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Background Lung nodules are a diagnostic challenge, with an estimated yearly incidence of 1.6 million in the United States. This study evaluated the accuracy of an integrated proteomic classifier in identifying benign nodules in patients with a pretest probability of cancer (pCA) ≤ 50%. Methods A prospective, multicenter observational trial of 685 patients with 8- to 30-mm lung nodules was conducted. Multiple reaction monitoring mass spectrometry was used to measure the relative abundance of two plasma proteins, LG3BP and C163A. Results were integrated with a clinical risk prediction model to identify likely benign nodules. Sensitivity, specificity, and negative predictive value were calculated. Estimates of potential changes in invasive testing had the integrated classifier results been available and acted on were made. Results A subgroup of 178 patients with a clinician-assessed pCA ≤ 50% had a 16% prevalence of lung cancer. The integrated classifier demonstrated a sensitivity of 97% (CI, 82-100), a specificity of 44% (CI, 36-52), and a negative predictive value of 98% (CI, 92-100) in distinguishing benign from malignant nodules. The classifier performed better than PET, validated lung nodule risk models, and physician cancer probability estimates (P

BACKGROUND Pulmonary nodules (PNs) are a common reason for referral to pulmonologists. The majority of data for the evaluation and management of PNs is derived from studies performed in academic medical centers. Little is known about the prevalence and diagnosis of PNs, the use of diagnostic testing, or the management of PNs by community pulmonologists. METHODS This multicenter observational record review evaluated 377 patients aged 40 to 89 years referred to 18 geographically diverse community pulmonary practices for intermediate PNs (8-20 mm). Study measures included the prevalence of malignancy, procedure/test use, and nodule pretest probability of malignancy as calculated by two previously validated models. The relationship between calculated pretest probability and management decisions was evaluated. RESULTS The prevalence of malignancy was 25% (n = 94). Nearly one-half of the patients (46%, n=175) had surveillance alone. Biopsy was performed on 125 patients (33.2%). A total of 77 patients (20.4%) underwent surgery, of whom 35% (n = 27) had benign disease. PET scan was used in 141 patients (37%). The false-positive rate for PET scan was 39% (95% CI, 27.1%-52.1%). Pretest probability of malignancy calculations showed that 9.5% (n = 36) were at a low risk, 79.6% (n = 300) were at a moderate risk, and 10.8% (n = 41) were at a high risk of malignancy. The rate of surgical resection was similar among the three groups (17%, 21%, 17%, respectively; P = .69). CONCLUSIONS A substantial fraction of intermediate-sized nodules referred to pulmonologists ultimately prove to be lung cancer. Despite advances in imaging and nonsurgical biopsy techniques, invasive sampling of low-risk nodules and surgical resection of benign nodules remain common, suggesting a lack of adherence to guidelines for the management of PNs. Materials and Methods This was a multicenter, community-based, retrospective observational study of patients with PNs, ranging from 8 to 20 mm in diameter, presenting to 18 geographically representative outpatient pulmonary clinics across the United States. The study was approved at 15 sites by a central institutional review board and at three sites by local institutional review board approval. Site Selection Four hundred forty sites were identified based on investigator databases and claims data from a large insurance carrier whose coverage population was representative of the overall US population. Of these, 77 sites expressed interest in participating, and 48 sites went on to sign confidentiality agreements. Of these, 17 did not request additional information, leaving 31 sites undergoing qualification review. Eighteen outpatient pulmonary clinics were chosen to participate based on the following criteria: (1) management of patients with PNs, (2) availability of medical records, and (3) ability to perform data abstraction. In addition, investigators targeted enrollment of geographically diverse patients to limit the potential bias associated with differences in practice patterns and to account for variation in disease prevalence (eg, endemic mycoses) that could alter management decisions. Patient Selection Patients were identified by querying databases (eg, billing and scheduling systems) using five International Classification of Diseases, Ninth Revision, Clinical Modification codes for PN (793.1, 786.6, 518.89, 519.8, 519.9) to ensure homogeneity in patient identification and inclusion. 17 Manual chart abstraction was then used to identify those who met the criteria. To minimize selection bias, the sites were not permitted to use additional codes during database query to identify patients. To ensure a systematic sample, patient eligibility was determined by examining consecutively referred patients to the site. Inclusion criteria included age ≥ 40 years and ≤ 89 years at the time of nodule finding, presentation to a pulmonologist, nodule size 8 to 20 mm, and definitive diagnosis ascertained by tissue diagnosis or radiographic follow-up for 2 years. Exclusion criteria included chest CT scan performed > 60 days prior to the initial visit, prior diagnosis of any cancer within 2 years of nodule detection, or incomplete chart data. Patients were categorized into three groups by the most invasive procedure performed during management, as follows: surveillance (serial imaging), biopsy (CT scan-guided transthoracic needle aspiration [TTNA] or bronchoscopy), or surgery (including mediastinoscopy, video-assisted thorascopic surgery, and/or thoracotomy). Data Collection Clinical data were abstracted retrospectively by designated study staff into an electronic data capture system from initial consultation through establishment of a definitive diagnosis (ie, pathology results) or a minimum 2-year follow-up. Data included patient demographic and clinical characteristics, PN characteristics, imaging tests, invasive testing, and surgery. PET scan reports were reviewed and abstracted where available for a subset of patients. To adjudicate a PET scan report as positive or negative, an algorithm was developed that prioritized the following components of the report from highest to lowest: final radiology impression, description of findings, and standard uptake values (SUVs) (e-Fig 1). PET scanning was defined as negative if the report included any of the following statements: no evidence of malignancy, no 18F-fluorodeoxyglucose uptake or hypermetabolic activity, or an SUV of > 2.5. A positive PET scan was defined as a report that included any of the following statements: concern or suggestion of malignancy, findings that described increased 18F-fluorodeoxyglucose uptake or hypermetabolic activity, or an SUV ≥ 2.5. In the 27 cases in which the findings were discordant with the final impression, adjudication was performed by two independent pulmonologists and agreement was reached in all cases. Data quality was ensured through ongoing site monitoring. Programmed edit checks were built into the electronic data capture system and at the conclusion of chart abstraction, each site provided access to a random 10% sample of deidentified patient records for review. Pretest Probability of Malignancy Two previously developed and validated models 9,10 were used to estimate the pCA in each patient. Model accuracy was determined by comparing the pCA with the final diagnosis. Receiver operating characteristic curves and the area under the curve were generated with 95% CIs. The pCA was calculated for each patient and categorized into three groups ( Data Analysis χ 2 or analysis of variance tests were used to compare subgroups, and P values > .05 were considered significant. A nodule was classified as benign based on confirmed benign pathology or the absence of radiographic change as determined by the managing physician during surveillance for at least 2 years. Multivariate logistic regression was performed to identify factors associated with the use of an invasive diagnostic procedure. All statistical analyses were performed using SAS/STAT, version 9.3 (SAS Institute inc).

Introduction Indeterminate pulmonary nodules (IPNs) lack clinical or radiographic features of benign etiologies and often undergo invasive procedures unnecessarily, suggesting potential roles for diagnostic adjuncts using molecular biomarkers. The primary objective was to validate a multivariate classifier that identifies likely benign lung nodules by assaying plasma protein expression levels, yielding a range of probability estimates based on high negative predictive values (NPVs) for patients with 8 to 30 mm IPNs. Methods A retrospective, multicenter, case-control study was performed using multiple reaction monitoring mass spectrometry, a classifier comprising five diagnostic and six normalization proteins, and blinded analysis of an independent validation set of plasma samples. Results The classifier achieved validation on 141 lung nodule-associated plasma samples based on predefined statistical goals to optimize sensitivity. Using a population based nonsmall-cell lung cancer prevalence estimate of 23% for 8 to 30 mm IPNs, the classifier identified likely benign lung nodules with 90% negative predictive value and 26% positive predictive value, as shown in our prior work, at 92% sensitivity and 20% specificity, with the lower bound of the classifier's performance at 70% sensitivity and 48% specificity. Classifier scores for the overall cohort were statistically independent of patient age, tobacco use, nodule size, and chronic obstructive pulmonary disease diagnosis. The classifier also demonstrated incremental diagnostic performance in combination with a four-parameter clinical model. Conclusions This proteomic classifier provides a range of probability estimates for the likelihood of a benign etiology that may serve as a noninvasive, diagnostic adjunct for clinical assessments of patients with IPNs.

We report on peptide‐based ligands matured through the protein catalyzed capture (PCC) agent method to tailor molecular binders for in vitro sensing/diagnostics and in vivo pharmacokinetics parameters. A vascular endothelial growth factor (VEGF) binding peptide and a peptide against the protective antigen (PA) protein of Bacillus anthracis discovered through phage and bacterial display panning technologies, respectively, were modified with click handles and subjected to iterative in situ click chemistry screens using synthetic peptide libraries. Each azide‐alkyne cycloaddition iteration, promoted by the respective target proteins, yielded improvements in metrics for the application of interest. The anti‐VEGF PCC was explored as a stable in vivo imaging probe. It exhibited excellent stability against proteases and a mean elimination in vivo half‐life (T 1/2) of 36 min. Intraperitoneal injection of the reagent results in slow clearance from the peritoneal cavity and kidney retention at extended times, while intravenous injection translates to rapid renal clearance. The ligand competed with the commercial antibody for binding to VEGF in vivo. The anti‐PA ligand was developed for detection assays that perform in demanding physical environments. The matured anti‐PA PCC exhibited no solution aggregation, no fragmentation when heated to 100°C, and > 81% binding activity for PA after heating at 90°C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives.

Background We have previously demonstrated that a peripheral blood transcriptional profile using 11 distinct genes predicts onset of cardiac allograft rejection weeks to months prior to the actual event. Methods In this analysis, we ascertained the performance of this transcriptional algorithm in a Bayesian representative population: 28 cardiac transplant recipients who progressed to moderate to severe rejection; 53 who progressed to mild rejection; and 46 who remained rejection-free. Furthermore, we characterized longitudinal alterations in the transcriptional gene expression profile before, during and after recovery from rejection. Results In this patient cohort, we found that a gene expression score (range 0 to 40) of ≤20 represents very low risk of rejection in the subsequent 12 weeks: 0 progressed to treatable (ISHLT Grade ≥3A) rejection; 16 of 53 (30%) from the intermediate group (those who progressed to ISHLT Grade 1B or 2) and 13 of 46 (28%) controls (who remained Grade 0 or 1A) had scores ≤20. A gene score of ≥30 was associated with progression to moderate to severe rejection in 58% of cases. These two extreme scores (≤20 or ≥30) represented 44% of the cardiac transplant population within 6 months post-transplant. In addition, longitudinal gene expression analysis demonstrated that baseline scores were significantly higher for those who went on to reject, remained high during an episode of rejection, and dropped post-treatment for rejection ( p Conclusions The use of gene expression profiling early after transplantation allows for separation into low-, intermediate- or high-risk categories for future rejection, permitting development of discrete surveillance strategies.

Gene expression profiling distinguishes the absence or presence of moderate to severe grades of acute cellular rejection in cardiac allograft recipients using a 20-gene classifier. We explored the hypothesis that the rejection classifier also differentiates various forms of mild rejection and we performed sub-analyses based on time post-transplant and confirmatory pathology interpretations.A post hoc analysis of 265 CARGO study patients and 714 clinical encounters focused on the correlation of rejection classifier-derived gene expression (GE) scores for blood samples accompanying endomyocardial biopsies. Biopsy grades assigned by a study center pathologist (center) were re-interpreted by three pathologists (panel) in a blinded manner.Mean GE scores not only differentiated Gradesor=3A from Grade 0 (p0.00001, center or panel), but also from Grades 1A or 2 (p0.05, center or panel), based on mild rejection sub-groups defined by the ISHLT 1990 grading system. In contrast, mean GE scores for Grades 1B andor=3A were indistinguishable, using either center or panel interpretation. Sub-group analyses of encounters from 2 to 6 months or6 months post-transplant showed similar results for the classifier's ability to discriminate moderate to severe rejection from Grades 1A and 2 mild rejection, but indistinguishable mean GE scores for Gradesor=3A and the Grade 1B sub-group. Of the classifier's 11 informative genes, expression of MIR and WDR40 showed statistically significant increases for both Grade 1B and Gradeor=3A rejection, while expression of PDCD1 or SEMA7A showed similar directional patterns without achieving statistical significance.These data demonstrate that GE scores discriminate moderate to severe rejection from Grades 1A and 2 mild rejection. However, a sub-group of mild rejection cases, defined as Grade 1B according to the 1990 grading system, share a molecular signature more consistent with moderate to severe rejection. The clinical relevance of these data remains to be defined.

Objectives Enzymatic activity of lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) mediates vascular inflammation in coronary heart disease (CHD). Calibration of Lp-PLA 2 activity measurements using a recombinant enzyme was performed to assess intra- and inter-laboratory assay precision and accuracy in routine clinical settings. Design and methods Test performance assessment included recovery, analytical sensitivity, linear range, within-lab and site-to-site precision, interference, and analyte stability. Results using the Beckman-Coulter AU400 analyzer were compared to other chemistry analyzers. Results Lp-PLA 2 activity ranged from 84 to 303 nmol/min/mL in 300 subjects, with 82.0% and 18.0% measurements below and at or above a cut-point of 225 nmol/min/mL, respectively. Results of matched K 2 -EDTA plasma and serum (n = 131) were similar with a slope of 1.00, y-intercept of 0.05, and R-value of 0.988. Mean recovery ranged from 90 to 106% of baseline after storage at different temperatures and time periods. Limit of detection was ≤ 10 nmol/min/mL, without deviation from linearity between 10 and 382 nmol/min/mL. Endogenous substances and medications did not interfere with the activity measurements. Overall intra- and inter-laboratory precision among three sites showed coefficients of variation of ≤ 3.8% and ≤ 5% respectively. Limit of quantitation was 1.3 nmol/min/mL. Method comparison studies for multiple analyzers demonstrated slopes, intercepts or R 2 coefficients ranging from 0.96 to 1.06, − 5.6 to 2.0, or 0.997 to 0.999, respectively. Conclusion Analytical performance of the calibrated PLAC ® test for Lp-PLA 2 enzyme activity assay in CHD is resistant to a wide variety of pre-analytical factors, with site-to-site reproducibility on multiple analyzers sufficient to standardize results in diverse laboratory settings.

Background Current quantification methods for mass spectrometry (MS)-based proteomics either do not provide sufficient control of variability or are difficult to implement for routine clinical testing. Results We present here an integrated quantification (InteQuan) method that better controls pre-analytical and analytical variability than the popular quantification method using stable isotope-labeled standard peptides (SISQuan). We quantified 16 lung cancer biomarker candidates in human plasma samples in three assessment studies, using immunoaffinity depletion coupled with multiple reaction monitoring (MRM) MS. InteQuan outperformed SISQuan in precision in all three studies and tolerated a two-fold difference in sample loading. The three studies lasted over six months and encountered major changes in experimental settings. Nevertheless, plasma proteins in low ng/ml to low μg/ml concentrations were measured with a median technical coefficient of variation (CV) of 11.9% using InteQuan. The corresponding median CV using SISQuan was 15.3% after linear fitting. Furthermore, InteQuan surpassed SISQuan in measuring biological difference among clinical samples and in distinguishing benign versus cancer plasma samples. Conclusions We demonstrated that InteQuan is a simple yet robust quantification method for MS-based quantitative proteomics, especially for applications in biomarker research and in routine clinical testing. Electronic supplementary material The online version of this article (doi:10.1186/1559-0275-12-3) contains supplementary material, which is available to authorized users.

Background— Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine expressed widely by vascular cells. However, scant in vivo evidence supports direct participation of MIF in atherogenesis. Therefore, we investigated whether deficiency of MIF modulates atherosclerotic lesion formation and composition in low-density lipoprotein receptor-deficient (LDLr −/− ) mice. Methods and Results— MIF −/− LDLr −/− and LDLr −/− mice were generated and consumed an atherogenic diet for 12 or 26 weeks. MIF −/− LDLr −/− mice had significantly reduced abdominal aorta lipid deposition and intimal thickening from aortic arch throughout the abdominal aorta compared with LDLr −/− mice. Marked retardation of atherosclerosis over time in MIF-deficient mice accompanied decreased lesion cell proliferation. At 26 weeks, 20% of MIF-deficient mice developed only early, fatty streak-like lesions, whereas >80% of LDLr −/− mice developed advanced lesions containing calcification and lipid cores. Analysis of smooth muscle cells from mouse aortae demonstrated that MIF deficiency reduced smooth muscle cell proliferation, cysteine protease expression, and elastinolytic and collagenolytic activities. Conclusions— Deficiency of MIF reduces atherogenesis in LDLr −/− mice. These results provide novel insight into inflammatory pathways operating in atheromata and identify a new potential target for modulating atherogenesis.

Transmembrane metalloproteinases of the disintegrin and metalloproteinase (ADAM) family control cell signaling interactions via hydrolysis of protein extracellular domains. Prior work has shown that the receptor tyrosine kinase, c-Kit (CD117), is essential for mast cell survival and that serum levels of c-Kit increase in proliferative mast cell disorders, suggesting the existence of c-Kit shedding pathways in mast cells. In the present work, we report that tumor necrosis factor alpha-converting enzyme (TACE; ADAM-17) mediates shedding of c-Kit. Stimulation of transfected cells with phorbol 12-myristate 13-acetate (PMA) induced metalloproteinase-mediated release of c-Kit ectodomain, which increased further upon TACE overexpression. By contrast, TACE-deficient fibroblasts did not demonstrate inducible release, thus identifying TACE as the metalloproteinase primarily responsible for PMA-induced c-Kit shedding. Surface expression of c-Kit by the human mast cell-1 line decreased upon phorbol-induced shedding, which involved metalloproteinase activity susceptible to inhibition by tissue inhibitor of metalloproteinase (TIMP)-3. To further explore the role of TACE in shedding of c-Kit from mast cells, we compared the behavior of mast cells derived from murine embryonic stem cells. In these studies, PMA decreased surface c-Kit levels on mast cells expressing wild-type (+/+) TACE but not on those expressing an inactive mutant (DeltaZn/DeltaZn), confirming the role of TACE in PMA-induced c-Kit shedding. Compared with TACE(+/+) cells, TACE(DeltaZn/DeltaZn) mast cells also demonstrated decreased constitutive shedding and increased basal surface expression of c-Kit, with diminished apoptosis in response to c-Kit ligand deprivation. These data suggest that TACE controls mast cell survival by regulating shedding and surface expression of c-Kit.

BACKGROUND: In rodent models, investigators have transplanted donor tracheas into a recipient rat's abdomen or s.c. tissue to study airway rejection. We describe a modification of this model, which provides improved histology to study the airway injury related to obliterative bronchiolitis. METHODS: The standard technique of implanting the donor trachea was compared to a model in which a tracheal Y graft was created by anastomosis of the donor trachea to the recipient airway. Syngeneic and allogeneic tracheal grafts (Lewis and Brown Norway rats) were harvested at 2 and 4 weeks using each model (eight groups). RESULTS: Gross patency at the tracheal anastomosis grafts was 100%. All donor tracheas, which were implanted without an anastomosis, were occluded with mucus (syngeneic) or granulation tissue (allogeneic). Syngeneic implant grafts demonstrated significantly less lumenal granulation tissue 35.3%+/-32 than the allograft implant group (95.3%+/-9.2, P=0.0005 at 4 weeks). The anastomotic allograft group demonstrated significantly less lumenal granulation tissue 48.3%+/-23.7 when compared with the implanted allograft group (P=0.003). The implanted allograft demonstrated a severe loss of epithelial integrity by 2 weeks (16.7%+/-38), which progressed to complete loss by 4 weeks (P=0.0001 and P=0.0001 vs. native). This loss was significantly more than that of the anastomotic group at 2 weeks (89.5%+/-13, P=0.004) and 4 weeks (88.3+/-29, P=0.005). CONCLUSIONS: The rat tracheal allograft anastomosed to the recipient airway demonstrated less lumenal granulation tissue obstruction and better preservation of epithelial integrity than an implant allograft, suggesting that an open airway improves assessment of transplant-related changes associated with rejection.

Pulmonologists frequently encounter indeterminate pulmonary nodules in practice, but it is unclear what clinical factors they rely on to guide the diagnostic evaluation.To assess the current approach to the management of indeterminate pulmonary nodules and to determine the extent to which the addition of a hypothetical diagnostic blood test will influence clinical decision making.Selected pulmonologists practicing in the United States were invited to participate in a conjoint exercise based on 20 randomly generated cases of varying age, smoking history, and nodule size. Some cases included the result of a hypothetical blood test. Each respondent chose from among three diagnostic options for a patient: noninvasive monitoring (i.e., serial CT or positron emission tomography scan), a minor procedure (i.e., biopsy or bronchoscopy), or a major procedure (i.e., video-assisted thorascopic surgery or thoracotomy). Multivariate logistic regression was used to assess the impact of the three risk factors and the diagnostic blood test on decision making.Four hundred nineteen physicians participated (response rate, 10%). One hundred fifty-three physician surveys met predetermined criteria and were analyzed (4% of all invitees). A diagnostic procedure was recommended for 23% of 6-mm nodules, versus 54, 66, 77, and 84% of nodules 10, 14, 18, and 22 mm, respectively (P 0.001). Older age limited recommendations for invasive testing: 54% of 80-year-olds versus 61, 64, 63, and 61% of patients 71, 62, 53, and 44 years of age, respectively (P 0.001). In multivariate analyses, nodule size, smoking history, age, and the blood test each influenced decision making (P 0.001).The pulmonologists who participated in this survey were more likely to proceed with invasive testing, instead of observation or additional imaging, as the size of the nodule increased. The use of a hypothetical blood test resulted in significant alterations in the decision to pursue invasive testing.

Induction of alveolar epithelial cell phenotypes via mesenchymal signaling regulates pneumocyte differentiation not only during lung development (1, 2) but also during the response to a myriad of injuries inciting fibrogenesis (3). Signals generated in the epithelium and the mesenchyme regulate critical functions of cells in both compartments reciprocally, with transduction mediated by soluble growth factors, membrane-anchored receptors, and extracellular matrix proteins (4). Such epithelial‐mesenchymal interactions characterize remodeling in a variety of tissues, including airways and lung parenchyma (3, 5, 6). Transition from injury to the repair phase in alveolar remodeling requires epithelialization to regenerate pneumocyte populations and the epithelial component of the alveolar barrier. Ordered layering of type I and type II pneumocytes, basement membranes, and capillary endothelial cells establishes compartmentalization that normalizes communication among cells and extracellular matrix components in distinct epithelial and mesenchymal layers. Restoration of alveolar quiescence reverses ventilation‐perfusion defects to equilibrate gas exchange. By contrast, unrelenting signaling between the compartments leads to continued invasion of fibroblasts and excessive deposition of matrix proteins, causing intra-alveolar fibrosis and the loss of surface area, which defines the pathophysiology of pulmonary fibrosis (3). Epithelial cells differentiate into subpopulations, which reconstruct a protective barrier and regulate clearance of alveolar exudates. Type II cells differentiate from cuboidal progenitor cells at septal intersections into thin type I cells that line approximately 90% of the alveolar unit. Subpopulations of type II pneumocytes synthesize surfactants needed to reduce alveolar surface tension and also produce matrix constituents such as fibronectin, collagen IV, and proteoglycan, essential for remodeling. Other type II cells help resorb proteinaceous exudates by releasing fibrinolytic proteases or by upregulating expression of water and ion channels. Some type II pneumocytes also serve as stem cells to replenish the type II epithelial cell population itself (3). Failure of the alveolar epithelium to perform these functions results both in the unchecked migration of fibroblasts onto the provisional matrix and the continued matrix protein deposition, which propagate fibrogenesis. Premature demise of the epithelial cell population may result from apoptosis due to incessant injury or from apoptotic signals arising from the mesenchyme (7). Thus, epithelial dysfunction permits sequential flooding and increased surface tension in the alveolar space, apposition and fusion of exposed basement membranes, and irreversible collapse and fibrosis—all of which diminish effective alveolar surface area (3).

Background: Bronchiolitis obliterans occurs in 30% to 80% of lung-transplant recipients and is a direct cause of death in more than 40% of patients with this complication. This study assessed the potential utility of measuring fibroblast-proliferative activity in bronchoalveolar lavage fluid from lung-transplant recipients to better understand the pathogenesis of this process. Methods The capacity of bronchoalveolar lavage fluid obtained from transplant recipients, during routine surveillance bronchoscopy, to stimulate the proliferation of human lung fibroblasts in vitro was assessed retrospectively and compared to that of control subjects. For each recipient, a correlation was made between the fibroblast-proliferative activity in serial lavage samples over time and the other modalities employed for detecting post-transplant complications including spirometry, transbronchial lung biopsy, and high-resolution computed tomography. Results There was a significant difference in fibroblast-proliferative activity between volunteer and transplant recipient groups ( p = 0.002). Further, for each transplant recipient, the decline in the forced expired flow rate between 25% and 75% of expired volume (FEF 25%–75% ) was correlated with the mean fibroblast-proliferative activity during the period of this study ( r = 0.83; p = 0.04). Conclusions A sustained increase in fibroblast-proliferative activity in lavage supernatant precedes both histologic and physiologic evidence of bronchiolitis obliterans. Relative to an increase in fibroblast-proliferative activity or abnormalities in FEF 25%–75% , a decrease in forced expiratory volume in 1 second is a late finding.

Our prior work shows that cultured BR cells derived from dog mastocytomas secrete the 92-kDa proenzyme form of gelatinase B. We provided a possible link between mast cell activation and metalloproteinase-mediated matrix degradation by demonstrating that α-chymase, a serine protease released from secretory granules by degranulating mast cells, converts progelatinase B to an enzymatically active form. The current work shows that these cells also secrete gelatinase A. Furthermore, gelatinases A and B both colocalize to α-chymase-expressing cells of canine airway, suggesting that normal mast cells are a source of gelatinases in the lung. In BR cells, gelatinase B and α-chymase expression are regulated, whereas gelatinase A expression is constitutive. Progelatinase B mRNA and enzyme expression are strongly induced by the critical mast cell growth factor, kit ligand, which is produced by fibroblasts and other stromal cells. Induction of progelatinase B is blocked by U-73122, Ro31-8220, and thapsigargin, implicating phospholipase C, protein kinase C, and Ca2+, respectively, in the kit ligand effect. The profibrotic cytokine TGF-β virtually abolishes the gelatinase B mRNA signal and also attenuates kit ligand-mediated induction of gelatinase B expression, suggesting that an excess of TGF-β in inflamed or injured tissues may alter mast cell expression of gelatinase B, which is implicated in extracellular matrix degradation, angiogenesis, and apoptosis. In summary, these data provide the first evidence that normal mast cells express gelatinases A and B and suggest pathways by which their regulated expression by mast cells can influence matrix remodeling and fibrosis.

In prior work we showed that a metallogelatinase is secreted from dog mastocytoma cells and directly activated by exocytosed mast cell alpha-chymase. The current work identifies the protease as a canine homologue of progelatinase B (92-kDa gelatinase, MMP-9), determines the sites cleaved by alpha-chymase, and explores the regulation of gelatinase expression in mastocytoma cells. To obtain a cDNA encoding the complete sequence of mastocytoma gelatinase B, a 2. 3-kilobase clone encoding progelatinase was isolated from a BR mastocytoma library. The sequenced cDNA predicts a 704-amino acid protein 80% identical to human progelatinase B. Regions thought to be critical for active site latency, such as the Cys-containing propeptide sequence, PRCGVPD, and the catalytic domain sequence, HEFGHALGLDHSS, are entirely conserved. Cleavage of progelatinase B by purified dog alpha-chymase yielded an approximately 84-kDa product that contained two NH2-terminal amino acid sequences, QTFEGDLKXH and EGDLKXHHND, which correspond to residues 89-98 and 92-101 of the cDNA predicted sequence, respectively. Thus, alpha-chymase cleaves the catalytic domain of gelatinase B at the Phe88-Gln89 and Phe91-Glu92 bonds. Like BR cells, the C2 line of dog mastocytoma cells constitutively secrete progelatinase B which is activated by alpha-chymase. By contrast, non-chymase-producing C1 cells secrete a gelatinase B (which remains in its proform) only in response to 12-O-tetradecanoylphorbol-13-acetate. Whereas 12-O-tetradecanoylphorbol-13-acetate stimulation of BR cells produced a approximately 15-fold increase in gelatinase B mRNA expression, dexamethasone down-regulated its expression by approximately 5-fold. Thus, extracellular stimuli may regulate the amount of mast cell progelatinase B expressed by mast cells. These data further support a role for mast cell alpha-chymase in tissue remodeling involving gelatinase B-mediated degradation of matrix proteins.

Each year, millions of pulmonary nodules are discovered by computed tomography and subsequently biopsied. Because most of these nodules are benign, many patients undergo unnecessary and costly invasive procedures. We present a 13-protein blood-based classifier that differentiates malignant and benign nodules with high confidence, thereby providing a diagnostic tool to avoid invasive biopsy on benign nodules. Using a systems biology strategy, we identified 371 protein candidates and developed a multiple reaction monitoring (MRM) assay for each. The MRM assays were applied in a three-site discovery study (n = 143) on plasma samples from patients with benign and stage IA lung cancer matched for nodule size, age, gender, and clinical site, producing a 13-protein classifier. The classifier was validated on an independent set of plasma samples (n = 104), exhibiting a negative predictive value (NPV) of 90%. Validation performance on samples from a nondiscovery clinical site showed an NPV of 94%, indicating the general effectiveness of the classifier. A pathway analysis demonstrated that the classifier proteins are likely modulated by a few transcription regulators (NF2L2, AHR, MYC, and FOS) that are associated with lung cancer, lung inflammation, and oxidative stress networks. The classifier score was independent of patient nodule size, smoking history, and age, which are risk factors used for clinical management of pulmonary nodules. Thus, this molecular test provides a potential complementary tool to help physicians in lung cancer diagnosis.

Gelatinolytic metalloproteinases implicated in connective tissue remodeling and tumor invasion are secreted from several types of cells in the form of inactive zymogens. In this report, characterization of gelatinase activity secreted by the BR line of dog mastocytoma cells reveals a phorbol-inducible, approximately 92-kD, Ca2+ - and Zn2+ -dependent proenzyme cleaved over time to smaller, active forms. Incubation of cells with the general serine protease inhibitor, PMSF, prevented proenzyme cleavage and permitted its purification free of activation products. The NH2-terminal 13 amino acids of the purified mastocytoma progelatinase are 50-67% identical to those of human, mouse, and rabbit 92-kD progelatinase (gelatinase B; matrix metalloproteinase-9). Degranulation of mastocytoma cells using ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 greatly accelerated proenzyme cleavage, suggesting that a serine protease present in secretory granules hydrolyzed the progelatinase to active fragments. To identify the activating protease, cells were coincubated with ionophore and a panel of selective serine protease inhibitors. Soybean trypsin inhibitor and succinyl-L-Ala-Ala-Pro-Phe-chloromethylketone, which inhibit mast cell chymase, prevented progelatinase activation. Inhibitors of tryptase and dog mast cell protease (dMCP)-3, i.e., aprotinin or bis(5-amidino-2-benzimidazolyl) methane (BABIM), did not. In further experiments using highly purified enzymes, mastocytoma cell chymase activated 92-kD progelatinase in the absence of other enzymes or cofactors; tryptase and dMCP-3, however, had no effect. These data demonstrate that dog mastocytoma cells secrete a metalloproteinase related to progelatinase B that is directly activated outside of the cell by exocytosed chymase, and provide the first demonstration of a cell that activates a matrix metalloproteinase it secretes by cosecreting an activating enzyme. In mastocytomas, this pathway may facilitate tumor invasion of surrounding tissues, and in normal mast cells, it could play a role in tissue remodeling and repair.

There have been some successes in qualifying biomarkers and applying them to drug development and clinical treatment of various diseases. A recent success is illustrated by a collaborative effort among the US Food and Drug Administration, the European Medicines Agency, and the pharmaceutical industry to provide a set of seven preclinical kidney toxicity biomarkers for drug development. Other successes include, but are not limited to, clinical biomarkers for cancer treatment and clinical management of heart transplant patients. The value of fully qualified surrogate endpoints in facilitating successful drug development is undisputed, especially for diseases in which the traditional clinical outcome can only be assessed in large, multi-year trials. Emerging biomarkers, including chemical genomic or imaging biomarkers, and measurement of circulating tumor cells hold great promise for early diagnosis of disease and as prognostic tests for managing treatment of chronic diseases such as osteoarthritis, Alzheimer disease, cardiovascular disease, and cancer. To advance the success of treating and managing these diseases, efforts are needed to establish the temporal relationship between changes in inflammatory or imaging biomarkers with the progression of the chronic disease, and in the case of cancer, between the extent of circulating cancer cells and tumor progression or remission.

Cardiac allografts induce host immune responses that lead to endomyocardial tissue injury and progressive graft dysfunction. Inflammatory cell infiltration and myocyte damage characterize acute cellular rejection (ACR) that presents episodically in either a subclinical or symptom-associated manner. Sampling of the endomyocardium by transvenous biopsy enables pathologic grading using light microscopic criteria to distinguish severity based on the focality or diffuseness of inflammation and associated myocyte injury. Monitoring for ACR utilizes endomyocardial biopsy in conjunction with history and physical examination and assessment of allograft function by echocardiography. However, procedural and interpretive issues limit the diagnostic certainty provided by endomyocardial biopsy. The dynamic profiling of genes expressed by peripheral blood mononuclear cells (PBMCs) enables quantitative assessments of intracellular mRNA whose levels fluctuate during systemic alloimmune responses. Gene expression profiling of PBMCs using a multi-gene ACR classifier enables the AlloMap® molecular expression test to distinguish moderate to severe ACR (p = 0.0018) in heart transplant patients. The AlloMap test provides molecular insights into a patient's risk for ACR by distilling the aggregate expression levels of its informative genes into a single score on a scale of 0 to 40. The selection of a score as a threshold value for clinical decision-making is based on its associated negative predictive value (NPV), which ranges from 98 to 99% for values in three post-transplant periods: >2 to ≤6 months, > 6to ≤ 12 months, and >12 months. Scores below the threshold value rule out ACR, while those above suggest increased ACR risk. Incorporating the AlloMap test into immunomonitoring protocols provides an opportunity for clinicians to enhance patient care and to define its role in immunodiagnostic strategies to optimize the clinical outcomes of heart transplant recipients. This summary highlights the concepts presented in an invited presentation at a conference focused on Immunodiagnostics and Immunomonitoring: From Research to Clinic, in San Diego, CA on November 7, 2006.

Profiling mRNA levels of 11 informative genes expressed by circulating immune effector cells identifies cardiac allograft recipients at low risk for current moderate-severe acute cellular rejection (ACR).We conducted a nested case-control study of 104 cardiac allograft recipients to investigate the association of transcriptional profiles of blood samples with either a future rejection episode within 12 weeks of a baseline clinical sample or persistent histologic quiescence for the same time period.The transcription profile yielded a score (0 to 40 scale) of 27.4 +/- 6.3 for future rejectors (n = 39) and 23.9 +/- 7.1 for controls (n = 65) (p = 0.01). In patients who wereor=180 days post-transplant, the gene expression score was 28.4 +/- 4.9 for rejectors (n = 28) and 22.4 +/- 7.5 for controls (n = 48) (p0.001). In this period, no samples from patients who went on to reject within 12 weeks had gene expression scores of20. Differential expression of the gene IL1R2 was significantly associated with future events. Of 33 additional genes profiled, 5 supported corticosteroid-sensitive constituents (IL1R2 and FLT3), whereas 6 supported T-cell activation (PDCD1).These data suggest that pathways regulating T-cell homeostasis and corticosteroid sensitivity are associated with future ACR in cardiac allografts and suggest that these signals are evident before histologically detectable rejection.

From purulent cystic fibrosis (CF) sputum, previous investigators partially purified a trypsinlike protease. A similar purified enzyme is available commercially as “human sputum trypsin.” To explore the nature and origin of this preparation, we purified and NH2terminally sequenced its major protein component. The resulting sequence, Ile-Val-Gly-Gly-Tyr-Thr-(Cys)-Ala-Ala-Asn-Ser-Val/Ile-Pro-Tyr-Gln-Val-Ser-Leu-Asn-Ser, differs from known human proteins but is identical to porcine trypsin, including the Val/Ile polymorphism at residue 12. Specific activity and electrophoretic and inhibition profiles and immunoreactivity of sputum and porcine pancreatic trypsin are nearly identical. Because porcine trypsin is a major ingredient of digestive enzyme supplements taken by CF patients with pancreatic dysfunction, we propose that one or more lots of human sputum trypsin derive from enzyme supplements and are of porcine origin. The path by which trypsin ends up in sputum is unknown. Because sputum trypsin is active but susceptible to inactivation by plasma α1-proteinase inhibitor, it is unlikely to derive from trypsin absorbed into the bloodstream. However, it may originate from tracheally aspirated stomach contents or from digestive supplement-contaminated saliva mixed with expectorated sputum. The imbalance between proteases and antiproteases in CF bronchial secretions allows trypsin to remain active despite sensitivity to serpins and secretory leukocyte proteinase inhibitor. Furthermore, because sputum trypsin activates human progelatinase B, it may be responsible in part for the reported presence of activated matrix metalloproteinases in CF sputum.

Diverse interstitial lung diseases (ILD) demonstrate mesenchymal infiltration by an abundance of activated mast cells whose role in parenchymal fibrogenesis remains unclear. Since mast cells differentiate in a dynamic, tissue-specific manner via signals transduced by c-Kit receptor, we examined the effect of ILD microenvironments on c-Kit expression and metalloproteinase phenotypes of mesenchymal mast cell populations. Immunohistochemical and flow cytometric analyses characterized surface expression of c-Kit on mast cells in tissues obtained from patients with idiopathic pulmonary fibrosis, systemic sclerosis, sarcoidosis, and lymphangioleiomyomatosis, thus identifying a unique immunophenotype not shared by normal lung mast cells. Isolation of c-Kit+/FcepsilonRI+/CD34- mast cells via immunocytometric sorting of heterogeneous cell populations from mechanically disaggregated lung tissues permitted analysis of gene expression patterns by two-step real-time polymerase chain reaction. Transcriptional profiling identified expression of c-Kit and the neutral serine proteases, tryptase and chymase, establishing the identity of sorted populations as mature mast cells. Mast cells harvested from ILD tissues demonstrated characteristic metalloproteinase phenotypes which included expression of matrix metalloproteinase (MMP)-1 and a disintegrin and metalloproteinase (ADAM)-9, -10, and -17. Immunohistochemical co-localization guided by gene profiling data confirmed expression of chymase, MMP-1, and ADAM-17 protein in subpopulations of mast cells in remodelling interstitium. Gene profiling of harvested mast cells also showed increased transcript copy numbers for TNFalpha and CC chemokine receptor 2, which play critical roles in lung injury. We conclude that ILD microenvironments induce unique c-Kit receptor and metalloproteinase mast cell phenotypes.

The pathogenesis of atherosclerosis and abdominal aortic aneurysm involves substantial proteolysis of the arterial extracellular matrix. The lysosomal cysteine proteases can exert potent elastolytic and collagenolytic activity. Human atherosclerotic plaques have increased cysteine protease content and decreased levels of the endogenous inhibitor cystatin C, suggesting an imbalance that would favor matrix degradation in the arterial wall. This study tested directly the hypothesis that impaired expression of cystatin C alters arterial structure. Cystatin C–deficient mice (Cyst C −/− ) were crossbred with apolipoprotein E–deficient mice (ApoE −/− ) to generate cystatin C and apolipoprotein E–double deficient mice (Cyst C −/− ApoE −/− ). After 12 weeks on an atherogenic diet, cystatin C deficiency yielded significantly increased tunica media elastic lamina fragmentation, decreased medial size, and increased smooth muscle cell and collagen content in aortic lesions of ApoE −/− mice. Cyst C −/− ApoE −/− mice also showed dilated thoracic and abdominal aortae compared with control ApoE −/− mice, although atheroma lesion size, intimal macrophage accumulation, and lipid core size did not differ between these mice. These findings demonstrate directly the importance of cysteine protease/protease inhibitor balance in dysregulated arterial integrity and remodeling during experimental atherogenesis.

Activated mast cells release stored and newly synthesized mediators that influence the caliber and responsiveness of inflamed airways. In this work, we show that alloimmune-mediated mechanisms induce mast cell activation and expression of CC chemokines in remodeling rat tracheal allografts. Decreased expression of rat mast cell protease (RMCP) I and II, in concert with tryptase release in tracheal allografts, identified degranulation of stored serine proteases as an early mast cell response to allotransplantation. Transient upregulation of c-Kit expression occurred in a synchronous manner, suggesting that c-Kit receptor signaling controls mast cell responses. Increased expression of CC chemokine ligand (CCL) 2 and CCL3 by RMCP I-positive cells identified mast cells as epithelial and mesenchymal sources of chemoattractant chemokines in allograft airways. Cyclosporin A immunosuppression both attenuated and delayed these changes in mast cell phenotypes. Incubation of rat basophil leukemia 2H3 cells with CCL2 or CCL3 decreased surface c-Kit expression, an effect blocked by protease inhibitors. By controlling surface receptor availability, CC chemokines may regulate c-Kit signaling via a novel proteolytic mechanism. These data suggest that targeting alloimmune responses and restoring quiescence of mast cells may attenuate the development of fibroproliferative and obstructive distortions of bronchiolar architecture in lung allografts.

In rodent models, investigators have transplanted donor tracheas into a recipient rat's abdomen or s.c. tissue to study airway rejection. We describe a modification of this model, which provides improved histology to study the airway injury related to obliterative bronchiolitis.The standard technique of implanting the donor trachea was compared to a model in which a tracheal Y graft was created by anastomosis of the donor trachea to the recipient airway. Syngeneic and allogeneic tracheal grafts (Lewis and Brown Norway rats) were harvested at 2 and 4 weeks using each model (eight groups).Gross patency at the tracheal anastomosis grafts was 100%. All donor tracheas, which were implanted without an anastomosis, were occluded with mucus (syngeneic) or granulation tissue (allogeneic). Syngeneic implant grafts demonstrated significantly less lumenal granulation tissue 35.3%+/-32 than the allograft implant group (95.3%+/-9.2, P=0.0005 at 4 weeks). The anastomotic allograft group demonstrated significantly less lumenal granulation tissue 48.3%+/-23.7 when compared with the implanted allograft group (P=0.003). The implanted allograft demonstrated a severe loss of epithelial integrity by 2 weeks (16.7%+/-38), which progressed to complete loss by 4 weeks (P=0.0001 and P=0.0001 vs. native). This loss was significantly more than that of the anastomotic group at 2 weeks (89.5%+/-13, P=0.004) and 4 weeks (88.3+/-29, P=0.005).The rat tracheal allograft anastomosed to the recipient airway demonstrated less lumenal granulation tissue obstruction and better preservation of epithelial integrity than an implant allograft, suggesting that an open airway improves assessment of transplant-related changes associated with rejection.

In sensitized individuals birch pollen induces an allergic response characterized by IgE-dependent mast cell degranulation of mediators, such as α-chymase and other serine proteases. In birch and other plant pollens, a major allergen is profilin. In mammals, profilin homologues are found in an intracellular form bound to cytoskeletal or cytosolic proteins or in a secreted form that may initiate signal transduction. IgE specific to birch profilin also binds human profilin I. This cross-reactivity between airborne and endogenous proteins may help to sustain allergy symptoms. The current work demonstrates that cultured mast cells constitutively secrete profilin I, which is susceptible to degranulation-dependent proteolysis. Coincubation of chymase-rich BR mastocytoma cells with Ala-Ala-Pro-Phe-chloromethylketone (a chymase inhibitor) blocks profilin cleavage, which does not occur in degranulated HMC-1 mast cells, which are rich in tryptase, but chymase deficient. These data implicate chymase as the serine protease cleaving secreted mast cell profilin. Sequencing of chymase-cleaved profilins reveals hydrolysis at Tyr6-Val7 and Trp35-Ala36 in birch profilin and at Trp32-Ala33 in human profilin, with all sites lying within IgE-reactive epitopes. IgE immunoblotting studies with sera from birch pollen-allergic individuals demonstrate that cleavage by chymase attenuates binding of birch profilin to IgE. Thus, destruction of IgE-binding epitopes by exocytosed chymase may limit further mast cell activation by this class of common plant allergens, thereby limiting the allergic responses in sensitized individuals.

We previously reported that mast cell α-chymase cleaves and activates progelatinase B (progel B). Outside of cells, progel B is complexed with tissue inhibitor of metalloproteinase (TIMP)-1, which hinders zymogen activation and inhibits activity of mature forms. The current work demonstrates that dog BR mastocytoma cells, HMC-1 cells, and murine bone marrow-derived mast cells secrete TIMP-1 whose electrophoretic profile in supernatants suggests degranulation-dependent proteolysis. α-Chymase cleaves uncomplexed TIMP-1, reducing its ability to inhibit gel B, whereas tryptase has no effect. Sequencing of TIMP-1’s α-chymase-mediated cleavage products reveals hydrolysis at Phe12-Cys13 and Phe23-Val24 in loop 1 and Phe101-Val102 and Trp105-Asn106 in loop 3 of the NH2-terminal domain. TIMP-1 in a ternary complex with progel B and neutrophil gelatinase-associated lipocalin is also susceptible to α-chymase cleavage, yielding products like those resulting from processing of free TIMP-1. Thus, α-chymase cleaves free and gel B-bound TIMP-1. Incubation of the progel B-TIMP-1-neutrophil gelatinase-associated lipocalin complex with α-chymase increases gel B activity 2- to 5-fold, suggesting that α-chymase activates progel B whether it exists as free monomer or as a complex with TIMP-1. Furthermore, inhibition of α-chymase blocks degranulation-induced TIMP-1 processing (absent in α-chymase-deficient HMC-1 cells). Purified α-chymase processes TIMP-1 in BR supernatants, generating products like those induced by degranulation. In summary, these results suggest that controlled exocytosis of mast cell α-chymase activates progel B even in the presence of TIMP-1. This is the first identification of a protease that overcomes inhibition by bound TIMP-1 to activate progel B without involvement of other proteases.

Purpose: Heart failure (HF) and the unloading of the myocardium by Left Ventricular Assist Device (LVAD) support have been associated with remodeling of cardiomyocytes and the extracellular matrix (ECM). Integrins (ITG) are membrane spanning proteins connecting the cytoskeleton of cardiomyocytes to the ECM, thereby functioning as mechanotransducers. Given the close connection between the ECM, ITG and cardiomyocytes we investigated changes in ITG expression during HF and LVAD support. Methods and Materials: Frozen left ventricular tissue was obtained during LVAD implantation (pre-LVAD) and at time of heart transplantation (post-LVAD) from 8 patients with dilated cardiomyopathy (DCM) and 8 patients with ischemic heart disease (IHD). Five healthy heart samples served as controls. mRNA-expression of several ITG (Alpha1,3,5,6,7,10,11 and Beta1,3,5,6) was investigated by Q-PCR using a low density array. Immunohistochemistry (IH) and Western blot (WB) analysis were used to localize and quantify protein expression of ITG-A5,-A6,-A7 and -B1. Results: In DCM and IHD combined ITG-A1,-A6,-A10 and -B6 mRNA levels increased during LVAD support (p 0.05). ITG-A7 was significantly elevated pre-LVAD and returned to control levels after LVAD support. In DCM alone ITG-A1,-A6 and -A10 increased significantly during LVAD support (1.1, 1.5 and 2.6 fold respectively, p 0.05) In IHD alone ITG-A6 increased 1.1 fold (p 0.05). IH showed a membraneous staining of ITG-A7 and ITG-B1, whereas ITG-A6 only stained capillaries and ITG-A5 only larger vessels and stroma. The staining pattern preand post-LVAD was not different, but less organized than in control samples. WB showed a low ITG protein level preand post-LVAD compared to controls. Interestingly, Q-PCR and WB showed an increase of ITG-A5 in DCM and a decrease in IHD after LVAD support. Conclusions: The changes in cardiomyocyte make-up and ECM composition during HF are associated with alterations in ITG expression pattern and are altered by ventricular unloading by LVAD, re-inforcing the important role of ITG in mechanotransduction. Supported by the NHF 2004T31