classified as indolent, a significant subset of patients with mastocytosis presents with aggressive forms of the disease and have a poor prognosis. This spectrum ...
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Acquired Activating NRAS Mutations Associated with Aggressive Systemic Mastocytosis T. M. Wilson1, Y. Wu1, W. Fu2, Y. Bai1, P. Noel2, I. Maric2, J. Robyn1, D. D. Metcalfe1; 1Laboratory of Allergic Diseases, NIAID, NIH, Bethesda, MD, 2Department of Laboratory Medicine, Clinical Center, NIH, Bethesda, MD. RATIONALE: Over 80% of patients with systemic mastocytosis have an acquired activating mutation (D816V) in the tyrosine kinase domain of the stem cell factor receptor, KIT. Although the majority of patients are classified as indolent, a significant subset of patients with mastocytosis presents with aggressive forms of the disease and have a poor prognosis. This spectrum of clinical manifestations is not explained by a single activating mutation. Recent animal models indicate that activation of the RAS pathway may play an important role in the pathogenesis of mastocytosis. Therefore, we hypothesized that RAS activating mutations may be contributing to this phenotypic diversity. METHODS: cDNA was prepared from the bone marrow mononuclear cells of 41 adult patients with indolent (31) or aggressive (10) systemic mastocytosis. NRAS, MRAS, KRAS and/or HRAS genes were amplified and sequenced. All patients possessed the c-kit D816V mutation identified by restriction fragment length polymorphism analysis. RESULTS: Heterozygous activating point mutations in NRAS were identified in two patients with aggressive systemic mastocytosis. NRAS mutations were absent in patients with indolent disease. CONCLUSIONS: This is the first report of acquired genetic mutations in NRAS potentially contributing to aggressive forms of systemic mastocytosis. Most agents in current clinical trials for mastocytosis target c-kit and, despite potent in vitro activity, have displayed modest clinical efficacy. Therefore, the finding of two independent acquired activating mutations with the potential to cooperate in disease pathogenesis suggests that strategies targeting only one mutation may not be sufficient. Funding: NIH
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Role for Mast Cell Chymase in Intestinal Epithelial Permeability K. R. Groschwitz1, R. Ahrens1, F. D. Finkelman1, J. P. Abonia1, M. F. Gurish2, M. E. Rothenberg1, S. P. Hogan1; 1Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA. RATIONALE: Employing mast cell (MC) neutralizing antibodies and stabilizing agents, we have previously demonstrated a critical role for MCs in the regulation of intestinal epithelial permeability in mice. We hypothesize that a MC-derived serine protease, chymase, is an important mediator of intestinal epithelial permeability. METHODS: Jejunal levels of mouse mast cell protease-4 (mMCP-4), the murine functional homologue to human chymase, were analyzed by quantitative real-time PCR and immunohistochemistry in wild-type BALB/c (WT) mice and in a murine model of intestinal mastocytosis using intestinal IL-9 Tg (iIL-9 Tg) mice. To examine the effect of chymase on intestinal epithelial permeability, confluent colonic adenocarcinoma Caco2-bbe cells were exposed to 0, 2.5 or 5U of human skin-derived chymase basolaterally for 24 hours and intestinal epithelial permeability measured in Ussing Chambers by monitoring the mucosal to serosal flux of FITC-dextran and the inversely correlating transepithelial resistance (TER). RESULTS: Intestinal mastocytosis was associated with a significant increase in jejunal mMCP-4 mRNA levels (10.1 6 1.0 fold; p < 0.0001) in iIL-9 Tg mice compared to WT. Furthermore, jejunum from iIL-9 Tg mice displayed strong and diffuse mMCP-4 positive immunohistochemical staining in comparison to very low levels of punctate staining in jejunum from WT mice. Exposure of Caco2-bbe monolayers to chymase induced a significant decrease in the TER (2.5U: 125.9 6 47.6, p < 0.05; 5U: 192.4 6 19.9, p < 0.005; vs control: 297.5 6 15.9 V/cm2) and increase in FITCdextran permeability (2.5U: 12926 6 2277, p < 0.05; 5U: 11752 6 2063, p < 0.01; vs control: 3474 6 457 ng/ml) compared to controls.
J ALLERGY CLIN IMMUNOL FEBRUARY 2008
CONCLUSIONS: Collectively, these data suggest that MC-derived chymase may play an important role in MC-mediated intestinal epithelial permeability. Funding: Cincinnati Digestive Health Center, NIH
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Mast Cell-Eosinophil Cross-Talk Regulates Function and Viability of Both Cells in Vitro M. Elishmereni, A. H. Nissim Ben-Efraim, I. Bachelet, F. Levi-Schaffer; Hebrew University of Jerusalem, Jerusalem, ISRAEL. RATIONALE: Mast cells (MC) and eosinophils (Eos), the key effector cells of allergic reactions, are both present in enhanced numbers in the late and chronic phases of the inflammatory process. Thus, MC-Eos interactions by physical contact and soluble mediators are thought to have a key role in regulating the allergic response at these stages. However, the characteristics of this cross-talk are not fully elucidated. METHODS: Human nasal polyp tissue sections were stained for MC and Eos. Human cord blood-derived MC were co-cultured with peripheral blood Eos in MEM-a medium with stem cell factor (SCF). Live-imaging microscopy was used to analyze couple formation dynamics. In co-cultures at different ratios, MC were untreated or activated using anti-IgE, and degranulation was measured by chromogenic assays for b-hexosaminidase. MC and Eos viability in long-term (3-day) co-culture was measured by propidium iodide staining and analyzed by FACS. RESULTS: MC and Eos physically interact and form well-defined couples, both in nasal polyps and in co-culture. MC and Eos interactions in vitro occur within 1-5 minutes, and couples are stable for a similar time frame. In the presence of Eos up to a 1:2 ratio, MC are 10% and 20% more releasable under baseline and IgE-activating conditions (n 5 5, p < 0.05), respectively. Eos significantly increase MC viability, and vice versa, in the presence or absence of SCF (n 5 3, p < 0.05). CONCLUSIONS: MC and Eos interactions result in augmented MC activation and increased survival of both cells, and may therefore enhance the hallmarks of late and chronic phases of allergic reactions. Funding: Aimwell Trust
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Mast Cell Chymase Can Disrupt the Human Bronchial Epithelium and Stimulate the Loss of Adhesion Molecules X. Zhou, C. Cox, S. Rajenthirar, A. A. Mahrous, B. Masters, W. R. Roche, A. F. Walls; University of Southampton - Medical School, Southampton, UNITED KINGDOM. RATIONALE: The airways of asthmatics are associated with an increased degree of mast cell degranulation and with damage to the epithelial lining. The actions on the epithelium of chymase secreted by mast cells have not been examined. METHODS: Bronchial tubes from freshly resected human lung tissue were incubated for various periods at 370C with chymase (recombinant or purified from human tissues). Tissue was paraffin-embedded and sections stained with haematoxylin and eosin were analyzed using a computerized image analysis system to assess relative degrees of epithelial damage. Primary epithelial cells and cells of the 16HBE line were cultured in the presence of chymase. Changes in cell proliferation and cell viability were determined and immunocytochemistry or western blotting applied to detect alterations in cytokeratin and adhesion molecule expression using antibodies against occludin, ZO-1, claudin 1, 2 and 4, E-cadherin and cytokeratins. RESULTS: Chymase provoked profound changes in the morphology of the epithelial layer. Following incubation with the maximal concentration of chymase (2 ug/ml) there was a reduction of 40% in the length of epithelium that was intact, with detachment of columnar epithelial cells and even of basal cells. At low concentrations, chymase stimulated an increase in proliferation of epithelial cells in culture, but at higher concentrations there was a decrease in epithelial cell numbers and evidence for cytotoxic responses. There was detachment of epithelial cells in culture, and in intact layers, loss of adhesion molecules and cytokeratin disruption. CONCLUSIONS: Mast cell chymase could play a key role in inducing damage to the bronchial epithelium in asthma. Funding: Thrasher Research Fund, Foundation for the Study of Infant Death, Asthma UK
