Detection and characterization of lung cancer using cell-free DNA fragmentomes.

Non-invasive approaches for cell-free DNA (cfDNA) assessment provide an opportunity for cancer detection and intervention. Here, we use a machine learning model for detecting tumor-derived cfDNA through genome-wide analyses of cfDNA fragmentation in a prospective study of 365 individuals at risk for lung cancer. We validate the cancer detection model using an independent cohort of 385 non-cancer individuals and 46 lung cancer patients. Combining fragmentation features, clinical risk factors, and CEA levels, followed by CT imaging, detected 94% of patients with cancer across stages and subtypes, including 91% of stage I/II and 96% of stage III/IV, at 80% specificity. Genome-wide fragmentation profiles across ~13,000 ASCL1 transcription factor binding sites distinguished individuals with small cell lung cancer from those with non-small cell lung cancer with high accuracy (AUC = 0.98). A higher fragmentation score represented an independent prognostic indicator of survival. This approach provides a facile avenue for non-invasive detection of lung cancer.

Assessment of Plasma Proteomics Biomarker's Ability to Distinguish Benign From Malignant Lung Nodules: Results of the PANOPTIC (Pulmonary Nodule Plasma Proteomic Classifier) Trial.

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 < .001). If the integrated classifier results were used to direct care, 40% fewer procedures would be performed on benign nodules, and 3% of malignant nodules would be misclassified. CONCLUSIONS: When used in patients with lung nodules with a pCA ≤ 50%, the integrated classifier accurately identifies benign lung nodules with good performance characteristics. If used in clinical practice, invasive procedures could be reduced by diverting benign nodules to surveillance. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01752114; URL: www.clinicaltrials.gov).

Protein catalyzed capture agents with tailored performance for in vitro and in vivo applications.

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 (T1/2 ) 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.

Evaluation of recombinant enzyme calibration to harmonize lipoprotein-associated phospholipase A2 activity results between instruments.

OBJECTIVES: Enzymatic activity of lipoprotein-associated phospholipase A2 (Lp-PLA2) mediates vascular inflammation in coronary heart disease (CHD). Calibration of Lp-PLA2 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-PLA2 activity ranged from 84 to 303nmol/min/mL in 300 subjects, with 82.0% and 18.0% measurements below and at or above a cut-point of 225nmol/min/mL, respectively. Results of matched K2-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 ≤10nmol/min/mL, without deviation from linearity between 10 and 382nmol/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 1.3nmol/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-PLA2 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.

Management of Pulmonary Nodules by Community Pulmonologists: A Multicenter Observational Study.

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.

An integrated quantification method to increase the precision, robustness, and resolution of protein measurement in human plasma samples.

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.

Validation of a multiprotein plasma classifier to identify benign lung nodules.

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.

Factors that influence physician decision making for indeterminate pulmonary nodules.

RATIONALE: Pulmonologists frequently encounter indeterminate pulmonary nodules in practice, but it is unclear what clinical factors they rely on to guide the diagnostic evaluation. OBJECTIVES: 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. METHODS: 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. MEASUREMENTS AND MAIN RESULTS: 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). CONCLUSIONS: 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.

A blood-based proteomic classifier for the molecular characterization of pulmonary nodules.

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.

Translational biomarkers: from preclinical to clinical a report of 2009 AAPS/ACCP Biomarker Workshop.

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.

Clinical implications and longitudinal alteration of peripheral blood transcriptional signals indicative of future cardiac allograft rejection.

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 or =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 < or =20. A gene score of > or =30 was associated with progression to moderate to severe rejection in 58% of cases. These two extreme scores (< or =20 or > 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 < 0.01). 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.

Transcriptional signals of T-cell and corticosteroid-sensitive genes are associated with future acute cellular rejection in cardiac allografts.

BACKGROUND: 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). METHODS: 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. RESULTS: 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 were

Gene expression profiling distinguishes a molecular signature for grade 1B mild acute cellular rejection in cardiac allograft recipients.

BACKGROUND: 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. METHODS: 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. RESULTS: Mean GE scores not only differentiated Grades >or=3A from Grade 0 (p < 0.00001, center or panel), but also from Grades 1A or 2 (p < 0.05, center or panel), based on mild rejection sub-groups defined by the ISHLT 1990 grading system. In contrast, mean GE scores for Grades 1B and >or=3A were indistinguishable, using either center or panel interpretation. Sub-group analyses of encounters from 2 to 6 months or >6 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 Grades >or=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 Grade >or=3A rejection, while expression of PDCD1 or SEMA7A showed similar directional patterns without achieving statistical significance. CONCLUSIONS: 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.

Clinical utilities of peripheral blood gene expression profiling in the management of cardiac transplant patients.

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 < or =6 months, > 6 to < or = 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.

c-Kit immunophenotyping and metalloproteinase expression profiles of mast cells in interstitial lung diseases.

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.

Induction of mast cell activation and CC chemokine responses in remodeling tracheal allografts.

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.

Tumor necrosis factor-alpha-converting enzyme controls surface expression of c-Kit and survival of embryonic stem cell-derived mast cells.

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.

A comparison of rat tracheal transplant models: implantation verses anastomotic techniques for the study of airway rejection.

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.

Mast cell alpha-chymase reduces IgE recognition of birch pollen profilin by cleaving antibody-binding epitopes.

In sensitized individuals birch pollen induces an allergic response characterized by IgE-dependent mast cell degranulation of mediators, such as alpha-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 Tyr(6)-Val(7) and Trp(35)-Ala(36) in birch profilin and at Trp(32)-Ala(33) 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.