or vehicle and eight hours of high tidal volume mechanical ventilation. S1P attenuated the formation of shunt fraction (32%) and both the presence of protein (72%) and neutrophils (95%) in bronchoalveolar lavage (BAL) fluid compared to vehicle controls. Although lung tissue inflammatory cytokine production was found to vary regionally throughout the LPS-injured lung, S1P did not alter the expression pattern. Similarly, BAL cytokine production was not significantly altered by intravenous S1P in this model. Interestingly, S1P potentiated the LPS-induced systemic production of three inflammatory cytokines, TNF-α (6-fold), KC (1.2-fold), and IL-6 (3-fold), without resulting in end-organ dysfunction. In conclusion, intravenous S1P reduces inflammatory lung injury when administered as rescue therapy in our canine model of LPS-induced ALI. This improvement is observed in the absence of changes in local pulmonary inflammatory cytokine production and an augmentation of systemic inflammation.

Background

Chronic obstructive pulmonary disease (COPD) is characterized by both airway remodeling and parenchymal destruction. The identification of unique biomarker patterns associated with airway dominant versus parenchymal dominant patterns would support the existence of unique phenotypes representing independent biologic processes. A cross-sectional study was performed to examine the association of serum biomarkers with radiographic airway and parenchymal phenotypes of COPD.

Methodology/Principal Findings

Serum from 234 subjects enrolled in a CT screening cohort was analyzed for 33 cytokines and growth factors using a multiplex protein array. The association of serum markers with forced expiratory volume in one second percent predicted (FEV1%) and quantitative CT measurements of airway thickening and emphysema was assessed with and without stratification for current smoking status. Significant associations were found with several serum inflammatory proteins and measurements of FEV1%, airway thickening, and parenchymal emphysema independent of smoking status. The association of select analytes with airway thickening and emphysema was independent of FEV1%. Furthermore, the relationship between other inflammatory markers and measurements of physiologic obstruction or airway thickening was dependent on current smoking status.

Conclusions/Significance

Airway and parenchymal phenotypes of COPD are associated with unique systemic serum biomarker profiles. Serum biomarker patterns may provide a more precise classification of the COPD syndrome, provide insights into disease pathogenesis and identify targets for novel patient-specific biological therapies.

Rationale: Ventilator-induced lung injury (VILI) leads to an unacceptably high mortality. In this regard, the antiinflammatory properties of inhaled carbon monoxide (CO) may provide a therapeutic option.

Objectives: This study explores the mechanisms of CO-dependent protection in a mouse model of VILI.

Methods: Mice were ventilated (12 ml/kg, 1–8 h) with air in the absence or presence of CO (250 ppm). Airway pressures, blood pressure, and blood gases were monitored. Lung tissue was analyzed for inflammation, injury, and gene expression. Bronchoalveolar lavage fluid was analyzed for protein, cell and neutrophil counts, and cytokines.

Measurements and Main Results: Mechanical ventilation caused significant lung injury reflected by increases in protein concentration, total cell and neutrophil counts in the bronchoalveolar lavage fluid, as well as the induction of heme oxygenase-1 and heat shock protein-70 in lung tissue. In contrast, CO application prevented lung injury during ventilation, inhibited stress-gene up-regulation, and decreased lung neutrophil infiltration. These effects were preceded by the inhibition of ventilation-induced cytokine and chemokine production. Furthermore, CO prevented the early ventilation-dependent up-regulation of early growth response-1 (Egr-1). Egr-1–deficient mice did not sustain lung injury after ventilation, relative to wild-type mice, suggesting that Egr-1 acts as a key proinflammatory regulator in VILI. Moreover, inhibition of peroxysome proliferator-activated receptor (PPAR)-γ, an antiinflammatory nuclear regulator, by GW9662 abolished the protective effects of CO.

Conclusions: Mechanical ventilation causes profound lung injury and inflammatory responses. CO treatment conferred protection in this model dependent on PPAR-γ and inhibition of Egr-1.

Rationale: Adaptive immune responses are present in patients with chronic obstructive pulmonary disease (COPD), and it has been postulated that these processes could be autoreactive.

Objectives: To ascertain if humoral autoimmunity could play a role in COPD pathogenesis.

Methods: Circulating IgG autoantibodies were detected by immunofluorescence and immunoprecipitation. Immunohistochemistry and immunofluorescence were used to evaluate intrapulmonary IgG and complement (C3) deposition in human lung explants. Autoantibody pathogenicity was also investigated with an antibody-dependent cell-mediated cytotoxicity assay.

Measurements and Main Results: The prevalence of anti–HEp-2 epithelial cell autoantibodies in 47 smokers/former smokers with COPD (GOLD stages 1–4) was greater than among 8 subjects with a smoking history but normal spirometry and 21 healthy control subjects who had never smoked (68 vs. 13 vs. 10%, respectively; P < 0.0001). Antibodies against primary pulmonary epithelial cells were found in 12 of 12 patients with COPD versus 3 of 12 never-smoked control subjects (P < 0.001). Self-antigens immunoprecipitated from 34 of 35 (97%) of COPD plasmas (vs. 0/12 never-smoked controls). Antibodies against a particular 130-kD autoantigen (n = 7) were associated with decreased body mass index (23.2 ± 2.1 vs. 29.5 ± 1.0 kg/m2, P = 0.007). Intrapulmonary immune complexes were present in six of six and C3 was seen in five of six COPD lung explants, unlike zero of six and one of six normals, respectively. Cytotoxicity of pulmonary epithelial cells by allogeneic mononuclear cells also increased 46% after incubation with COPD plasmas (n = 10), compared with identical treatments with eight normal specimens (P = 0.03).

Conclusions: IgG autoantibodies with avidity for pulmonary epithelium, and the potential to mediate cytotoxicity, are prevalent in patients with COPD. Autoreactive adaptive immune responses may be important in the etiology of this disease.

Traumatic injuries of the inferior gluteal artery are rare, the majority of which are aneurysms due to sharp or blunt trauma. We report the rare case of a "near miss" event of a patient with an acute hemorrhagic mass in the right buttock caused by blunt trauma to the inferior gluteal artery without "hard" clinical signs of vascular injury. Despite the unusual presentation, diffuse injury of the inferior gluteal artery branches was diagnosed by ultrasonography and angiography. This article highlights the importance of considering an arterial injury following blunt trauma to the buttock with subsequent pain and swelling. Appreciation of this rare injury pattern is necessary in order to facilitate rapid diagnosis and appropriate treatment.

Background

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear.

Methodology and Principal Findings

Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7). Cigarette smoke extract (CSE) is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC) inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1) and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1−/− mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema.

Conclusions

We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.

Background

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease associated with substantial morbidity and mortality. The objective of this study was to determine whether there is a peripheral blood protein signature in IPF and whether components of this signature may serve as biomarkers for disease presence and progression.

Methods and Findings

We analyzed the concentrations of 49 proteins in the plasma of 74 patients with IPF and in the plasma of 53 control individuals. We identified a combinatorial signature of five proteins—MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A—that was sufficient to distinguish patients from controls with a sensitivity of 98.6% (95% confidence interval [CI] 92.7%–100%) and specificity of 98.1% (95% CI 89.9%–100%). Increases in MMP1 and MMP7 were also observed in lung tissue and bronchoalveolar lavage fluid obtained from IPF patients. MMP7 and MMP1 plasma concentrations were not increased in patients with chronic obstructive pulmonary disease or sarcoidosis and distinguished IPF compared to subacute/chronic hypersensitivity pneumonitis, a disease that may mimic IPF, with a sensitivity of 96.3% (95% CI 81.0%–100%) and specificity of 87.2% (95% CI 72.6%–95.7%). We verified our results in an independent validation cohort composed of patients with IPF, familial pulmonary fibrosis, subclinical interstitial lung disease (ILD), as well as with control individuals. MMP7 and MMP1 concentrations were significantly higher in IPF patients compared to controls in this cohort. Furthermore, MMP7 concentrations were elevated in patients with subclinical ILD and negatively correlated with percent predicted forced vital capacity (FVC%) and percent predicted carbon monoxide diffusing capacity (DLCO%).

Conclusions

Our experiments provide the first evidence for a peripheral blood protein signature in IPF to our knowledge. The two main components of this signature, MMP7 and MMP1, are overexpressed in the lung microenvironment and distinguish IPF from other chronic lung diseases. Additionally, increased MMP7 concentration may be indicative of asymptomatic ILD and reflect disease progression.

Naftali Kaminski and colleagues find increased levels of specific proteins in the bloodstream of individuals with idiopathic pulmonary fibrosis, and suggest that these proteins may ultimately provide a biomarker for the disease.

Editors' Summary

Background.

Idiopathic pulmonary fibrosis (IPF) is a serious disease in which the lungs become progressively scarred or thickened for unknown reasons. In healthy people, air is taken in through the mouth or nose and travels down the windpipe into tubes in the lungs called the airways. Each airway has many small branches that end in alveoli, tiny air sacs with thin walls that are surrounded by small blood vessels called capillaries. When air reaches the alveoli, the oxygen in it passes into the bloodstream and is taken to the organs of the body to keep them working. In IPF, the alveoli and the space around them (the “interstitial” area) gradually become scarred and thickened, which stops oxygen's movement into the bloodstream. When only small areas of the lung are scarred, IPF may cause no symptoms. But, as more of the lung becomes damaged, IPF eventually causes breathlessness, even when resting. There is no effective treatment for IPF, although steroids and drugs that suppress the body's immune system are often tried in an attempt to slow its progression. On average, half of the people with IPF die within three years of diagnosis, often from respiratory or heart failure.

Why Was This Study Done?

It can be difficult to diagnose IPF—there are many lung diseases with similar symptoms, including numerous other interstitial lung diseases—and currently, physicians can only follow the progression of IPF by repeatedly testing their patients' lung function or by doing multiple chest X-rays. If proteins could be identified whose level in blood indicated disease activity (so-called “peripheral blood biomarkers”), it would be easier to diagnose and monitor patients. In addition, the identification of such biomarkers might suggest new drug targets for the treatment of IPF. In this study, the researchers look for peripheral blood biomarkers in IPF by using a “multiplex analysis” system to measure the level of several proteins in patient blood samples simultaneously.

What Did the Researchers Do and Find?

The researchers measured the levels of 49 plasma proteins (plasma is the fluid part of blood) in 74 patients with IPF and 53 healthy people (controls) and used a technique called “recursive partitioning” to define a five-protein signature that distinguished patients from unaffected study participants (controls). Matrix metalloproteinase 7 (MMP7) and MMP1—the two plasma proteins whose levels were most increased in patients with IPF compared to controls—were key components of this signature. Concentrations of MMP7 and MMP1 were higher in bronchoalveolar lavage samples (fluid obtained by washing out the lungs with saline) and in lung tissue samples from patients with IPF than in similar samples taken from healthy individuals. Plasma concentrations of MMP7 and MMP1 were significantly higher in patients with IPF than in patients with hypersensitivity pneumonitis, an interstitial lung disease that mimics IPF, but not increased in patients with chronic obstructive pulmonary disease or sarcoidosis, two other lung diseases. In an independent validation group, patients with IPF and familial pulmonary fibrosis had increased plasma concentrations of MMP7 and MMP1 that correlated with the severity of their disease. In addition, MMP7 concentrations were raised in close relatives of people with familial pulmonary fibrosis who had normal lung function tests but some lung scarring.

What Do These Findings Mean?

These findings provide evidence for a protein signature in the blood for IPF and suggest MMP1 and MMP7 may be useful as biomarkers for IPF. These two matrix metalloproteinases have previously been suggested to be involved in the development of IPF. However, additional work is probably needed to confirm that increased plasma concentrations MMP7 and MMP1 are specific for IPF, since it may be that these markers will not distinguish IPF from other interstitial lung diseases.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050093.

Read a related PLoS Medicine Perspective article

The MedlinePlus Encyclopedia has a page on idiopathic pulmonary fibrosis (in English and Spanish) and on pulmonary fibrosis

The US National Heart Lung and Blood Institute and the British Lung Foundation also provide information on IPF for patients and relatives

Some of the researchers involved in this study provide more details about what might go wrong in IPF in a recent PLoS Medicine article

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by activation of fibroblasts and overproduction of extracellular matrix (ECM). Caveolin-1 (cav-1), a principal component of caveolae, has been implicated in the regulation of numerous signaling pathways and biological processes. We observed marked reduction of cav-1 expression in lung tissues and in primary pulmonary fibroblasts from IPF patients compared with controls. We also demonstrated that cav-1 markedly ameliorated bleomycin (BLM)-induced pulmonary fibrosis, as indicated by histological analysis, hydroxyproline content, and immunoblot analysis. Additionally, transforming growth factor β1 (TGF-β1), the well-known profibrotic cytokine, decreased cav-1 expression in human pulmonary fibroblasts. cav-1 was able to suppress TGF-β1–induced ECM production in cultured fibroblasts through the regulation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, highly activated JNK was detected in IPF- and BLM-instilled lung tissue samples, which was dramatically suppressed by ad–cav-1 infection. Moreover, JNK1-null fibroblasts showed reduced smad signaling cascades, mimicking the effects of cav-1. This study indicates a pivotal role for cav-1 in ECM regulation and suggests a novel therapeutic target for patients with pulmonary fibrosis.

Background

Chronic obstructive pulmonary disease (COPD) is a heterogeneous syndrome characterized by varying degrees of airflow limitation and diffusion impairment. There is increasing evidence to suggest that COPD is also characterized by systemic inflammation. The primary goal of this study was to identify soluble proteins in plasma that associate with the severity of airflow limitation in a COPD cohort with stable disease. A secondary goal was to assess whether unique markers associate with diffusion impairment, based on diffusion capacity of carbon monoxide (DLCO), independent of the forced expiratory volume in 1 second (FEV1).

Methods

A cross sectional study of 73 COPD subjects was performed in order to examine the association of 25 different plasma proteins with the severity of lung function impairment, as defined by the baseline measurements of the % predicted FEV1 and the % predicted DLCO. Plasma protein concentrations were assayed using multiplexed immunobead-based cytokine profiling. Associations between lung function and protein concentrations were adjusted for age, gender, pack years smoking history, current smoking, inhaled corticosteroid use, systemic corticosteroid use and statin use.

Results

Plasma concentrations of CCL2/monocyte chemoattractant protein-1 (CCL2/MCP-1), CCL4/macrophage inflammatory protein-1β (CCL4/MIP -1β), CCL11/eotaxin, and interleukin-13 (IL-13) were inversely associated with the % FEV1. Plasma concentrations of soluble Fas were associated with the % DLCO, whereas CXCL9/monokine induced by interferon-γ (CXCL9/Mig), granulocyte- colony stimulating factor (G-CSF) and IL-13 showed inverse relationships with the % DLCO.

Conclusion

Systemic inflammation in a COPD cohort is characterized by cytokines implicated in inflammatory cell recruitment and airway remodeling. Plasma concentrations of IL-13 and chemoattractants for monocytes, T lymphocytes, and eosinophils show associations with increasing severity of disease. Soluble Fas, G-CSF and CXCL9/Mig may be unique markers that associate with disease characterized by disproportionate abnormalities in DLCO independent of the FEV1.

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by activation of fibroblasts and overproduction of extracellular matrix (ECM). Caveolin-1 (cav-1), a principal component of caveolae, has been implicated in the regulation of numerous signaling pathways and biological processes. We observed marked reduction of cav-1 expression in lung tissues and in primary pulmonary fibroblasts from IPF patients compared with controls. We also demonstrated that cav-1 markedly ameliorated bleomycin (BLM)-induced pulmonary fibrosis, as indicated by histological analysis, hydroxyproline content, and immunoblot analysis. Additionally, transforming growth factor β1 (TGF-β1), the well-known profibrotic cytokine, decreased cav-1 expression in human pulmonary fibroblasts. cav-1 was able to suppress TGF-β1–induced ECM production in cultured fibroblasts through the regulation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, highly activated JNK was detected in IPF- and BLM-instilled lung tissue samples, which was dramatically suppressed by ad–cav-1 infection. Moreover, JNK1-null fibroblasts showed reduced smad signaling cascades, mimicking the effects of cav-1. This study indicates a pivotal role for cav-1 in ECM regulation and suggests a novel therapeutic target for patients with pulmonary fibrosis.

The presence of endotoxin (detected by the Limulus amebocyte lysate assay) was compared to the presence of viable Haemophilus influenzae and Moraxella catarrhalis (detected by PCR) in 106 middle-ear effusions from pediatric patients with chronic otitis media. Endotoxin was found in 81 of the 106 specimens. Of these 81 specimens, 66 (81.5%) also tested positive for one or both of the gram-negative bacteria H. influenzae and M. catarrhalis. The data suggest that viable gram-negative bacteria, detectable by PCR but often undetectable by culture, may be the source of endotoxin in middle-ear effusions.