Mast cells (MCs) play critical roles in allergy and inflammation, yet their development remains controversial due to limitations posed by traditional animal models. The zebrafish provides a highly efficient system for studying vertebrate hematopoiesis. We have identified zebrafish MCs in the gill and intestine, which resemble their mammalian counterparts both structurally and functionally. Carboxypeptidase A5 (cpa5), a MCspecific enzyme, is expressed in zebrafish blood cells beginning at 24 hours post fertilization (hpf). At 28 hpf, colocalization is observed with pu.1, mpo, l-plastin, and lysozyme C, but not fms or cepb␣, identifying these early MCs as a distinct myeloid population arising from a common granulocyte/ monocyte progenitor. Morpholino "knockdown" studies demonstrate that transcription factors gata-2 and pu.1, but not gata-1 or fog-1, are necessary for early MC development. These studies validate the zebrafish as an in vivo tool for studying MC ontogeny and function with future capacity for modeling human MC diseases. (Blood. 2008;112:2969-2972) IntroductionMast cells (MCs) play central roles in allergic and inflammatory reactions. 1,2 Stimulation of cell-surface receptors, such as C-KIT and the high-affinity IgE receptor, 1,2 results in the release of mediators from cytoplasmic granules, including tryptase and histamine. 2 MC number and function are regulated by their development, proliferation, migration, and survival. 1 Barriers to understanding these processes include accessibility and imaging limitations posed by traditional animal models. The zebrafish has proven itself to be a robust and highly conserved model for studying vertebrate hematopoiesis. 3 Here, we provide the first evidence that the zebrafish possesses MC equivalents that share structural and functional characteristics with their mammalian counterparts. Furthermore, we demonstrate the utility of the zebrafish as an in vivo tool in dissecting the contribution of transcription factors to MC development. MethodsZebrafish were maintained, bred, and developmentally staged according to Westerfield. 4 Use of zebrafish in this study was approved by the Dalhousie University Animal Care Committee. Zebrafish gills and intestine were fixed in 10% neutral buffered formalin, and standard staining procedures were applied ( Figure 1A-F). Immunohistochemistry was facilitated by antigen retrieval ( Figure 1I,J). For electron microscopy, tissues were fixed overnight in 2% glutaraldehyde in 0.1 M caccodylate and postfixed in 1% osmium tetroxide. Thin sections (90 nm) were stained in 25% uranyl acetate in methanol and lead citrate.Bromophenol blue and 10 g compound 48/80 or saline were injected intraperitoneally, and blood was extracted by cardiac puncture after 2.5 minutes. Tryptase activity was measured in plasma spectrophotometrically at 415 nm by the release of p-nitroanilide from N-benzoyl-DL-argininep-nitroanilide (BAPNA), a tryptase substrate.Digoxogenin-or fluorescein isothiocyanate (FITC)-labeled antisense mRNA probes for zebrafish carboxypep...

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The isolation and analysis of circulating tumor cells (CTC) has the potential to provide minimally invasive diagnostic, prognostic and predictive information. Widespread clinical implementation of CTC analysis has been hampered by a lack of comparative investigation between different analytic methodologies in clinically relevant settings. The objective of this study was to evaluate four different CTC isolation techniques–those that rely on surface antigen expression (EpCAM or CD45 using DynaBeads ® or EasySep™ systems) or the biophysical properties (RosetteSep™ or ScreenCell ® ) of CTCs. These were evaluated using cultured cells in order to calculate isolation efficiency at various levels including; inter-assay and inter-operator variability, protocol complexity and turn-around time. All four techniques were adequate at levels above 100 cells/mL which is commonly used for the evaluation of new isolation techniques. Only the RosetteSep™ and ScreenCell ® techniques were found to provide adequate sensitivity at a level of 10 cells/mL. These techniques were then applied to the isolation and analysis of circulating tumor cells blood drawn from metastatic breast cancer patients where CTCs were detected in 54% (15/28) of MBC patients using the RosetteSep™ and 75% (6/8) with ScreenCell ® . Overall, the ScreenCell ® method had better sensitivity.

ObjectiveTo identify serum biomarkers of papillary thyroid cancer.MethodsProspective analysis was performed of banked tumor and serum specimens from 99 patients with thyroid masses. Enzyme-linked immunosorbent assay (ELISA) was employed to measure levels of five serum proteins previously demonstrated to be up-regulated in papillary thyroid cancer (PTC): angiopoietin-1 (Ang-1), cytokeratin 19 (CK-19), tissue inhibitor of metalloproteinase-1 (TIMP-1), chitinase 3 like-1 (YKL-40), and galectin-3 (GAL-3). Serum levels were compared between patients with PTC and those with benign tumors.ResultsA total of 99 patients were enrolled in the study (27 men, 72 women), with a median age of 54 years. Forty-three patients had PTC and 58 cases were benign tumors. There were no statistically significant differences when comparing all five different biomarkers between PTC and other benign thyroid tumors. The p-values were 0.94, 0.48, 0.72, 0.48, and 0.90 for YKL-40, Gal-3, CK19, TIMP-1, and Ang-1, respectively.ConclusionSerum levels of four of the five proteins were elevated in patients with thyroid masses relative to normal values. However, the difference between benign and PTC was not significant. Two of the markers (Gal-3 & TIMP-1) displayed a greater potential difference, which may warrant further investigation. This study suggests that other serum markers should be sought. This is the first study to investigate potential serum biomarkers based on over-expressed proteins in thyroid cancer versus benign pathology.