We performed a genome-wide association study in non-Hispanic white subjects with fibrotic idiopathic interstitial pneumonias (N=1616) and controls (N=4683); replication was assessed in 876 cases and 1890 controls. We confirmed association with TERT and MUC5B on chromosomes 5p15 and 11p15, respectively, the chromosome 3q26 region near TERC, and identified 7 novel loci (PMeta = 2.4×10−8 to PMeta = 1.1×10−19). The novel loci include FAM13A (4q22), DSP (6p24), OBFC1 (10q24), ATP11A (13q34), DPP9 (19p13), and chromosomal regions 7q22 and 15q14-15. Our results demonstrate that genes involved in host defense, cell-cell adhesion, and DNA repair contribute to the risk of fibrotic IIP.
Frizzled homolog 1 (FZD1) is a transmembrane receptor that mediates Wnt signaling. The transcriptional regulation of FZD1 and the role of FZD1 in osteoblast biology are not well understood. We examined the role of E2F1 in FZD1 promoter activation and osteoblast differentiation and mineralization. A putative E2F1 binding site in the FZD1 promoter region was initially identified in silico and characterized further in Saos2 cells in vitro by chromatin immunoprecipitation (ChIP), electrophoretic mobility shift (EMSA) and promoter reporter assays. Over-expression of E2F1 transactivated the FZD1 promoter and increased endogenous FZD1 mRNA and protein levels in Saos2 cells. Over-expression of E2F1 in Saos2 cells up-regulated osteoblast differentiation markers alkaline phosphatase (ALP), type I collagen α (COL1A), and osteocalcin (OCN). Furthermore, E2F1 over-expression enhanced mineralization of differentiated Saos2 cells, whereas siRNA knockdown of FZD1 diminished the effects of E2F1 on osteoblast mineralization. The effects of E2F1 on FZD1 expression and osteoblast mineralization were further confirmed in normal human FOB osteoblasts. Taken together, our experiments demonstrate a role of E2F1 in osteoblast differentiation and mineralization and suggest that FZD1 is required, in part, for E2F1 regulation of osteoblast mineralization.
Frizzled homology1 (FZD1); E2F1; Osteoblasts; Mineralization
Uncontrolled activation of tumor necrosis factor receptor-associated factor (TRAF) proteins may result in profound tissue injury by linking surface signals to cytokine release. Here we show that a ubiquitin E3 ligase component, Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by destabilizing a sentinel TRAF inhibitor, Fbxl2. Fbxo3 and TRAF protein in circulation positively correlated with cytokine responses in septic subjects and we furthermore identified a hypofunctional Fbxo3 human polymorphism. A small molecule inhibitor targeting Fbxo3 was sufficient to lessen severity of cytokine-driven inflammation in several murine disease models. These studies identify a pathway of innate immunity that may characterize subjects with altered immune responses during critical illness or provide a basis for therapeutic intervention targeting TRAF protein abundance.
This study aimed to evaluate the preliminary clinical and radiographic outcomes of acute displaced femoral neck fracture treated by closed reduction and internal fixation (CRIF) with free iliac bone block grafting with comparison to a routine protocol of CRIF without bone grafting.
From December 2008 to February 2010, 220 adult patients with acute displaced femoral neck fractures were enrolled in this study. In study group, there were 124 patients (57 males, 67 females) with a mean age of 44.8 years (range, 20-64 years). There were 70 transcervical fractures and 54 subcapital fractures. The patients were treated by CRIF and free iliac bone block grafting. The control group consisted of 96 adult patients (46 males, 50 females) with a mean age of 46.3 years (range, 23-64 years). There were 61 transcervical fractures and 35 subcapital fractures. The patients in control group were treated by CRIF without bone grafting.
In study group, 112 patients were followed up for an average of 27.4 months (range, 24-34 months). All fractures healed within 5 months. However, 10 patients presented AVN of the femoral heads. The mean Harris score was 88.6 (range, 41-100). In control group, 68 patients were followed up for an average of 31.2 months (range, 24-42 months). The rates of AVN of the femoral head and fracture nonunion in control group were 26.5% (18/68) and 16.2% (11/68), respectively, significantly higher than those in study group (both P<0.05). The mean Harris score in control group was 83.8 (41–100), significantly lower than that in study group (P<0.05).
Acute displaced femoral neck fractures can be treated by CRIF and free iliac bone block grafting in a minimally invasive manner. This technique can guarantee uneventful fracture healing and significantly reduce the rate of femoral head osteonecrosis.
The surgical technique, medial patellar retinaculum plasty, can almost restore both static and dynamic stability and verge on anatomical repair for the treatment of habitual patellar dislocation in adolescents.
In accordance with the injury patterns of the medial patellar retinaculum through knee MRI, we repaired different injury sites with this surgical procedure. We reviewed this technique in 16 patients with an average age of 15 years. Retrospective review of charts and radiographs immediately after the surgery up to the latest follow-up (range 12–36 months) was undertaken.
All patients were evaluated clinically and radiologically over an average of 20.7 months. The recovery of knee mobility results were good. No recurrence of patellar instability has been found.
We think this could be a valid technique to treat habitual patellar dislocation in adolescents.
B7-H3 is a member of the B7-family of co-stimulatory molecules, which has been shown to be broadly expressed in various tumor tissues, and which plays an important role in adaptive immune responses. The role of B7-H3 in osteosarcoma, however, remains unknown. In this study we used immunohistochemistry to analyze B7-H3 expression in 61 primary osteosarcoma tissues with case-matched adjacent normal tissues, and 37 osteochondroma and 20 bone fibrous dysplasia tissues. B7-H3 expression was expressed in 91.8% (56/61) of the osteosarcoma lesions, and the intensity of B7-H3 expression in osteosarcoma was significantly increased compared with adjacent normal tissues, osteochondroma and bone fibrous dysplasia tissues (p<0.001). Patients with high tumor B7-H3 levels had a significantly shorter survival time and recurrence time than patients with low tumor B7-H3 levels (p<0.001). Moreover, tumor B7-H3 expression inversely correlated with the number of tumor-infiltrating CD8+ T cells (p<0.05). In vitro, increasing expression of B7-H3 promotes osteosarcoma cell invasion, at least in part by upregulating matrix metalloproteinase-2 (MMP-2). In conclusion, our study provides the first evidence of B7-H3 expression in osteosarcoma cells as a potential mechanism controlling tumor immunity and invasive malignancy, and which is correlated with patients’ survival and metastasis.
The less invasive stabilization system (LISS) can effectively treat tibial fractures. However, the LISS is technically demanding, has a long learning curve, and presents a heavy economic burden to patients. The U-grooved locking compression plate (U-LCP), characterized by a U-groove at each end, is designed to treat tibial fractures. This paper reports the outcomes of tibial fractures treated using the U-LCP compared with the LISS.
Seventy-eight patients with unilateral tibial fractures treated with either the U-LCP (group I) or LISS (group II) were enrolled. In group I, a U-LCP was inserted subcutaneously with two Kirschner wires embedded into the U-grooves to temporarily secure the plate. A second identical plate was placed over the first to guide screw insertion. In group II, the LISS was used to fix the tibial fractures. Patient age, sex, fracture type, severity of soft tissue injury, operative time, fluoroscopic time, complications, and functional recovery of affected limbs were recorded.
The two groups were comparable in age, sex, fracture type, and severity of soft tissue injury (p > 0.05). The average operation and fluoroscopic times in group I were significantly less than those in group II (p < 0.05). At follow-up, all fractures healed. There were no significant differences between both groups in time to bony union, wound complication rate, or functional recovery of injured limbs (p > 0.05).
The U-LCP can yield good outcomes in the treatment of proximal tibial fractures, with less radiation exposure, a shorter operation time, and a sustainable price compared with the LISS.
Platelets are activated in sickle cell disease (SCD), and particularly during vaso-occlusive episodes (VOE). Thrombospondin-1 (TSP1), a major secretory product of activated platelets, is increased in the circulation in VOE and binds to sickle red blood cells (RBC) promoting vascular adhesion. Thus, we hypothesized that TSP1 may represent a plasma biomarker of disease severity in SCD. We tested the plasma collected from patients in steady state (n = 27) and VOE (n = 14), as well as healthy controls (n = 17) at the University of Pittsburgh Medical Center (UPMC), and from patients in steady state enrolled in the walk-PHaSST clinical trial (n = 483). We found that TSP1 levels were increased in VOE in the UPMC cohort. Among steady-state patients at UPMC, TSP1 values correlated positively with lifetime history of acute chest syndrome (r = 0.72, P < 0.0001) and hemoglobin concentration (r = 0.49, P = 0.01), and negatively with markers of hemolysis, such as LDH (r = −0.50, P = 0.009). Analysis of the walk-PHaSST cohort also showed a positive association between TSP1 levels and hydroxyurea use (r = 0.14, P = 0.003), and confirmed the negative associations with the severity of hemolysis. Our results suggest that TSP1 levels are associated with more VOE, hydroxyurea use and lower rates of hemolysis. High TSP1 concentrations may indicate higher risk of the viscosity/vaso-occlusion phenotype of SCD.
To determine relationship of echocardiographic measures of pulmonary hypertension to lung function and inflammatory biomarkers in HIV-infected individuals.
Cross-sectional study of 116 HIV-infected outpatients.
Doppler-echocardiography and pulmonary function testing were performed. Induced sputum and plasma cytokines, sputum cell counts and differentials, markers of peripheral T cell activation, and serum N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured. Univariate and multivariate analyses determined relationship of echocardiographic variables to pulmonary function, inflammation, and NT-proBNP.
Mean estimated pulmonary artery systolic pressure (PASP) was 34.3 mmHg (SD 6.9) and mean tricuspid regurgitant jet velocity (TRV) was 2.5 m/sec (SD 0.32). Eighteen participants (15.5%) had PASP of at least 40 mmHg, and 9 (7.8%) had TRV of at least 3.0 m/sec. Elevated TRV was significantly associated with CD4 cell counts below 200 cells/μl and higher log HIV RNA levels. Forced expiratory volume in one second (FEV1) percent predicted, FEV1/forced vital capacity (FVC), and diffusing capacity for carbon monoxide (DLco) percent predicted were significantly lower in those with elevated PASP or TRV. Sputum interleukin-8, peripheral interleukin-8, peripheral interferon-γ levels, and CD8+ T-cell expression of CD69+ were associated increased with increasing PASP and TRV. Log NT-proBNP was significantly higher with increasing PASP and TRV. Left ventricular function was not associated with PASP or TRV.
Echocardiographic manifestations of pulmonary hypertension are common in HIV and are associated with respiratory symptoms, more advanced HIV disease, airway obstruction, abnormal DLco, and systemic and pulmonary inflammation. Pulmonary hypertension and COPD coexist in HIV and may arise secondary to common inflammatory mechanisms.
HIV; pulmonary hypertension; emphysema; COPD; inflammation
To investigate the long-term impacts of different posterior operations on curvature, neurological improvement and axial symptoms for multilevel cervical degenerative myelopathy (CDM), and to study the relationship among loss of cervical lordosis, recovery rate and axial symptom severity.
We retrospectively reviewed 98 patients with multilevel CDM who had undergone laminoplasty (Group LP, 36 patients), laminectomy (Group LC, 30 patients), or laminectomy with lateral mass screw fixation (Group LCS, 32 patients) between January 2000 and January 2005. Loss of curvature index (CI) was measured according to the preoperative and final follow-up radiographic parameters. The recovery rate was calculated based on the Japanese Orthopedic Association (JOA) score. Axial symptom severity was quantified by Neck Disability Index (NDI).
Analysis of final follow-up data showed significant differences among the three groups regarding loss of CI (F = 41.46, P < 0.001) between preoperative and final follow-up JOA scores (P < 0.001), final follow-up JOA score (F = 7.81, P < 0.001), recovery rate (F = 12.98, P < 0.001) and axial symptom severity (χ2 = 18.04, P < 0.001). Loss of CI showed negative association with neurological recovery (r = −0.555, P < 0.001) and positive correlation with axial symptom severity (r = 0.696, P < 0.001).
Excellent neurological improvement was obtained by LP and LCS for patients with multilevel CDM, while loss of CI in groups LP and LC caused a high incidence of axial symptoms. Loss of CI was correlated with poor neurological recovery and axial symptom severity. Lateral mass screw fixation can effectively prevent loss of postoperative cervical curvature and reduce incidence of axial symptoms.
Multilevel cervical degenerative myelopathy; Posterior operations; Curvature index; Neurological recovery; Axial symptoms
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).
To determine the prevalence of asthma diagnosis and related pulmonary function abnormalities in an HIV-infected cohort and to identify potential mechanisms.
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.
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).
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.
HIV; asthma; airway obstruction; allergy
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.
Neurofibromatosis type 1; Spinal deformity; Osteopathy; Osteoclast; Osteoblast
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.
MMP-7; adhesion molecules; biomarkers; personalized medicine; mortality prediction
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.
chronic obstructive pulmonary disease; Pneumocystis jirovecii; lung
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.
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.
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).
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.
Deep vein thrombosis; pelvic and acetabular fractures; risk assessment; thromboprophylaxis
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.
pulmonary disease, chronic obstructive; emphysema; osteoporosis
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.
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.
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).
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.
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.
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.
frizzled-1; haplotype; osteoblast; WNT; osteoporosis
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.
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).
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.
Osteoporosis; Genetics; BMD; men; HMGA2; race
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.
CCNA2; α-defensins; microarray; apoptosis; viral infection
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.