Collagen Turnover Biomarkers Associate with Active Psoriatic Arthritis and Decrease with Guselkumab Treatment in a Phase 3 Clinical Trial (DISCOVER-2).

INTRODUCTION: Guselkumab, a novel interleukin-23p19 subunit monoclonal antibody, has been shown to effectively improve the diverse manifestations of active psoriatic arthritis (PsA) in two phase 3 trials (DISCOVER-1, DISCOVER-2). Serum concentrations of extracellular matrix (ECM) biomarkers at baseline and following treatment with guselkumab were evaluated in patients with active PsA, and the relationship of these biomarkers with baseline PsA characteristics and clinical response to guselkumab treatment was explored. METHODS: Serum samples were collected at weeks 0, 4, 24, and 52 from a selected subset (N = 260) of the 739 biologic-naïve patients with PsA treated with guselkumab 100 mg every 4 or 8 weeks or placebo in DISCOVER-2. Demographically matched healthy controls (N = 76) were used for comparison. The samples were analyzed for ECM biomarkers associated with collagen degradation (C1M, C2M, C3M, C4M, C6M, C10C) and collagen formation (PRO-C1, PRO-C2, PRO-C3, PRO-C4, PRO-C6). RESULTS: Baseline concentrations of collagen degradation biomarkers C1M, C3M, C4M, and C6M and collagen formation biomarkers PRO-C3 and PRO-C6 were significantly higher (i.e., ≥ 1.25-fold and false discovery rate adjusted p < 0.05) in PsA patients than in healthy controls. Serum C1M, C3M, C4M, and C6M levels declined from baseline in guselkumab-treated patients in both dosing regimens. In addition, guselkumab-treated ACR20 responders (≥ 20% improvement in American College of Rhematology response criteria) had significantly lower C1M levels than ACR20 nonresponders. CONCLUSION: These data demonstrate that serum collagen biomarkers are elevated in patients with PsA compared with healthy controls and that treatment with guselkumab decreases levels of C1M, C3M, C4M, and C6M. Importantly, C1M serves as a biomarker that associates with improvement of joint signs and symptoms. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03158285.

Guselkumab induces robust reduction in acute phase proteins and type 17 effector cytokines in active psoriatic arthritis: results from phase 3 trials.

OBJECTIVE: To investigate serum protein expression in participants with psoriatic arthritis (PsA) and changes after guselkumab treatment. METHODS: Participants with PsA were treated with guselkumab or placebo in the DISCOVER-1 and DISCOVER-2 studies. Serum levels of acute phase reactants C reactive protein (CRP) and serum amyloid A (SAA) and inflammatory cytokines/chemokines were measured at weeks 0, 4 and 24 in 300 study participants and 34 healthy controls (HCs). The PSUMMIT studies measured serum interleukin (IL)-17A, IL-17F and CRP after ustekinumab treatment and levels with ustekinumab versus guselkumab treatment were compared. RESULTS: Baseline serum levels of CRP, SAA, IL-6, IL-17A and IL-17F were elevated in participants with active PsA vs HCs (p<0.05, geometric mean (GM) ≥40% higher). Baseline T-helper cell 17 (Th17) effector cytokines were significantly associated with baseline psoriasis but not joint disease activity. Compared with placebo, guselkumab treatment resulted in decreases in serum CRP, SAA, IL-6, IL-17A, IL-17F and IL-22 as early as week 4 and continued to decrease through week 24 (p<0.05, GM decrease from baseline ≥33%). At week 24, IL-17A and IL-17F levels were not significantly different from HCs, suggesting normalisation of peripheral IL-23/Th17 axis effector cytokines postguselkumab treatment. Reductions in IL-17A/IL-17F levels were greater in guselkumab-treated versus ustekinumab-treated participants, whereas effects on CRP levels were similar. CONCLUSION: Guselkumab treatment reduced serum protein levels of acute phase and Th17 effector cytokines and achieved comparable levels to those in HCs. In participants with PsA, reductions of IL-17A and IL-17F were of greater magnitude after treatment with guselkumab than with ustekinumab.

The CSF-1-receptor inhibitor, JNJ-40346527 (PRV-6527), reduced inflammatory macrophage recruitment to the intestinal mucosa and suppressed murine T cell mediated colitis.

BACKGROUND & AIMS: Originally believed to be primarily a disorder of T-cell signaling, evidence shows that macrophage-lineage cells also contribute to the pathogenesis of Crohn's disease (CD). Colony stimulating factor-1 (CSF-1) is a key regulator of the macrophage lineage, but its role in CD has not been well established. We examined transcriptional data from CD mucosa for evidence of CSF-1 pathway activation and tested JNJ-40346527 (PRV-6527), a small molecule inhibitor of CSF-1 receptor kinase (CSF-1R), for its ability to inhibit disease indices in murine colitis. METHODS: A CSF-1 pathway gene set was created from microarray data of human whole blood cultured ex vivo with CSF-1 and compared to a TNFα-induced gene set generated from epithelial-lineage cells. Gene set variation analysis was performed using existing Crohn's mucosa microarray data comparing patients who either responded or failed to respond to anti-TNFα therapy. Commencing day 14 or day 21, mice with T-cell transfer colitis were treated with vehicle or JNJ-40346527 until study termination (day 42). Endpoints included colon weight/length ratios and histopathology scores, and macrophage and T cells were assessed by immunohistochemistry. Mucosal gene expression was investigated using RNAseq. RESULTS: Both the CSF-1 and the TNFα gene sets were enriched in the colonic mucosal transcriptomes of Crohn's disease and in mouse colitis, and expression of both gene sets was highest in patients who did not respond to anti-TNFα therapy. In these patients neither set was reduced by therapy. In the mouse model, JNJ-40346527 inhibited the increase in colon weight/length ratio by ∼50%, reduced histological disease scores by ∼60%, and reduced F4/80+ mononuclear cell and CD3+ lymphocyte numbers. RNAseq analysis confirmed the CSF-1 gene set was sharply reduced in treated mice, as were gene sets enriched in "M1" inflammatory and "M0" resident macrophages and in activated T cells. CONCLUSIONS: CSF-1 biology is activated in Crohn's disease and in murine T cell transfer colitis. Inhibition of CSF-1R by JNJ-40346527 was associated with attenuated clinical disease scores and reduced inflammatory gene expression in mice. These data provide rationale for testing JNJ-40346527 (PRV-6527) in human inflammatory bowel disease.

Responsiveness of Serum C-Reactive Protein, Interleukin-17A, and Interleukin-17F Levels to Ustekinumab in Psoriatic Arthritis: Lessons From Two Phase III, Multicenter, Double-Blind, Placebo-Controlled Trials.

OBJECTIVE: To evaluate the associations of C-reactive protein (CRP) and circulating Th17-associated cytokine levels with psoriatic arthritis (PsA) disease activity and therapeutic response to ustekinumab. METHODS: Interleukin-17A (IL-17A), IL-17F, IL-23, and CRP concentrations were measured in serum samples collected as part of the 2 PSUMMIT phase III studies of ustekinumab in PsA (n = 927). In post hoc analyses, relationships of IL-17A, IL-17F, and CRP levels at baseline, week 4, and week 24 with baseline skin and joint disease activity and response to therapy were evaluated using generalized linear models and Pearson's product-moment correlation tests. RESULTS: Baseline serum levels of IL-17A and IL-17F were positively correlated with baseline skin disease scores (r = 0.39-0.62). IL-23 levels were correlated with skin disease scores to a lesser extent (r = 0.26-0.31). No significant correlations were observed between these cytokine or CRP levels and baseline joint disease activity. There was no significant association of baseline levels of IL-17A, IL-17F, IL-23, or CRP with therapeutic response to ustekinumab in either the skin or joints. Significant reductions from baseline in levels of IL-17A, IL-17F, and CRP were seen in patients treated with ustekinumab compared to those treated with placebo. Ustekinumab-treated patients in whom 75% improvement in the Psoriasis Area and Severity Index score or 20% improvement according to the American College of Rheumatology criteria was achieved after 24 weeks of treatment had greater reductions in CRP level (geometric mean decreases of 51-58% versus 32-33%; P < 0.05), but not IL-17A or IL-17F levels, than nonresponders. CONCLUSION: Baseline serum IL-23/IL-17 levels correlated with skin, but not joint, disease activity, suggesting tissue-specific variation. However, neither baseline Th17-associated cytokine levels nor CRP level were predictive of therapeutic response to ustekinumab in the skin or joints, despite rapid reductions in their levels following ustekinumab therapy.

I_MDS: an inflammatory bowel disease molecular activity score to classify patients with differing disease-driving pathways and therapeutic response to anti-TNF treatment.

Crohn's disease and ulcerative colitis are driven by both common and distinct underlying mechanisms of pathobiology. Both diseases, exhibit heterogeneity underscored by the variable clinical responses to therapeutic interventions. We aimed to identify disease-driving pathways and classify individuals into subpopulations that differ in their pathobiology and response to treatment. We applied hierarchical clustering of enrichment scores derived from gene set variation analysis of signatures representative of various immunological processes and activated cell types, to a colonic biopsy dataset that included healthy volunteers, Crohn's disease and ulcerative colitis patients. Patient stratification at baseline or after anti-TNF treatment in clinical responders and non-responders was queried. Signatures with significantly different enrichment scores were identified using a general linear model. Comparisons to healthy controls were made at baseline in all participants and then separately in responders and non-responders. Fifty-nine percent of the signatures were commonly enriched in both conditions at baseline, supporting the notion of a disease continuum within ulcerative colitis and Crohn's disease. Signatures included T cells, macrophages, neutrophil activation and poly:IC signatures, representing acute inflammation and a complex mix of potential disease-driving biology. Collectively, identification of significantly enriched signatures allowed establishment of an inflammatory bowel disease molecular activity score which uses biopsy transcriptomics as a surrogate marker to accurately track disease severity. This score separated diseased from healthy samples, enabled discrimination of clinical responders and non-responders at baseline with 100% specificity and 78.8% sensitivity, and was validated in an independent data set that showed comparable classification. Comparing responders and non-responders separately at baseline to controls, 43% and 70% of signatures were enriched, respectively, suggesting greater molecular dysregulation in TNF non-responders at baseline. This methodological approach could facilitate better targeted design of clinical studies to test therapeutics, concentrating on patient subsets sharing similar underlying pathobiology, therefore increasing the likelihood of clinical response.

Combined Blockade of TNF-α and IL-17A Alleviates Progression of Collagen-Induced Arthritis without Causing Serious Infections in Mice.

The cytokines TNF-α and IL-17A are elevated in a variety of autoimmune diseases, including rheumatoid arthritis. Both cytokines are targets of several biologic drugs used in the clinic, but unfortunately many patients are refractory to these therapies. IL-17A and TNF-α are known to mediate signaling synergistically to drive expression of inflammatory genes. Hence, combined blockade of TNF-α and IL-17A represents an attractive treatment strategy in autoimmune settings where monotherapy is not fully effective. However, a major concern with this approach is the potential predisposition to opportunistic infections that might outweigh any clinical benefits. Accordingly, we examined the impact of individual versus combined neutralization of TNF-α and IL-17A in a mouse model of rheumatoid arthritis (collagen-induced arthritis) and the concomitant susceptibility to infections that are likely to manifest as side effects of blocking these cytokines (oral candidiasis or tuberculosis). Our findings indicate that combined neutralization of TNF-α and IL-17A was considerably more effective than monotherapy in improving collagen-induced arthritis disease even when administered at a minimally efficacious dose. Encouragingly, however, dual cytokine blockade did not cooperatively impair antimicrobial host defenses, as mice given combined IL-17A and TNF-α neutralization displayed infectious profiles and humoral responses comparable to mice given high doses of individual anti-TNF-α or anti-IL-17A mAbs. These data support the idea that combined neutralization of TNF-α and IL-17A for refractory autoimmunity is likely to be associated with acceptable and manageable risks of opportunistic infections associated with these cytokines.

Epithelial IL-6 trans-signaling defines a new asthma phenotype with increased airway inflammation.

BACKGROUND: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) to asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthmatic patients is unclear. OBJECTIVE: We sought to explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthmatic patients. METHODS: An IL-6TS gene signature obtained from air-liquid interface cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R was used to stratify lung epithelial transcriptomic data (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes [U-BIOPRED] cohorts) by means of hierarchical clustering. IL-6TS-specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis, and immunohistochemical analysis of bronchial biopsy specimens. RESULTS: Activation of IL-6TS in air-liquid interface cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of patients with IL-6TS-high asthma with increased epithelial expression of IL-6TS-inducible genes in the absence of systemic inflammation. The IL-6TS-high subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings Toll-like receptor pathway genes were upregulated, whereas expression of cell junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, matrix metalloproteinase 3, macrophage inflammatory protein 1ß, IL-8, and IL-1ß. CONCLUSIONS: Local lung epithelial IL-6TS activation in the absence of type 2 airway inflammation defines a novel subset of asthmatic patients and might drive airway inflammation and epithelial dysfunction in these patients.

Enhanced oxidative stress in smoking and ex-smoking severe asthma in the U-BIOPRED cohort.

Oxidative stress is believed to be a major driver of inflammation in smoking asthmatics. The U-BIOPRED project recruited a cohort of Severe Asthma smokers/ex-smokers (SAs/ex) and non-smokers (SAn) with extensive clinical and biomarker information enabling characterization of these subjects. We investigated oxidative stress in severe asthma subjects by analysing urinary 8-iso-PGF2α and the mRNA-expression of the main pro-oxidant (NOX2; NOSs) and anti-oxidant (SODs; CAT; GPX1) enzymes in the airways of SAs/ex and SAn. All the severe asthma U-BIOPRED subjects were further divided into current smokers with severe asthma (CSA), ex-smokers with severe asthma (ESA) and non-smokers with severe asthma (NSA) to deepen the effect of active smoking. Clinical data, urine and sputum were obtained from severe asthma subjects. A bronchoscopy to obtain bronchial biopsy and brushing was performed in a subset of subjects. The main clinical data were analysed for each subset of subjects (urine-8-iso-PGF2α; IS-transcriptomics; BB-transcriptomics; BBr-transcriptomics). Urinary 8-iso-PGF2α was quantified using mass spectrometry. Sputum, bronchial biopsy and bronchial brushing were processed for mRNA expression microarray analysis. Urinary 8-iso-PGF2α was increased in SAs/ex, median (IQR) = 31.7 (24.5-44.7) ng/mmol creatinine, compared to SAn, median (IQR) = 26.6 (19.6-36.6) ng/mmol creatinine (p< 0.001), and in CSA, median (IQR) = 34.25 (24.4-47.7), vs. ESA, median (IQR) = 29.4 (22.3-40.5), and NSA, median (IQR) = 26.5 (19.6-16.6) ng/mmol creatinine (p = 0.004). Sputum mRNA expression of NOX2 was increased in SAs/ex compared to SAn (probe sets 203922_PM_s_at fold-change = 1.05 p = 0.006; 203923_PM_s_at fold-change = 1.06, p = 0.003; 233538_PM_s_at fold-change = 1.06, p = 0.014). The mRNA expression of antioxidant enzymes were similar between the two severe asthma cohorts in all airway samples. NOS2 mRNA expression was decreased in bronchial brushing of SAs/ex compared to SAn (fold-change = -1.10; p = 0.029). NOS2 mRNA expression in bronchial brushing correlated with FeNO (Kendal's Tau = 0.535; p< 0.001). From clinical and inflammatory analysis, FeNO was lower in CSA than in ESA in all the analysed subject subsets (p< 0.01) indicating an effect of active smoking. Results about FeNO suggest its clinical limitation, as inflammation biomarker, in severe asthma active smokers. These data provide evidence of greater systemic oxidative stress in severe asthma smokers as reflected by a significant changes of NOX2 mRNA expression in the airways, together with elevated urinary 8-iso-PGF2α in the smokers/ex-smokers group. Trial registration ClinicalTrials.gov-Identifier: NCT01976767.

Subcutaneous golimumab for children with active polyarticular-course juvenile idiopathic arthritis: results of a multicentre, double-blind, randomised-withdrawal trial.

OBJECTIVE: This report aims to determine the safety, pharmacokinetics (PK) and efficacy of subcutaneous golimumab in active polyarticular-course juvenile idiopathic arthritis (polyJIA). METHODS: In this three-part randomised double-blinded placebo-controlled withdrawal trial, all patients received open-label golimumab (30 mg/m2 of body surface area; maximum: 50 mg/dose) every 4 weeks together with weekly methotrexate during Part 1 (weeks 0-16). Patients with at least 30% improvement per American College of Rheumatology Criteria for JIA (JIA ACR30) in Part 1 entered the double-blinded Part 2 (weeks 16-48) after 1:1 randomisation to continue golimumab or start placebo. In Part 3, golimumab was continued or could be restarted as in Part 1. The primary outcome was JIA flares in Part 2; secondary outcomes included JIA ACR50/70/90 responses, clinical remission, PK and safety. RESULTS: Among 173 patients with polyJIA enrolled, 89.0% (154/173) had a JIA ACR30 response and 79.2%/65.9%/36.4% demonstrated JIA ACR50/70/90 responses in Part 1. At week 48, the primary endpoint was not met as treatment groups had comparable JIA flare rates (golimumab vs placebo: 32/78=41% vs 36/76=47%; p=0.41), and rates of clinical remission were comparable (golimumab vs placebo: 10/78=12.8% vs 9/76=11.8%). Adverse event and serious adverse event rates were similar in the treatment groups during Part 2. Injection site reactions occurred with <1% of all injections. PK analysis confirmed adequate golimumab dosing for polyJIA. CONCLUSION: Although the primary endpoint was not met, golimumab resulted in rapid, clinically meaningful, improvement in children with active polyJIA. Golimumab was well tolerated, and no unexpected safety events occurred. CLINICAL TRIAL REGISTRATION: NCT01230827; Results.

Sputum transcriptomics reveal upregulation of IL-1 receptor family members in patients with severe asthma.

BACKGROUND: Sputum analysis in asthmatic patients is used to define airway inflammatory processes and might guide therapy. OBJECTIVE: We sought to determine differential gene and protein expression in sputum samples from patients with severe asthma (SA) compared with nonsmoking patients with mild/moderate asthma. METHODS: Induced sputum was obtained from nonsmoking patients with SA, smokers/ex-smokers with severe asthma, nonsmoking patients with mild/moderate asthma (MMAs), and healthy nonsmoking control subjects. Differential cell counts, microarray analysis of cell pellets, and SOMAscan analysis of sputum analytes were performed. CRID3 was used to inhibit the inflammasome in a mouse model of SA. RESULTS: Eosinophilic and mixed neutrophilic/eosinophilic inflammation were more prevalent in patients with SA compared with MMAs. Forty-two genes probes were upregulated (>2-fold) in nonsmoking patients with severe asthma compared with MMAs, including IL-1 receptor (IL-1R) family and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NRLP3) inflammasome members (false discovery rate < 0.05). The inflammasome proteins nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 1 (NLRP1), NLRP3, and nucleotide-binding oligomerization domain (NOD)-like receptor C4 (NLRC4) were associated with neutrophilic asthma and with sputum IL-1ß protein levels, whereas eosinophilic asthma was associated with an IL-13-induced TH2 signature and IL-1 receptor-like 1 (IL1RL1) mRNA expression. These differences were sputum specific because no activation of NLRP3 or enrichment of IL-1R family genes in bronchial brushings or biopsy specimens in patients with SA was observed. Expression of NLRP3 and of the IL-1R family genes was validated in the Airway Disease Endotyping for Personalized Therapeutics cohort. Inflammasome inhibition using CRID3 prevented airway hyperresponsiveness and airway inflammation (both neutrophilia and eosinophilia) in a mouse model of severe allergic asthma. CONCLUSION: IL1RL1 gene expression is associated with eosinophilic SA, whereas NLRP3 inflammasome expression is highest in patients with neutrophilic SA. TH2-driven eosinophilic inflammation and neutrophil-associated inflammasome activation might represent interacting pathways in patients with SA.

Inhaled Steroids and Active Smoking Drive Chronic Obstructive Pulmonary Disease Symptoms and Biomarkers to a Greater Degree Than Airflow Limitation.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease, and development of novel therapeutics requires an understanding of pathophysiologic phenotypes. OBJECTIVES: The purpose of the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study was to correlate clinical features and biomarkers with molecular characteristics in a well-profiled COPD cohort. METHODS: A total of 67 COPD subjects (forced expiratory volume in the first second of expiration [FEV1]: 45%-80% predicted) and 63 healthy smoking and nonsmoking controls underwent multiple assessments including patient questionnaires, lung function, and clinical biomarkers including fractional exhaled nitric oxide (FENO), induced sputum, and blood. MEASUREMENTS AND MAIN RESULTS: The impact of inhaled corticosteroids (ICSs), and to a lesser extent current smoking, was more associated with symptom control, exacerbation rates, and clinical biomarkers, than severity by FEV1. The ICS-treated smoking subjects were most symptomatic, with significantly elevated scores on patient-reported outcomes and more annual exacerbations (P < .05). Inhaled corticosteroid users had greater airflow obstruction and air trapping compared with non-ICS users, regardless of smoking status. Smoking, regardless of ICS use, was associated with significantly lower FENO (P < .05). Smoking, in non-ICS users, was associated with an elevated proportion of sputum neutrophils and reduced sputum macrophages. Increased serum C-reactive protein was observed in smokers but not in ICS and nonsmoking ICS users (P < .05). In contrast, only air trapping and neutrophilic inflammation increased with severity, defined by postbronchodilator FEV1. CONCLUSIONS: Compared with COPD severity by FEV1, ICS use and current smoking were better determinants of clinical characteristics and biomarkers. Use of the ADEPT COPD data promises to prove useful in defining biological phenotypes to facilitate personalized therapeutic approaches.

U-BIOPRED clinical adult asthma clusters linked to a subset of sputum omics.

BACKGROUND: Asthma is a heterogeneous disease in which there is a differential response to asthma treatments. This heterogeneity needs to be evaluated so that a personalized management approach can be provided. OBJECTIVES: We stratified patients with moderate-to-severe asthma based on clinicophysiologic parameters and performed an omics analysis of sputum. METHODS: Partition-around-medoids clustering was applied to a training set of 266 asthmatic participants from the European Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes (U-BIOPRED) adult cohort using 8 prespecified clinic-physiologic variables. This was repeated in a separate validation set of 152 asthmatic patients. The clusters were compared based on sputum proteomics and transcriptomics data. RESULTS: Four reproducible and stable clusters of asthmatic patients were identified. The training set cluster T1 consists of patients with well-controlled moderate-to-severe asthma, whereas cluster T2 is a group of patients with late-onset severe asthma with a history of smoking and chronic airflow obstruction. Cluster T3 is similar to cluster T2 in terms of chronic airflow obstruction but is composed of nonsmokers. Cluster T4 is predominantly composed of obese female patients with uncontrolled severe asthma with increased exacerbations but with normal lung function. The validation set exhibited similar clusters, demonstrating reproducibility of the classification. There were significant differences in sputum proteomics and transcriptomics between the clusters. The severe asthma clusters (T2, T3, and T4) had higher sputum eosinophilia than cluster T1, with no differences in sputum neutrophil counts and exhaled nitric oxide and serum IgE levels. CONCLUSION: Clustering based on clinicophysiologic parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.

Severe asthma exists despite suppressed tissue inflammation: findings of the U-BIOPRED study.

The U-BIOPRED study is a multicentre European study aimed at a better understanding of severe asthma. It included three steroid-treated adult asthma groups (severe nonsmokers (SAn group), severe current/ex-smokers (SAs/ex group) and those with mild-moderate disease (MMA group)) and healthy controls (HC group). The aim of this cross-sectional, bronchoscopy substudy was to compare bronchial immunopathology between these groups.In 158 participants, bronchial biopsies and bronchial epithelial brushings were collected for immunopathologic and transcriptomic analysis. Immunohistochemical analysis of glycol methacrylate resin-embedded biopsies showed there were more mast cells in submucosa of the HC group (33.6 mm-2) compared with both severe asthma groups (SAn: 17.4 mm-2, p<0.001; SAs/ex: 22.2 mm-2, p=0.01) and with the MMA group (21.2 mm-2, p=0.01). The number of CD4+ lymphocytes was decreased in the SAs/ex group (4.7 mm-2) compared with the SAn (11.6 mm-2, p=0.002), MMA (10.1 mm-2, p=0.008) and HC (10.6 mm-2, p<0.001) groups. No other differences were observed.Affymetrix microarray analysis identified seven probe sets in the bronchial brushing samples that had a positive relationship with submucosal eosinophils. These mapped to COX-2 (cyclo-oxygenase-2), ADAM-7 (disintegrin and metalloproteinase domain-containing protein 7), SLCO1A2 (solute carrier organic anion transporter family member 1A2), TMEFF2 (transmembrane protein with epidermal growth factor like and two follistatin like domains 2) and TRPM-1 (transient receptor potential cation channel subfamily M member 1); the remaining two are unnamed.We conclude that in nonsmoking and smoking patients on currently recommended therapy, severe asthma exists despite suppressed tissue inflammation within the proximal airway wall.

Systemic inflammatory profile and response to anti-tumor necrosis factor therapy in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by progressive worsening of airflow limitation associated with abnormally inflamed airways in older smokers. Despite correlative evidence for a role for tumor necrosis factor-alpha in the pathogenesis of COPD, the anti-tumor necrosis factor-alpha, infliximab did not show clinical efficacy in a double-blind, placebo-controlled, phase II clinical trial. This study sought to evaluate the systemic inflammatory profile associated with COPD and to assess the impact of tumor necrosis factor neutralization on systemic inflammation. METHODS: Serum samples (n = 234) from the phase II trial were collected at baseline and after 24 weeks of placebo or infliximab. Additionally, baseline serum samples were obtained from an independent COPD cohort (n = 160) and 2 healthy control cohorts (n = 50; n = 109). Serum concentrations of a broad panel of inflammation-associated analytes were measured using a 92-analyte multiplex assay. RESULTS: Twenty-five proteins were significantly elevated and 2 were decreased in COPD, including highly elevated CD40 ligand, brain-derived neurotrophic factor, epidermal growth factor, acute-phase proteins, and neutrophil-associated proteins. This profile was largely independent of smoking status, age, and clinical phenotype. The majority of these associations of serum analytes with COPD are novel findings. Increased serum creatine kinase-muscle/brain and myoglobin correlated modestly with decreased forced expiratory volume at 1 second, suggesting cardiac involvement. Infliximab did not affect this systemic inflammatory profile. CONCLUSIONS: A robust systemic inflammatory profile was associated with COPD. This profile was generally independent of disease severity. Because anti-tumor necrosis factor-alpha did not influence systemic inflammation, how to control the underlying pathology beyond symptom suppression remains unclear.

T-cell specific defect in expression of the NTPDase CD39 as a biomarker for lupus.

Regulatory T cells (T(regs)) are critical for maintenance of peripheral tolerance via suppression of T-cell responses, and absence of T(regs) results in autoimmunity. The role of aberrations in the T(reg) pool for the development of systemic lupus erythematosus (SLE, lupus) remains uncertain. T(reg)-mediated generation of adenosine, dependent on the ectonucleotidase CD39, is an important mechanism for suppression of T-cell responses. We tested whether decreases in numbers of T(regs), and specifically CD39-expressing T(regs), are associated with human lupus. We studied 15 SLE patients, six patients with rheumatoid arthritis (RA) and 24 healthy controls. T(reg) phenotypic markers, including CD39 expression, were studied by flow cytometry. Varying numbers of sorted T(regs) cells were co-cultured with responder T (T(resp)) cells, with proliferation assessed by (3)H-thymidine incorporation. The proportion of T(regs) as defined by Foxp3(+) CD25(+high) CD127(-/low) was similar in lupus and control populations. CD39-expressing T(regs) comprised 37±13% of the T(reg) population in healthy controls and 36±21% in lupus subjects using nonsteroidal immunosuppressants to control active disease, but was nearly absent in five of six lupus subjects with minimally active disease. In contrast to healthy controls and lupus subjects without the CD39 defect, in SLE subjects with the CD39 defect, adenosine-dependent T(reg)-mediated suppression was nearly absent. These results suggest that functional defects in T(regs), rather than reduced T(reg) numbers, are important for the loss of peripheral tolerance in lupus. Presentation of this defect may serve as a biomarker for untreated disease.

Inflammatory profile and response to anti-tumor necrosis factor therapy in patients with chronic pulmonary sarcoidosis.

Sarcoidosis is an inflammatory, granulomatous disease of unknown etiology that most commonly afflicts the lungs. Despite aggressive immunosuppressive therapies, many sarcoidosis patients still chronically present significant symptoms. Infliximab, a therapeutic tumor necrosis factor alpha (TNF-α) monoclonal antibody (MAb), produced a small but significant improvement in forced vital capacity (FVC) in sarcoidosis patients in a double-blind, placebo-controlled, phase II clinical trial. In the current study, serum samples from this clinical trial were assessed to evaluate the underlying hypothesis that treatment with infliximab would reduce systemic inflammation associated with sarcoidosis, correlating with the extent of clinical response. A 92-analyte multiplex panel was used to assess the expression of serum proteins in 134 sarcoidosis patients compared with sera from 50 healthy controls. A strong systemic inflammatory profile was associated with sarcoidosis, with 29 analytes significantly elevated in sarcoidosis (false-discovery rate, <0.05 and >50% higher than controls). The associated analytes included chemokines, neutrophil-associated proteins, acute-phase proteins, and metabolism-associated proteins. This profile was evident despite patients receiving corticosteroids and immunosuppressive therapies. Following infliximab treatment, sarcoidosis patients expressing the highest levels of TNF-α, who had more severe disease, had the greatest improvement in FVC and reduction in serum levels of the inflammatory proteins MIP-1ß and TNF-RII. This study supports the need for further exploration of anti-TNF therapy for chronic sarcoidosis patients, particularly for those expressing the highest serum levels of TNF-α.

Outcome after percutaneous endoscopic gastrostomy in children and young adults.

BACKGROUND AND OBJECTIVES: Factors predicting outcome after percutaneous endoscopic gastrostomy (PEG) in large pediatric cohorts are not well defined. We hypothesized that definable preoperative clinical factors predict the need for further intervention to provide enteral access after PEG. Our aim was to identify factors associated with PEG outcome. MATERIALS AND METHODS: A retrospective review of 760 (407 boys and 353 girls) patients was performed after PEG at the Johns Hopkins Children's Center from 1994 to 2005. Logistic or multiple linear regression was used to analyze indication; diagnosis; age; prematurity; neurological impairment; weight-for-age z scores; modified barium swallow; postoperative complications; need for fundoplication (FP), gastrojejunal tube, or jejunostomy; and length of hospital stay. RESULTS: The median age was 1 year (range 0-26 years). The most common indications given for PEG were failure to thrive (n = 373) and dysphagia (n = 27). Postoperative FP, gastrojejunal tube, or jejunostomy were performed in 66 (10%), 24 (4%), and 9 (1%) patients, respectively. Preoperative report indicated that dysphagia and direct aspiration on modified barium swallow was strongly associated with patients undergoing FP after PEG, 10.6% of patients (P = 0.008, odds ratio 2.4) and 11.2% of patients (P = 0.013, odds ratio 2.8), respectively. Younger preoperative age was also associated with the need for FP (P = 0.0006; median age of 5.8 vs 14 months). Patients with preoperative dysphagia had a longer median length of hospital stay: 8 versus 3 days (P < 0.00001). Patients with neurological impairment demonstrated greater weight gain than neurologically normal patients after PEG (P = 0.04). Minor postoperative complications (most commonly wound infection) were observed in 4% (27/747) of children before hospital discharge from PEG and in 20% of children (138/682) after discharge. There were only 2 major complications (gastric separation and gastrocolonic fistula.). There were no fatalities. CONCLUSIONS: Preoperative diagnosis, indication, prematurity, and neurological impairment did not influence postoperative complications.

Genetic and epigenetic inactivation of TNFRSF10C in human prostate cancer.

BACKGROUND: TNFRSF10C, is located on 8p21.3, one of the most frequently deleted loci in the genome of prostate cancer (PCa). Hypermethylation of TNFRSF10C promoter CpG island (CGI) had been reported in many tumors including PCa. However, the interplay between somatic deletion and promoter hypermethylation of TNFRSF10C on PCa development has not been investigated. METHODS: Methylation status of promoter CGI and deletion status of the TNFRSF10C locus was investigated by bisulfite sequencing and Affymetrix SNP array, respectively, in 59 pairs of PCa tumor and matched normal samples with three PCa cell lines. TNFRSF10C gene expression changes in relation to cancer-associated genetic/epigenetic changes in clinical specimens, and change of TNFRSF10C expression before and after 5-aza-2'-deoxycytidine treatment in the PC3 PCa cell line was assessed by real-time RT-PCR. RESULTS: We found that TNFRSF10C promoter CGI was differentially methylated in 46 of 59 primary cancers (78.0%). Hemizygous deletion at TNFRSF10C was found in 44 of the 59 prostate tumors (74.5%). Interestingly, in 94.9% of the tumors (56 out of 59), TNFRSF10C was either hemizygously deleted or its promoter CGI hypermethylated. Deletion and/or methylation of the TNFRSF10C gene were correlated with decreased mRNA expression of the gene in clinical specimens. Demethylation of the TNFRSF10C promoter CGI was accompanied by transcriptional re-activation of TNFRSF10C in the PCa cell line PC3. CONCLUSION: We found a notably high frequency of promoter CGI methylation and deletion of TNFRSF10C in PCa tissues. Our results indicated that inactivation of TNFRSF10C by chromosomal deletion and promoter methylation may play an important role in PCa development.

Glucocorticoid- and protein kinase A-dependent transcriptome regulation in airway smooth muscle.

Glucocorticoids (GCs) and protein kinase A (PKA)-activating agents (beta-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma--excessive ASM growth--are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1 beta and TNF-alpha mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2-dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1 beta stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1 beta, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein-coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of beta-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies.

beta-Agonist enhances type 2 T-cell survival and accumulation.

BACKGROUND: Neurohumoral modulation of immune system function is poorly understood. beta-Adrenergic receptor ligands (beta-agonists) subserve numerous physiologic processes but also function as pathogenic or therapeutic agents in numerous diseases with inflammatory components. OBJECTIVES: We sought to establish the effects of beta-agonists and prostaglandin E(2) (PGE(2)) on antigen-dependent and antigen-independent accumulation of IL-13(+) (type 2) and IFN-gamma(+) (type 1) T cells. We also sought to clarify the mechanisms mediating the effects of these G protein-coupled receptor agonists. METHODS: Effects of beta-agonists or PGE(2) on T-cell subtype accumulation were assessed in peripheral blood lymphocytes cultured with alphaCD3/CD28 or IL-2 by using flow cytometry. The role of cyclic AMP-dependent protein kinase (PKA) in mediating agonist effects was assessed by means of characterization of (1) phosphorylation of an intracellular PKA substrate and (2) T cells from patients with lupus possessing a natural defect in PKA activation. RESULTS: beta-Agonists, in contrast to PGE(2), increased IL-2-induced accumulation of human type 2 T cells, an effect attributable to differential activation of PKA affecting regulation of cell proliferation and apoptosis. In T cells from patients with lupus exhibiting defective PKA activation, both beta-agonists and PGE(2) promoted an increase in type 2 T-cell accumulation. CONCLUSION: G(s)-coupled receptors have the capacity to elicit prosurvival signaling in type 2 T cells, which, in most instances, is obscured by concomitant and antimitogenic PKA activation. CLINICAL IMPLICATIONS: beta-Agonists and other G(s)-coupled receptor agonists have the potential to regulate T-cell development to affect disease pathogenesis or the efficacy of therapies, and variability of effect relates to the ability to stimulate PKA activity.