Search

Udgivelsesdato: 2008-May-22 BACKGROUND: We hypothesized that percutaneous coronary intervention (PCI) preceded by early treatment with abciximab plus half-dose reteplase (combination-facilitated PCI) or with abciximab alone (abciximab-facilitated PCI) would improve outcomes in patients with acute ST-segment elevation myocardial infarction, as compared with abciximab administered immediately before the procedure (primary PCI). METHODS: In this international, double-blind, placebo-controlled study, we randomly assigned patients with ST-segment elevation myocardial infarction who presented 6 hours or less after the onset of symptoms to receive combination-facilitated PCI, abciximab-facilitated PCI, or primary PCI. All patients received unfractionated heparin or enoxaparin before PCI and a 12-hour infusion of abciximab after PCI. The primary end point was the composite of death from all causes, ventricular fibrillation occurring more than 48 hours after randomization, cardiogenic shock, and congestive heart failure during the first 90 days after randomization. RESULTS: A total of 2452 patients were randomly assigned to a treatment group. Significantly more patients had early ST-segment resolution with combination-facilitated PCI (43.9%) than with abciximab-facilitated PCI (33.1%) or primary PCI (31.0%; P=0.01 and P=0.003, respectively). The primary end point occurred in 9.8%, 10.5%, and 10.7% of the patients in the combination-facilitated PCI group, abciximab-facilitated PCI group, and primary-PCI group, respectively (P=0.55); 90-day mortality rates were 5.2%, 5.5%, and 4.5%, respectively (P=0.49). CONCLUSIONS: Neither facilitation of PCI with reteplase plus abciximab nor facilitation with abciximab alone significantly improved the clinical outcomes, as compared with abciximab given at the time of PCI, in patients with ST-segment elevation myocardial infarction. (ClinicalTrials.gov number, NCT00046228 [ClinicalTrials.gov].)

Background: Facial lesions including lupus pernio are often a form of chronic cutaneous sarcoidosis. Objective: To evaluate the intra- and inter-observer consistency of objective measures of chronic facial lesions. Method: This was a retrospective study of patients with chronic cutaneous facial lesions including lupus pernio. The lesions were evaluated using two methods. Results: Of the 25 patients studied, 23 were women and 24 were African American. Lungs (24 patients), sinuses (11 patients), and eyes (7 patients) were also affected. The Sarcoidosis Activity and Severity Index (SASI) characterized individual areas of the face, with 95% of the observations being less than 2 points from the median. A facial SASI total gave a score for the entire face and 93.2% of the scores were within 3 points of the median. Conclusion: Patients with sarcoidosis and chronic facial lesions often have lung, sinus, and eye involvement. The SASI is a reproducible scoring system for chronic facial lesions.

Background and Purpose— A previous randomized, placebo-controlled, double-blind study suggested that abciximab may be safe and effective in treatment of acute ischemic stroke. The current phase 3 study was planned to test the relative efficacy and safety of abciximab in patients with acute ischemic stroke with planned treatment within 5 hours since symptoms onset. Methods— An international, randomized, placebo-controlled, double-blind phase 3 trial tested intravenous administration of abciximab in 2 study cohorts using stratification variables of time since onset and stroke severity. The planned enrollment was 1800 patients. The primary cohort enrolled those patients who could be treated within 5 hours of onset of stroke. A companion cohort enrolled patients that were treated 5 to 6 hours after stroke as well as a smaller cohort of patients who could be treated within 3 hours of stroke present on awakening. The primary efficacy measure was the dichotomous modified Rankin Scale score at 3 months as adjusted to the baseline severity of stroke among subjects in the primary cohort. The primary safety outcome was the rate of symptomatic or fatal intracranial hemorrhage that occurred within 5 days of stroke. Results— The trial was terminated prematurely after 808 patients in all cohorts were enrolled by recommendation of an independent safety and efficacy monitoring board due to an unfavorable benefit-risk profile. At 3 months, approximately 33% of patients assigned placebo (72/218) and 32% of patients assigned abciximab (71/221; P =0.944) in the primary cohort were judged to have a favorable response to treatment. The distributions of outcomes on the modified Rankin Scale were similar between the treated and control groups. Within 5 days of enrollment, ≈5.5% of abciximab-treated and 0.5% of placebo-treated patients in the primary cohort had symptomatic or fatal intracranial hemorrhage ( P =0.002). The trial also did not demonstrate an improvement in outcomes with abciximab among patients in the companion and wake-up cohorts. Although the number of patients was small, an increased rate of hemorrhage was noted within 5 days among patients in the wake-up population who received abciximab (13.6% versus 5% for placebo). Conclusions— This trial did not demonstrate either safety or efficacy of intravenous administration of abciximab for the treatment of patients with acute ischemic stroke regardless of end point or population studied. There was an increased rate of symptomatic or fatal intracranial hemorrhage in the primary and wake-up cohorts.

Chronic obstructive pulmonary disease (COPD) is a progressive, smoking-related, inflammatory lung disease in which tumor necrosis factor-alpha is overexpressed and has been suggested to play a pathogenic role.To determine if infliximab, an anti-TNF-alpha antibody, results in clinical benefit and has an acceptable safety profile in patients with moderate to severe COPD.In a multicenter, randomized, double-blind, placebo-controlled, parallel-group, dose-finding study, subjects with moderate to severe COPD received infliximab (3 mg/kg [n = 78] or 5 mg/kg [n = 79]) or placebo (n = 77) at Weeks 0, 2, 6, 12, 18, and 24. Efficacy, health status, and safety were assessed through Week 44.Infliximab was generally well tolerated, but showed no treatment benefit as measured by the primary endpoint, Chronic Respiratory Questionnaire total score. Similarly, there was no change in secondary measures, including prebronchodilator FEV(1), 6-min walk distance, SF-36 physical score, transition dyspnea index, or moderate-to-severe COPD exacerbations. Post hoc analysis revealed that subjects who were younger or cachectic showed improvement in the 6-min walk distance. Malignancies were diagnosed during the study in 9 of 157 infliximab-treated subjects versus 1 of 77 placebo-treated subjects. No opportunistic infections were observed, and there were no differences in the occurrence of antibiotic-requiring infections, although the incidence of pneumonia was higher in infliximab-treated subjects. No infection-related mortality was observed. Higher proportions of infliximab-treated subjects discontinued the study agent due to adverse events (20-27%) than did placebo-treated subjects (9%).Subjects with moderate to severe COPD did not benefit from treatment with infliximab. Although not statistically significant, more cases of cancer and pneumonia were observed in the infliximab-treated subjects. The impact of infliximab on malignancy risk in patients with COPD needs to be further elucidated.

Rationale: The ADEPT study aimed to correlate clinical features and accessible biomarkers with molecular characteristics by profiling asthmatics across severities and healthy nonatopic volunteers (HVs). This report focuses on the identification of mucosal Th2 phenotype (based on IL-13 driven gene expression), using accessible biomarkers, such as exhaled nitric oxide (FENO), blood eosinophils (bEOS) and serum analytes. Methods: Assessments included questionnaires, pulmonary function tests, airway hyperresponsiveness, FENO, and biomarkers from sputum, blood, and endobronchial biopsy (EBBX). Th2 phenotype was evaluated by EBBX gene expression of CCL26, periostin, or an IL-13 in vitro gene signature (IVS). Results: Relative to HVs, EBBX gene expression of CCL26 provided the best segregation into Th2-high and Th2-low asthmatics compared to periostin or IL-13 IVS. Most EBBX-Th2-CCL26-high subjects with moderate-severe asthma were FENO≥35ppb (69%) and bEOS≥300/ul (77%), compared to a minority of EBBX-Th2-CCL26-low subjects (24% for each). Classifying subjects to EBBX-Th2-CCL26-high status based on FENO-high or bEOS-high status gave 100% sensitivity but only 64% specificity. Serum CCL17-high status added to FENO/bEOS model improved specificity to 93% for identification of Th2 status, with 85% sensitivity. Conclusions: Combinations of accessible biomarkers (FENO, bEOS, and serum CCL17) can accurately predict Th2 status measured by EBBX expression of CCL26. Eosinophilic inflammation was associated with but not limited to persistent airway Th2 gene expression. The ability to identify distinct asthma phenotypes using accessible biomarkers will be important in developing novel therapeutic agents.

Patient-reported outcome (PRO) measures have been developed to measure symptoms and other aspects of health-related quality of life.The Sarcoidosis Assessment Tool (SAT), a sarcoidosis-specific PRO, was administered in a lung and skin sarcoidosis treatment trial. We explored SAT performance characteristics and correlation with standard clinical measurements to validate it as a useful clinical sarcoidosis-specific PRO.The SAT analyses focused on baseline and Week 16 assessments. Besides the SAT, participants underwent clinical and physician assessments plus additional PROs that were used as anchor variables and were compared with the SAT. Reliability was evaluated by using Cronbach α coefficient. Spearman correlation coefficients were used to evaluate the association between SAT scores with clinical and other PRO measures. Changes between assessments in the clinical and PRO "anchor" variables were classified as improved, stable, or worsened. Mean differences between adjacent categories of the known groups and mean changes from the ability to detect change analyses were reviewed for appropriate clinically important difference estimates.Results from 173 patients were analyzed. Each SAT module reflected appropriate anchor variables at baseline and in terms of change. The Cronbach α for each of these modules was at least 0.87. In addition, we successfully established a clinically important difference range for each SAT module.We demonstrated that the SAT is a reliable and consistent sarcoidosis-specific PRO. It has excellent internal consistency and reliability. A range of clinically important differences has been established for the SAT modules. Clinical trial registered with www.clinicaltrials.gov (NCT 00955279).

The objective of this study was to determine the safety and efficacy of carlumab in the treatment of idiopathic pulmonary fibrosis (IPF).A phase 2, randomised, double-blind placebo-controlled dose-ranging study was conducted in patients with IPF (n=126). Patients were randomised to carlumab (1 mg·kg−1, 5 mg·kg−1, or 15 mg·kg−1) or placebo every 4 weeks. The primary endpoint was the rate of percentage change in forced vital capacity (FVC). Secondary endpoints were time to disease progression, absolute change in FVC, relative change in diffusing capacity of the lung for carbon monoxide (DLCO), and St George's Respiratory Questionnaire (SGRQ) total score.Due to a pre-planned, unfavourable interim benefit–risk analysis, dosing was suspended. The rate of percentage change in FVC showed no treatment effect (placebo −0.582%, 1 mg·kg−1−0.533%, 5 mg·kg−1−0.799% and 15 mg·kg−1−0.470%; p=0.261). All active treatment groups showed a greater decline in FVC (1 mg·kg−1−290 mL, 5 mg·kg−1−370 mL and 15 mg·kg−1−320 mL) compared with placebo (−130 mL). No effect on disease progression,DLCO, infection rates or mortality was observed. SGRQ scores showed a nonsignificant trend toward worsening with active treatment. Unexpectedly, free CC-chemokine ligand 2 levels were elevated above baseline at both 24 and 52 weeks. A higher proportion of patients with one or more serious adverse events was observed in the 5 mg·kg−1group (53.1%) compared with 1 mg·kg−1(15.2%), 15 mg·kg−1(21.9%) and placebo (46.4%), although no unexpected serious adverse events were noted.Although dosing was stopped prematurely, it is unlikely that carlumab provides benefit to IPF patients.

Background Platelet glycoprotein IIb/IIIa inhibitors are administered during percutaneous coronary intervention as a bolus followed by infusion. The need for an infusion was established by the Evaluation of 7E3 for the Prevention of Ischemic Complications (EPIC) trial conducted during the percutaneous transluminal coronary balloon angioplasty (PTCA) era, when the threat of acute thrombotic complications prevailed over concerns regarding bleeding, and stenting was considered an adverse event. Methods The EPIC trial randomized high-risk PTCA patients to 3 arms: placebo, abciximab bolus only, and abciximab bolus plus infusion. The present analysis of the EPIC outcomes was done at 6-hour intervals during the first 24 hours after PTCA to identify any early benefit derived from the abciximab bolus–only arm. Results At 6 hours after randomization, the primary composite end point of death, myocardial infarction, or urgent intervention was significantly reduced by 46% with abciximab bolus-only compared with placebo (2.9% vs 5.3%; P = .022), which is mainly due to a reduced rate of urgent intervention. There was also a numerical but not statistically significant reduction in myocardial infarction rate using abciximab bolus-only compared with placebo. A lower bleeding rate in the bolus-only arm compared with bolus plus infusion has been reported. Conclusions As stenting and thienopyridine use have become routine, there has been a decrease in the incidence of acute closure and an increasing concern for bleeding complications after percutaneous coronary intervention, which potentially may be addressed by adopting a bolus-only glycoprotein IIb/IIIa inhibitor strategy. The early protective ischemic effect of abciximab bolus-only observed in the EPIC trial may be relevant in this regard.

Rationale: Neutralization of tumor necrosis factor-α (TNF-α) is an effective antiinflammatory therapy for several chronic inflammatory diseases.Methods and Objectives: We undertook a double-blind, placebo-controlled, parallel-group design study in 38 patients with moderate asthma treated with inhaled corticosteroids but symptomatic during a run-in phase. Infliximab (5 mg/kg) or placebo was administered by intravenous infusion at Weeks 0, 2, and 6. We assessed clinical response by monitoring lung function, symptoms, and inhaled β2-agonist usage using hand-held electronic devices.Results: The primary endpoint, change in morning PEF at Days 50–56 compared with the last 7 d of the run-in, was not significantly different on treatment. However, infliximab was associated with a decrease in mean diurnal variation of PEF at Week 8 (p = 0.02; 95% confidence interval [CI], −8.1 to −0.72). Furthermore, there was a decrease in the number of patients with exacerbations of asthma (p = 0.01; 95% CI, 4.4 to 52.7) and an i...

RATIONALE: Evidence suggests that tumor necrosis factor (TNF)-alpha plays an important role in the pathophysiology of sarcoidosis. OBJECTIVES: To assess the efficacy of infliximab in sarcoidosis. METHODS: A phase 2, multicenter, randomized, double-blind, placebo-controlled study was conducted in 138 patients with chronic pulmonary sarcoidosis. Patients were randomized to receive intravenous infusions of infliximab (3 or 5 mg/kg) or placebo at Weeks 0, 2, 6, 12, 18, and 24 and were followed through Week 52. MEASUREMENTS AND MAIN RESULTS: The primary endpoint was the change from baseline to Week 24 in percent of predicted FVC. Major secondary efficacy parameters included Saint George's Respiratory Questionnaire, 6-min walk distance, Borg's CR10 dyspnea score, and the proportion of Lupus Pernio Physician's Global Assessment responders for patients with facial skin involvement. Patients in the combined infliximab groups (3 and 5 mg/kg) had a mean increase of 2.5% from baseline to Week 24 in the percent of predicted FVC, compared with no change in placebo-treated patients (p = 0.038). No significant differences between the treatment groups were observed for any of the major secondary endpoints at Week 24. Results of post hoc exploratory analyses suggested that patients with more severe disease tended to benefit more from infliximab treatment. CONCLUSIONS: Infliximab therapy resulted in a statistically significant improvement in % predicted FVC at Week 24. The clinical importance of this finding is not clear. The results of this Phase 2 clinical study support further evaluation of anti-TNF-alpha therapy in severe, chronic, symptomatic sarcoidosis.

We postulated that patients with recent acute coronary syndromes and antibodies to the platelet factor 4/heparin complex would have an increased risk of myocardial infarction (MI), even in the absence of thrombocytopenia. We analyzed sera from patients enrolled in the placebo/unfractionated heparin arm of the GUSTO IV-ACS trial who had a high likelihood of prior heparin exposure. We selected 109 patients without thrombocytopenia with the 30-day primary endpoint (death, MI, or revascularization) and 109 age-, gender-, and race-matched controls who did not achieve the primary endpoint. Twenty-three of 218 patients (10.6%) had anti-PF4/heparin antibodies. Patients with anti-PF4/heparin were more likely to have death or MI (30.4% vs. 11.3%, p = 0.011) or MI (21.7% vs. 6.2%, p = 0.008) than patients who were negative for the antibody. Antibody-positive patients had higher levels of sVCAM-1 (892 +/- 263 microg/L vs. 780 +/- 228 microg/L; p = 0.04) and sICAM-1 (246 +/- 50 microg/L vs. 222 +/- 71 microg/L; p = 0.02) than antibody-negative patients. In a multiple logistic regression model that included inflammatory markers and clinical risk factors, antibodies to PF4/heparin were a strong predictor of 30-day MI (odds ratio, 9.0; 95% confidence intervals, 2.1 to 38.6; p < 0.01), with IL-6 being the only other predictor (odds ratio, 1.1; 95% confidence intervals, 1.0 to 1.2; p = 0.03). Antibodies to the platelet factor 4/heparin complex are a novel, independent predictor of MI at 30 days in patients presenting with acute coronary ischemic syndromes. Antibodies to PF4/heparin are a stronger predictor of MI than clinical characteristics or inflammation markers.

Because of its potential for antigenicity, theoretical concerns related to readministration of abciximab have been raised. We conducted the ReoPro Readministration Registry to assess the efficacy and safety of abciximab readministration. A total of 1,342 patients who underwent percutaneous coronary intervention and who received abciximab for at least a second time were recruited. Safety end points were hypersensitivity reactions, major bleeding, and thrombocytopenia (TCP). Human antichimeric antibody (HACA) titers were determined before and after readministration. Procedural success was 98% and was not influenced by the number of courses of abciximab or the presence of HACA. There were no cases of anaphylaxis. There were 5 minor allergic reactions, none of which required termination of the infusion. Clinically significant bleeding occurred in 31 patients (2.3%), including 1 (0.07%) with intracranial hemorrhage. TCP (

ADEPT (Airways Disease Endotyping for Personalized Therapeutics) and U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcome Consortium) are independent asthma biomarker studies that aim to enable personalization of therapies.Patients in both studies were identified by similar criteria, and similar clinical parameters and biomarkers were assessed in blood, sputum, and airway samples. Fuzzy partition-around-medoid clustering was performed on the ADEPT dataset (n = 154) and independently on the U-BIOPRED asthma dataset (n = 82), filtered to match ADEPT inclusion criteria. For both studies, the same eight easily measurable clinical variables were used, and ADEPT also included methacholine airway hyperresponsiveness. Models for cluster classification probabilities were derived and applied to the 12-month longitudinal ADEPT data and the full U-BIOPRED adult asthma dataset (n = 397) as independent external validation.Four clusters were identified in the ADEPT-asthma study population with distinct clinical and biomarker profiles. In general, Cluster 1 consists of patients with mild asthma not treated with steroids and well controlled with preserved lung function and a low-inflammatory phenotype; Cluster 2 is partially controlled, with mild airflow obstruction but severe airway hyperresponsiveness and a Th2 phenotype (brittle phenotype); Cluster 3 is partially controlled with mild airflow obstruction but reduced vital capacity, less bronchodilator reversibility, and a non-Th2 phenotype with neutrophilic inflammation (chronic obstructive pulmonary disease-like); and Cluster 4 is poorly controlled, with marked airflow obstruction, marked bronchodilator reversibility, and a mixed inflammatory phenotype. Overall, the ADEPT clusters were stable over 12 months and reproduced by identifying four analogous clusters in the U-BIOPRED asthma dataset, with distributions for most clustering and nonclustering variables similar to ADEPT.We report four clinical clusters in ADEPT and confirmed these by external validation in U-BIOPRED. The ADEPT clusters have distinct clinical and molecular characteristics, are stable over 12 months, and present opportunities for the development of tailored therapeutics for asthma.

Background— Biochemical markers are useful for prediction of cardiac events in patients with non–ST-segment-elevation acute coronary syndrome (ACS). The associations between N -terminal pro–brain natriuretic peptide (NT-proBNP) and other biochemical and clinical risk indicators, as well as their prognostic value concerning the individual end points of death and myocardial infarction (MI), were elucidated in a large cohort of ACS patients. Methods and Results— NT-proBNP, troponin T, and C-reactive protein (CRP) were analyzed in blood samples obtained at a median of 9.5 hours from symptom onset in 6809 of 7800 ACS patients in the Global Utilization of Strategies To Open occluded arteries-IV (GUSTO-IV) trial. Levels of NT-proBNP were correlated independently with age, female gender, low body weight, diabetes, renal dysfunction, history of MI, heart failure, heart rate, ongoing myocardial damage, and time since onset of ischemia. Increasing quartiles of NT-proBNP were related to short- and long-term mortality that reached 1.8%, 3.9%, 7.7%, and 19.2%, ( P Conclusions— The use of NT-proBNP appears to add critical prognostic insight to the assessment of patients with ACS.

Background— We postulated that antibodies to platelet factor 4/heparin complex might contribute to recurrent ischemic events in patients with acute coronary syndrome. Methods and Results— We analyzed serum from patients enrolled in the placebo/unfractionated heparin arm of the GUSTO IV-ACS trial who had high likelihood of prior heparin exposure. We selected 109 patients without thrombocytopenia with the 30-day primary end point (death, myocardial infarction [MI], or revascularization) and 109 age-, gender-, and race-matched controls who did not achieve the primary end point. An ELISA for anti-platelet factor 4/heparin antibodies was performed using 48-hour serum samples. The analyses were done by blinded investigators, and the results were correlated with clinical outcomes. Twenty-three of 218 patients (10.6%) had anti-PF4/heparin antibodies. Patients with anti-PF4/heparin antibodies were more likely to have death or MI (30.4% versus 11.3%, P =0.011) or MI (21.7% versus 6.2%, P =0.008) than patients who were negative for the antibody. After multiple logistic regression analysis, anti-PF4/heparin antibodies remained a predictor of 30-day death or MI (odds ratio, 4.0; 95% CI, 1.4 to 11.3; P =0.0093) and MI (odds ratio, 4.6; 95% CI, 1.4 to 15.0; P =0.0108). The antibody was not associated with the composite end point (death, MI, or revascularization) or with death or revascularization alone. Conclusions— Antibodies to the platelet factor 4/heparin complex are a novel, independent predictor of myocardial infarction at 30 days in patients presenting with acute coronary ischemic syndromes. This finding may explain the previous association between thrombocytopenia and adverse events in patients with acute coronary syndrome and may have important implications for the choice of anticoagulant regimens.

Objective The purpose of the study was to establish a rebolus regimen for abciximab that restores pharmacologic glycoprotein (GP) IIb/IIIa receptor blockade within a short time frame (up to 48 hours) after completion of an initial treatment. Methods and Results The study was a single-center, nonrandomized, open-label dose escalation trial in healthy volunteers (n = 30). Each subject received a 0.25 mg/kg bolus and a 0.125 μg/kg per minute infusion of abciximab, followed by incremental bolus doses of the agent at 15-minute intervals up to 48 hours (10 per group) after completion of the infusion, (maximal cumulative rebolus dose of 0.25 mg/kg). Pharmacodynamic measurements (GP IIb/IIIa receptor blockade, turbidimetric and whole blood platelet aggregation with use of a rapid platelet function assay [RPFA]) were obtained at periodic intervals during and after administration of the abciximab bolus and infusion. At the time of the first rebolus, pharmacodynamic measurements were attained immediately before administration of each rebolus and 15 minutes after the last rebolus dose. In subjects who received reboluses 12 hours after infusion, a cumulative dose of 0.05 mg/kg restored >80% blockade of GP IIb/IIIa receptors and >80% inhibition of turbidimetric (5 and 20 μmol/L adenosine diphosphate) and RPFA aggregation in 10 of 10 subjects. At 24 hours after treatment, a cumulative abciximab bolus dose of 0.1 mg/kg restored >80% blockade of all 4 pharmacodynamic measurements in 10 of 10 subjects. At 48 hours after treatment, a cumulative bolus dose of 0.15 mg/kg restored >80% blockade of all 4 pharmacodynamic measurements in 10 of 10 subjects. Conclusions A fraction of the bolus of abciximab restored pharmacologic (>80%) GP IIb/IIIa receptor blockade when readministered at various postinfusion time points. These observations suggest that in the setting where acute readministration of abciximab is required less than a full bolus dose of the agent is warranted. (Am Heart J 2002;143:87-94.)

Limited evidence exists demonstrating an effective treatment for chronic cutaneous sarcoidosis.To determine infliximab's effectiveness in sarcoidosis.We conducted a subset analysis from a randomized, double-blind, placebo-controlled trial for chronic pulmonary sarcoidosis to determine infliximab's effectiveness. Patients with chronic cutaneous sarcoidosis received infliximab (3 or 5 mg/kg) or placebo over 24 weeks. Of 138 patients, the subset analysis evaluated 17 patients with chronic facial and another 9 patients with nonfacial skin involvement. The SASI evaluated lesions for degree of erythema, desquamation, induration, and percentage of area involved. Facial and nonfacial lesions were scored in a blinded manner.Among 5 placebo-treated and 12 infliximab-treated patients, an improvement was observed with infliximab versus placebo in change from baseline to weeks 12 and 24 in desquamation (P0.005) and induration (P0.01) at week 24. Erythema, percentage of area involved and the evaluation of paired photographs did not reveal significant differences.Sample size; more extensive disease in placebo patients; chronic therapy upon enrollment; lung as primary organ of sarcoidosis involvement; limited investigator experience with SASI.Infliximab appears to be a beneficial treatment for chronic cutaneous sarcoidosis. The SASI scoring system demonstrated significant improvement versus placebo in lesion desquamation and induration.

Recent advances in genetics have spurred rapid progress towards the systematic identification of genes involved in complex diseases. Still, the detailed understanding of the molecular and physiological mechanisms through which these genes affect disease phenotypes remains a major challenge. Here, we identify the asthma disease module, i.e. the local neighborhood of the interactome whose perturbation is associated with asthma, and validate it for functional and pathophysiological relevance, using both computational and experimental approaches. We find that the asthma disease module is enriched with modest GWAS P-values against the background of random variation, and with differentially expressed genes from normal and asthmatic fibroblast cells treated with an asthma-specific drug. The asthma module also contains immune response mechanisms that are shared with other immune-related disease modules. Further, using diverse omics (genomics, gene-expression, drug response) data, we identify the GAB1 signaling pathway as an important novel modulator in asthma. The wiring diagram of the uncovered asthma module suggests a relatively close link between GAB1 and glucocorticoids (GCs), which we experimentally validate, observing an increase in the level of GAB1 after GC treatment in BEAS-2B bronchial epithelial cells. The siRNA knockdown of GAB1 in the BEAS-2B cell line resulted in a decrease in the NFkB level, suggesting a novel regulatory path of the pro-inflammatory factor NFkB by GAB1 in asthma.

Leukotriene B4 (LTB 4 ) is a chemotactic mediator implicated in the pathogenesis of asthma. JNJ-40929837 is an oral inhibitor of LTA 4 hydrolase, which catalyzes LTB 4 production. We evaluated the effects of JNJ-40929837 in a human bronchial allergen challenge (BAC) model. In this double-blind, 3-period crossover study, 22 patients with mild, atopic asthma were randomized to one of three treatments per period: 100 mg/day JNJ-40929837 for 6 days followed by 50 mg/day on day 7; 10 mg/day montelukast for 6 days; and matched placebo. The BAC was performed on day 6 of each treatment period. Primary outcome was BAC-induced late asthmatic response (LAR) measured by maximal percent reduction in forced expiratory volume (FEV 1 ) in one second. Secondary outcomes included early asthmatic response (EAR) by maximal percent reduction in FEV 1 , EAR and LAR evaluated by area under the FEV 1 /time curve (AUC 0-2 , AUC 3-10 , respectively), change in baseline FEV 1 after 5-day treatment, safety, and correlation of JNJ-40929837 to the divalent cation ionophore A23187-stimulated whole blood LTB 4 levels and sputum basal LTB 4 levels. No significant differences were observed in the primary or secondary FEV 1 endpoints with JNJ-40929837 versus placebo. Compared with placebo ( n = 17, LS mean = 27.7), there was no significant attenuation of the maximal percent reduction in the LAR FEV 1 with JNJ-40929837 ( n = 16, LS mean = 28.6, P = 0.63) but montelukast ( n = 17, LS mean = 22.6, P = 0.01) significantly attenuated the LAR. JNJ-40929837 substantially inhibited LTB 4 production in whole blood, decreased sputum LTB 4 levels and was well-tolerated. The number of adverse events leading to study withdrawal was the same in JNJ-40929837 and placebo groups. In conclusion, JNJ-40929837 demonstrated target engagement in blood and sputum. No significant impact in response to allergen inhalation was observed with JNJ-40929837 versus placebo. Registration This study is registered at ClinicalTrials.gov: NCT01241422 .

Sarcoidosis is characterised by non-caseating granulomas that secrete pro-inflammatory cytokines, including interleukin (IL)-12, IL-23, and tumour necrosis factor (TNF)-α. Ustekinumab and golimumab are monoclonal antibodies that specifically inhibit IL-12/IL-23 and TNF-α, respectively.Patients with chronic pulmonary sarcoidosis (lung group) and/or skin sarcoidosis (skin group) received either 180 mg ustekinumab at week 0 followed by 90 mg every 8 weeks, 200 mg golimumab at week 0 followed by 100 mg every 4 weeks, or placebo. Patients underwent corticosteroid tapering between weeks 16 and 28. The primary end-point was week 16 change in percentage predicted forced vital capacity (ΔFVC % pred) in the lung group. Major secondary end-points were: week 28 for ΔFVC % pred, 6-min walking distance, St George's Respiratory Questionnaire (lung group), and Skin Physician Global Assessment response (skin group).At week 16, no significant differences were observed in ΔFVC % pred with ustekinumab (-0.15, p = 0.13) or golimumab (1.15, p = 0.54) compared with placebo (2.02). At week 28, there were no significant improvements in the major secondary end-points, although a nonsignificant numerically greater Skin Physician Global Assessment response was observed following golimumab treatment (53%) when compared with the placebo (30%). Serious adverse events were similar in all treatment groups.Although treatment was well tolerated, neither ustekinumab nor golimumab demonstrated efficacy in pulmonary sarcoidosis. However, trends towards improvement were observed with golimumab in some dermatological end-points.

Background — Previous investigators have shown that systemic markers of inflammation may be increased in patients with acute ischemic syndromes or after percutaneous coronary revascularization and that persistent elevation in these markers is predictive of excess risk of subsequent adverse cardiac events. By virtue of its cross-reactivity with the glycoprotein IIb/IIIa, avβ3, and αMβ2 receptors, abciximab may reduce inflammatory processes. Methods and Results — Assays for the inflammatory markers C-reactive protein, interleukin-6, and tumor necrosis factor-α were performed on serum samples obtained from 160 patients in a placebo-controlled, randomized trial of abciximab during angioplasty. Eighty patients each had received a placebo or abciximab bolus plus a 12-hour infusion. Serum samples were drawn at baseline (before revascularization), 24 to 48 hours after study drug administration, and 4 weeks after study drug administration. Between baseline and 24 to 48 hours, the increase in C-reactive protein was 32% less in patients receiving abciximab than placebo ( P =0.025); the rise in interleukin-6 levels was 76% less in the abciximab group ( P P =0.112). By 4 weeks, most marker levels had returned to baseline, with no significant differences between placebo and abciximab groups. Conclusions — Systemic markers of inflammation increase in the first 24 to 48 hours after angioplasty, but the magnitude of that rise is diminished by periprocedural abciximab. Some of the long-term clinical benefit derived from this agent may be related to an anti-inflammatory effect.

Background —We evaluated platelet activation and aggregation in patients with acute myocardial infarction (AMI) treated with thrombolytic therapy alone or with reduced-dose thrombolysis and concomitant abciximab. Methods and Results —The study was performed in 20 control subjects and 51 patients with AMI before and after reperfusion with either alteplase or reteplase or reduced doses of these agents with concomitant abciximab. Platelet activation was assayed by platelet surface expression of P-selectin. Turbidometric platelet aggregation in response to ADP was measured in patients before thrombolytic therapy and 90 minutes and 24 hours after the beginning of thrombolytic therapy. P-selectin expression was greater at baseline in patients than normal control subjects (30.4% versus 9.8%, P P =0.37). However, at 24 hours, basal P-selectin expression declined in patients ( P =0.0025 versus baseline), whereas ADP-stimulated P-selectin expression was lower in patients than in control subjects (48% versus 69%, P =0.0004). When combined with reduced doses of either alteplase or reteplase, abciximab achieved 91% and 83% inhibition of 5 and 20 μmol/L ADP–induced platelet aggregation, which decreased to 46% and 40%, respectively, at 24 hours. No appreciable difference in the platelet inhibition profile of abciximab was observed between the 2 thrombolytics. Conclusions —Platelet activation and aggregation are heightened in the setting of thrombolysis for AMI. Despite this enhanced level of platelet activation, abciximab, combined with a reduced-dose thrombolytic, inhibited platelet aggregation similarly to the level reported in elective settings.

It has been shown that deletion of the chemokine receptor, CXCR4, causes disordered angiogenesis in mouse models. In the present studies, we examined the distribution and trafficking of CXCR4 in human endothelial cells, tested their responses to the CXCR4 ligand, SDF-1, and asked whether endothelial cell CXCR4 can serve as a cell surface receptor for the binding of viruses. The results show that CXCR4 is present on endothelial cells from coronary arteries, iliac arteries and umbilical veins (HUVEC), but expression was heterogeneous, with some cells expressing CXCR4 on their surface, while others did not. Addition of SDF-1 caused a rapid decrease in CXCR4 surface expression. It also caused CXCR4-mediated activation of MAPK, release of PGI2, endothelial migration, and the formation of capillary-like structures by endothelial cells in culture. Co-culture of HUVEC with lymphoid cells that were chronically infected with a CD4-independent/CXCR4-tropic variant of HIV-2 resulted in the formation of multinucleated syncytia. Formation of the syncytia was inhibited by each of several different CXCR4 antibodies. Thus, our findings indicate: (1) that CXCR4 is widely expressed on human endothelial cells; (2) the CXCR4 ligand, SDF-1, can evoke a wide variety of responses from human endothelial cells; and (3) CXCR4 on endothelial cells can serve as a receptor for isolates of HIV that can utilize chemokine receptors in the absence of CD4.

The ability to overexpress physiologically important proteins in cultured mammalian cells after delivering the encoding mRNAs could have important applications for analyzing their in vivo functions. To explore the potential of this approach, urokinase-type plasminogen activator receptor (uPAR), a membrane protein extensively modified post-translationally, was selected. The uPAR-encoding mRNAs, containing different 5' and 3' untranslated regions (UTR) were tested in cultured human osteosarcoma (HOS) cells following a cationic lipid-mediated delivery. The most effective structure was the capped and polyadenylated transcript containing Xenopus beta-globin 5' and 3' UTRs. Delivering this mRNA to HOS cells resulted in a significant increase of uPAR expression in 89% of the cells, measured by flow cytometry. Using a radioligand binding assay, the increase in functional uPAR levels was found to be up eight- to 11-fold between 8 and 48 h and up three-fold at 72 h after delivery. A similar increase in uPAR levels was achievable in a number of mammalian cell lines. Surprisingly, poly(A)-tailed mRNA leading to a uPAR production highest in magnitude and duration did not demonstrate increased intracellular stability compared with other tested mRNAs. Thus, the exceptional translational performance is not likely the result of an increased mRNA half-life. These results demonstrate that, after delivery of selected mRNAs into mammalian cells, immediate and significant overexpression of a post-translationally modified protein is achievable.

Abstract —The plasminogen activator system has been implicated in the modulation of the response to vascular injury. Although urokinase-type plasminogen activator (uPA) and its receptor (uPAR) may enhance matrix degradation as well as migration and invasion by smooth muscle cells (SMCs), their roles in cell adhesion are uncertain. Therefore, we examined the ability of uPA and uPAR to modulate adhesion of cultured human vascular SMCs to various matrices. We demonstrated a dose-dependent stimulation of adhesion by single-chain uPA (scuPA) to vitronectin (maximum 1.55-fold [±0.04-fold] increase, 10 nmol/L, P 40% of control in the presence of scuPA ( P =0.001 and 0.046, respectively). Adhesion to vitronectin was also significantly enhanced by the amino-terminal fragment of uPA ( P =0.007) and two-chain, high-molecular-weight uPA ( P P =0.01), as did pretreatment of SMCs with phosphatidylinositol-specific phospholipase C, which removes glycophosphatidylinositol-anchored proteins, including uPAR. Antibodies to both α v β 3 and α v β 5 integrin inhibited baseline adhesion but not scuPA stimulation. Finally, coating plates with scuPA alone enabled cell adhesion, which could be inhibited by both soluble uPAR and anti-uPAR antibodies. These data suggest that uPA stimulates adhesion of SMCs specifically to vitronectin and that it is mediated by an interaction with uPAR. Upregulation of both proteins after vascular injury may facilitate migration through stimulation of both matrix degradation and cell adhesion.

Cationic lipid-mediated gene transfer has been shown to be a competent albeit inefficient mechanism of promoting cellular gene transfer. One way to improve the efficacy of cationic lipid-mediated transgene expression is to optimize conditions for complex formation between the lipids and nucleic acids. In this report we describe the beneficial effects of using phosphate buffer to precondition lipofectin (a 1:1 (w/w) mixture of N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphatidylethanolamine (DOPE)) prior to complexing with plasmid DNA or mRNA. Under such optimized conditions we studied the kinetics of DNA- and RNA-mediated transgene expression in a human osteosarcoma cell line (HOS). Preincubation of lipofectin in phosphate buffer resulted in up to 26- and 56-fold increases in luciferase expression from plasmid DNA and mRNA, respectively. Addition of chloroquine (50μM), which enhanced plasmid-mediated gene delivery 3-fold, was synergistic with phosphate resulting in an additional 46-fold increase in luciferase expression. The preincubation with phosphate shortened both the time required for cellular uptake and the time to achieve maximal transgene expression. Optimal transfection was achieved in the presence of 30–80mM phosphate, at pH 5.6–6.8 under which the phosphate anion is divalent. The effect of phosphate anion was specific in that monovalent Cl- and acetate anions were not stimulatory. These results demonstrate that divalent phosphate anion plays a stimulatory role during complex formation and transfection when cationic lipids come in contact with negatively charged nucleic acids and cell membranes. These findings delineate specific conditions which dramatically enhance transfection efficiency for both DNA and mRNA, and provide an effective procedure for gene transfection studies.

SummaryInteraction of proteases with cell surface receptors may modulate cell adhesion, migration, invasion, and matrix degradation. Since the plasminogen activator system has been hypothesized to play a role in intimal thickening after various types of vascular injury, we first studied the expression of urokinase receptor (u-PAR) protein and mRNA by smooth muscle cells (SMC) grown in explant cultures from normal and diseased vessels. Using equilibrium binding studies with radiolabeled 125I-labeled single chain urokinase-type plasminogen activator (scu-PA), we determined that SMC cultured from atherosclerotic arteries expressed a higher maximal number of binding sites/cell (3.6 ± 0.4 × 105 sites/cell vs. 2.1 ± 0.3 × 105, ± SEM, p

Heparin has been recognized as possessing a large variety of cell-modulating activities. Using compositionally simple, structurally rigid, and low molecular weight saccharide molecules (cyclodextrins), we demonstrated that these activities depend primarily on a single, gross compositional parameter: a minimum intramolecular density of neighboring anionic (sulfate) groups. This same critical parameter is shown to be involved in achieving cell-modulating behavior as diverse as angiogenesis, endothelial proliferation, inhibition of smooth muscle cell growth, and cell protection against virus invasion. Physical chemical evidence is presented that associates this property with multi-ionic complex formation between the clusters of anionic and cationic sites on the complexing partners. These observations revive early suggestions of the decisive role of electrostatic complexation capabilities of glycosaminoglycans like heparin; taken together with numerous observations on heparinoids and other agents reported in diverse specialized fields of cell biology and medicine, they provide evidence that molecular agents of critical anionic (sulfate) density (MACADs) represent a broad class of molecules that, in contrast to proteins, do not rely on structural detail for their cell biological activities, but function by ionic complexation with proteinic agents (e.g. growth factors), thereby modifying their structure-specific activities.

Human endothelial cells express thrombin receptors and PAR-2, the two known members of the family of protease-activated G protein-coupled receptors. Because previous studies have shown that the biology of the human thrombin receptor varies according to the cell in which it is expressed, we have taken advantage of the presence of both receptors in endothelial cells to examine the enabling and disabling interactions with candidate proteases likely to be encountered in and around the vascular space to compare the responses elicited by the two receptors when they are present in the same cell and to compare the mechanisms of thrombin receptor and PAR-2 clearance and replacement in a common cellular environment. Of the proteases that were tested, only trypsin activated both receptors. Cathepsin G, which disables thrombin receptors, had no effect on PAR-2, while urokinase, kallikrein, and coagulation factors IXa, Xa, XIa, and XIIa neither substantially activated nor noticeably disabled either receptor. Like thrombin receptors, activation of PAR-2 caused pertussis toxin-sensitive phospholipase C activation as well as activation of phospholipase A2, leading to the release of PGI2. Concurrent activation of both receptors caused a greater response than activation of either alone. It also abolished a subsequent response to the PAR-2 agonist peptide, SLIGRL, while only partially inhibiting the response to the agonist peptide, SFLLRN, which activates both receptors. After proteolytic or nonproteolytic activation, PAR-2, like thrombin receptors, was cleared from the endothelial cell surface and then rapidly replaced with new receptors by a process that does not require protein synthesis. Selective activation of either receptor had no effect on the clearance of the other. These results suggest that the expression of both thrombin receptors and PAR-2 on endothelial cells serves more to extend the range of proteases to which the cells can respond than it does to extend the range of potential responses. The results also show that proteases that can disable these receptors can distinguish between them, just as do most of the proteases that activate them. Finally, the residual response to SFLLRN after activation of thrombin receptors and PAR-2 raises the possibility that a third, as yet unidentified member of this family is expressed on endothelial cells, one that is activated by neither thrombin nor trypsin.

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.

SummaryBackgroundInfliximab, a TNF-alpha antagonist, has shown efficacy in the treatment of sarcoidosis. Since corticosteroids inhibit TNF-alpha expression, we postulated that sarcoidosis patients receiving a sufficient corticosteroid dose may have an attenuated response to the addition of infliximab.MethodsWe analyzed data from a previous randomized double blind prospective trial of infliximab versus placebo for chronic pulmonary sarcoidosis. The effect of the maintenance corticosteroid dose on the change in FVC % predicted between 0 and 24 weeks (ΔFVC%pred0-24) was analyzed in two ways. First, the mean ΔFVC%pred0-24 was calculated for the placebo and infliximab groups using three different daily prednisone equivalent dose thresholds: a) 15 mg of prednisone and ≥20 mg of prednisone did not demonstrate a significant ΔFVC%pred0-24. For the placebo group, there was no significant correlation between the corticosteroid dose and the ΔFVC%pred0-24. For the infliximab group, there was a significant correlation (p = 0.0097) between higher corticosteroid dose and less improvement in FVC%pred0-24.ConclusionOur results suggest that infliximab adds minimal potential benefit to corticosteroids for pulmonary sarcoidosis at doses above 15–20 mg/day of prednisone.

Smooth muscle cell (SMC) migration is an early response to vascular injury and contributes to the development of intimal thickening. Upregulation of several components of the plasminogen activator (PA) system has been documented after vascular injury. Utilizing a Transwell filter assay system and human umbilical vein SMCs, we sought to define the role of four different PA system components on SMC migration and matrix invasion: (1) PAs, (2) plasmin, (3) PA receptors, and (4) PA clearance receptors (ie, low density lipoprotein receptor–related protein [LRP]). Addition of active two-chain urokinase-type PA (UPA) stimulated random migration (192±30% of control, 0.36 nmol/L, P P

Intimal thickening after vascular injury may be modulated in part by heparin binding growth factors. We hypothesized that placement of a therapeutic polymer in the periadventitial space capable of tightly binding growth factors might alter the vascular response to injury. We first demonstrated that incubation of rat aortic smooth muscle cells with an insoluble, sulfated polymer of beta-cyclodextrin (P-CDS) was associated with a dose-dependent inhibition of proliferation induced by fetal calf serum, fibroblast growth factor-2 (FGF-2), platelet-derived growth factor BB, or epidermal growth factor. Preincubation studies of P-CDS with FGF-2 revealed a very rapid removal of mitogenic activity. Using radiolabeled FGF-2 (0.25 microg/ml), we observed a very rapid association rate (0.34 +/- 0.07 min-1, n=4) and a very slow dissociation rate (3.3 +/- 0.2 X 10(-7) min-1) at 37 degrees C, suggesting a high affinity interaction. Using both Transwell and linear under-agarose assays, we demonstrated a significant inhibition of random migration (chemokinesis) by P-CDS. Unsulfated polymeric beta-cyclodextrin (P-CD) had little if any of these effects, suggesting that the high negative charge density of P-CDS was important for the effects. Finally, rats undergoing carotid artery balloon injury were randomized to treatment with periadventitial P-CDS or no treatment, and were killed at 4 (n=20), 14 (n=59), and 88 d (n=14). Morphometric analysis demonstrated significant and sustained inhibition of intimal thickening in P-CDS-treated rats at 14 (P < 0.01) and 88 d (P < 0.05) using absolute intimal area or intima/media area ratios. No inhibition was seen in a group of rats treated with P-CD. In P-CDS-treated rats, bromodeoxyuridine labeling studies revealed fewer labeled smooth muscle cells in the intima at 14 d (P=0.01), while staining with Evans blue revealed enhanced late endothelial cell regrowth. Thus, periadventitially applied sulfated beta-cyclodextrin polymer, which can tightly bind heparin binding growth factors, inhibits intimal thickening in vivo in a sustained fashion without using an additional delivery system. These studies suggest that cellular processes mediated by heparin binding growth factors may be modulated by P-CDS.

Abstract Altered coronary artery expression of plasminogen activator (PA) system components may predispose to thrombosis and modulate the vascular response to injury. By immunohistochemistry, we studied the expression of PAs (tPA and uPA), their major physiological inhibitor (PAI-1), and a receptor for uPA (uPAR) in human coronary arteries with either pure fibrointimal proliferation (n=15) or developed atherosclerotic plaques (n=10). Overall, the degree of staining showed the following rank order: PAI-1>tPA>uPAR>uPA. A similar pattern was seen in two normal coronary arteries. There were no significant differences in the extent of staining in any vascular compartment between atherosclerotic arteries and those with only fibrointimal proliferation. However, the ratio of intimal to medial expression of tPA ( P =.001) and uPAR ( P =.004) was significantly increased in atherosclerotic arteries, with a similar trend for uPA ( P =.069) but not for PAI-1 ( P =.73). Four of 10 atherosclerotic arteries had higher uPAR expression in the intima than in the media, whereas none of the 15 arteries with only fibrointimal proliferation had this pattern ( P

To determine whether unsaturated fatty acids induce changes in the mRNA level of plasminogen activator inhibitor type-1 (PAI-1), Northern analyses were performed on human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells that were treated with two common fatty acids. Supplementation of cultured HUVEC with docosahexanoic acid (DHA) or with dihomogamma linolenic acid (DGLA), resulted in a concentration dependent, specific increase of the PAI-1 transcript levels, which was detectable within 2 h. DHA and DGLA treatment of smooth muscle cells did not result in changes in the PAI-1 mRNA levels. Homology search of the upstream regulatory region of the PAI-1 gene sequences identified a consensus nucleotide sequence for a fatty acid-responsive element. Our results indicate that unsaturated fatty acids selectively increase PAI-1 mRNA levels in endothelial cells, the primary source of circulating PAI-1 in vivo.

Randomized trials directly comparing angioplasty with bypass surgery for complex multivessel disease are under way. Preliminary results suggest that angioplasty can be effective in some patient groups, so that neither procedure will predominate. The early results also suggest criteria to guide physicians in selecting the most appropriate method for individual patients.

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, 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-1β 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.

The plasminogen activator (PA), urokinase, (UK) plays an important role in tissue remodelling by mediating the conversion of plasminogen into the serine-protease, plasmin. UK can bind to a specific cell surface receptor which serves to accelerate plasminogen activation. The present study was undertaken to determine the effect of the chemical agent N,N-dimethylformamide (DMF), on the expression of UK and its receptor in a colon cancer cell line, CBS. DMF increased radioactive UK binding to CBS in a dose-dependent fashion. A level of 0.75% (v/v) has a maximal effect in this regard producing a 150% increase in the amount of radioligand bound. Crosslinking of a radioactive aminoterminal (ATF) of UK, corresponding to the receptor-binding sequence of the PA, to the surface of CBS cells indicated a radioactive complex of molecular mass 66 kDa. This binding could be abolished with an excess of unlabelled ATE The amount of this 66 kDa complex increased over an identical concentration range used to elevate radioligand binding in the receptor assays. Augmented radioligand binding to CBS cells, treated with DMF, was a consequence of an increase in the number of UK receptors from 7.6X 103 to 19.9 × 103 as determined by Scatchard analysis. Northern blotting for UK receptor transcript revealed that this increase in the amount of receptor binding protein was a consequence of a more abundant transcript. In contrast to the inductive effect on UK receptor expression, DMF, over an identical concentration range, suppressed, by up to 80%, the level of steady-state mRNA encoding the PA. Maximal suppression of UK mRNA occurred after 4 days suggesting that this effect of DMF was not coordinated with the induction of UK receptor expression which required only a 24h period. In conclusion, this is the first study to indicate a divergent effect of an agent, on the expression of UK and its receptor.

The FINESSE-ANGIO (Facilitated Intervention with Enhanced Reperfusion Speed to Stop Events-Angiographic) study evaluated acute treatment effects on infarct-related artery (IRA) patency and angiographic correlates of coronary microcirculatory function.The FINESSE trial evaluated the effects on clinical outcomes of primary percutaneous coronary intervention (PCI) facilitated with pre-catheterization laboratory administration of abciximab with half-dose reteplase (combination-facilitated group), abciximab alone (abciximab-facilitated group), or with abciximab administered immediately before the procedure (primary PCI).The FINESSE-ANGIO substudy compared the effects of the 3 treatment strategies on patency (TIMI [Thrombolysis In Myocardial Infarction] flow grade 2/3) of the IRA at basal coronary angiography. The secondary efficacy end points were corrected TIMI frame count, percentage of patients achieving TIMI flow grade 3, and the percentage achieving myocardial blush grade 2/3 of the IRA at post-PCI angiography. All angiographies were evaluated at a central core laboratory.Of the 2,452 FINESSE patients, 637 were included in the FINESSE-ANGIO substudy. Patients in the combination-facilitated group exhibited significantly higher rates of baseline IRA patency compared with the abciximab-facilitated and the primary PCI groups (76.1% vs. 43.7% and 32.7%, respectively; p0.0001 for both; p = 0.025 abciximab-facilitated vs. primary PCI). There were no significant differences in the post-PCI corrected TIMI frame count (17.1 ± 15.8, 17.4 ± 17.3, and 15.8 ± 14.1) or the rates of post-PCI TIMI flow grade 3 (79.8%, 77.7%, and 76.6%), myocardial blush grade 2/3 (85.6%, 79.5%, and 86.4%), respectively.Pre-catheterization laboratory administration of abciximab alone and especially in combination with half-dose reteplase resulted in higher rates of IRA patency at baseline coronary angiography compared with no pre-treatment. However, post-procedural angiographic and microcirculatory variables were unaffected by facilitation therapy.

Human umbilical vein endothelial cells (HUVEC) in culture express two classes of binding sites for tissue-type plasminogen activator (t-PA). The high-affinity binding site has been identified as PA inhibitor type 1 (PAI-1), which binds to the catalytic portion of the molecule, while the second site binds t-PA through an active-site independent domain. Because recombinant t-PA (rt-PA) is often administered concomitantly with heparin, we investigated the effects of heparin on rt-PA binding to HUVEC. Preincubation of HUVEC with heparin at 4 degrees C increased the binding of radiolabeled rt-PA in a time- and dose-dependent manner. One-half maximal increase in binding was observed within 10 minutes of heparin addition. When HUVEC were preincubated with optimal concentrations (5 U/mL) of heparin for 4 hours at 4 degrees C, a 2.5- +/- 0.2-fold increase in specific binding was observed (mean +/- SEM, n = 12, P less than .01). Other highly sulfated glycosaminoglycans and fucoidan (a sulfated polymer of fucose) stimulated rt-PA binding as well, whereas glycosaminoglycans with lower sulfate content than heparin did not. Several results suggested that heparin increased the binding of rt-PA to “cell-associated” PAI-1. First, only active-site- dependent binding was enhanced by heparin, whereas binding of active- site blocked rt-PA was not affected. Second, extracts from HUVEC preincubated with heparin contained increased amounts of rt-PA-PAI-1 complexes as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Third, antibodies to PAI-1 blocked the increased binding entirely. HUVEC preincubated with heparin also bound increased amounts of enzymatically active radiolabeled urokinase-type PAs. However, HUVEC preincubated with heparin did not express increased amounts of immunoreactive PAI-1. Therefore, heparin, at therapeutic concentrations, may enhance or stabilize the association of PAs with endothelial cell-associated PAI-1.

Endothelial cells synthesize and secrete urokinase-type plasminogen activators (u-PA) in response to various stimuli. To modulate the local expression of u-PA activity both intravascularly and pericellularly during angiogenesis, endothelial cells express both inhibitors (primarily PAI-1) and receptors (u-PAR) for u-PA. The interaction of u-PA with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Using radioligand binding studies, we and others have demonstrated that human umbilical vein endothelial cells (HUVEC) express high affinity receptors for urokinase on the cell surface. We have demonstrated that single chain urokinase (scu-PA, prourokinase) binds only via the growth factor domain, while two chain high molecular weight urokinase (tcu-PA) can bind to the receptor or to cell- or matrix-associated PAI-1. We have isolated a ∼46kDa glycoprotein from HUVEC using affinity chromatography which retains the ability to specifically bind u-PA. At least three post -translational modifications appear to occur including removal of an N-terminal signal peptide; N-linked glycosylation, and C-terminal cleavage with addition of a phosphatidylinositol-glycan moiety which links the externally oriented protein to the cell surface. Using the polymerase chain reaction and published sequence information of the u-PAR cloned from a transformed fibroblast cDNA library, we amplified cDNAs of u-PAR from HUVEC and PMA-treated U937 cells. The specificity of the cDNAs was confirmed by restriction mapping and direct sequence analysis. Using these probes and radioligand binding studies we have demonstrated that at least two independent protein kinase pathways exist in endothelial cells for upregulating u-PA receptor expression. Down regulation of receptors may be pathophysiologic in thrombosic disorders whereas up regulation may be important in promoting wound healing, vascular repair, and protection from thrombosis. Up regulation could be harmful as well in such conditions as pathologic neovascularization (e.g., diabetic retinopathy) and in tumour metastasis as well as in tumour-related angiogenesis. Understanding the control and functional significance of u-PA binding to cells in general will hopefully enable the design of therapies to optimize the beneficial aspects and minimize any harmful effects of this interaction. Changes in the local expression of u-PA, PAM and u-PAR by endothelial cells may affect the extent of angiogenesis or the degree of local intravascular fibrinolysis, which might be critical in conditions such as unstable angina and myocardial infarction.

We have prepared and purified the amino terminal fragment (ATF) or urokinase-type plasminogen activator (u-PA), as well as the kringle and the growth factor domain (GFD) of which it is composed. Structural studies on these materials are currently under way. In addition, we have demonstrated that either the ATF or its GFD portion can inhibit tumour cell invasion in vitro, and that both can likewise act as mitogens and induce differentiation in certain cell lines. Our results also suggest that the fucose attached to Thr18 may be essential for at least some of these activities, since defucosylated GFD becomes inactive in mitogenesis. Clearly the ATF has activities beyond simply anchoring u-PA to the cell surface. Availability of separated, purified domains will allow both structural and functional dissection of these activities.

The aim of this study was to assess the risk-benefit of enoxaparin (Sanofi-Aventis, Paris, France) in primary percutaneous coronary intervention (PCI).Randomized studies have demonstrated the superiority of enoxaparin over unfractionated heparin (UFH) in acute ST-segment elevation myocardial infarction (STEMI) treated with fibrinolytics.In the FINESSE (Facilitated INtervention with Enhanced Reperfusion Speed to Stop Events) trial--a double-blind, placebo-controlled study-2,452 patients with STEMI were randomized to primary PCI or facilitated PCI with abciximab alone or with half-dose reteplase. In this prospective FINESSE substudy, centers pre-specified use of either enoxaparin (0.5 mg/kg intravenous [IV], 0.3 mg/kg subcutaneous [SC]) or UFH (40 U/kg IV, 3,000 U maximum) with PCI. A logistic-regression model and a propensity multivariate model, both adjusted for baseline variables, were used to evaluate primary safety and secondary efficacy end points for enoxaparin versus UFH.Enoxaparin was administered to 759 patients and UFH to 1,693 patients. Nonintracranial Thrombolysis In Myocardial Infarction (TIMI) major/minor bleeding was not significantly different, but lower nonintracranial TIMI major bleeding was found with enoxaparin (2.6% vs. UFH 4.4%, logistic-regression adjusted odds ratio [OR]: 0.55; 95% confidence interval [CI]: 0.31 to 0.99, p = 0.045), whereas intracranial hemorrhage was similar (0.27% vs. 0.24%, adjusted OR: 1.03; 95% CI: 0.11 to 9.68, p = 0.980). Lower death, myocardial infarction, urgent revascularization, or refractory ischemia through 30 days was also associated with enoxaparin (5.3%) versus UFH (8.0%, adjusted OR: 0.47, 95% CI: 0.31 to 0.72, p = 0.0005) as was all-cause mortality through 90 days (3.8% vs. 5.6%, respectively, adjusted OR: 0.59, 95% CI: 0.35 to 0.99, p = 0.046). End points evaluating the net clinical benefit also significantly favored enoxaparin over UFH.Enoxaparin seems to be associated with a lower risk of cardiovascular outcomes compared with UFH in patients with STEMI undergoing primary PCI. Confirmation of these findings in a randomized study is warranted. (A Study of Abciximab and Reteplase When Administered Prior to Catheterization After a Myocardial Infarction [Finesse]; NCT00046228).

The best method to interpret the chest roentgenogram and its sensitivity to detect effect of treatment for sarcoidosis remains unclear. In a double-blind, randomized trial of infliximab for chronic pulmonary sarcoidosis, changes in serial chest roentgenograms were examined by radiologists, blinded to order or treatment.Chest roentgenograms were obtained at 0, 6, and 24 weeks of therapy with either placebo, 3 mg/kg infliximab, or 5 mg/kg infliximab. Films were reviewed in random order by two independent radiologists, unaware of treatment. The films were compared using two methods: the prespecified objective assessment, a scoring system previously proposed by Muers; and the post hoc assessment, a 5-point Likert scale global assessment between two films.Of 138 patients enrolled in the study, chest roentgenograms for all studies were available on 130 patients. There was only fair agreement between the two radiologists in the original stage of the chest roentgenogram (weighted kappa = 0.43; 95% confidence interval [CI], 0.32 to 0.54). For the Likert scale of global assessment of change, there was good agreement between the two readers (weighted kappa = 0.61; 95% CI, 0.51 to 0.71). There was good correlation between the two readers for the various components of the Muers score, especially the reticulonodular (R) score (R = 0.578; p0.05). The initial R score was positively correlated with improvement in FVC with infliximab therapy (R = 0.239; p0.05).Global assessment and the Muers scoring system were associated with good agreement between two expert readers. Improvement in both scores correlated with improvement in FVC.ClinicalTrials.gov Identifier: NCT00073437.