A transgenic zebrafish model expressing KIT-D816V recapitulates features of aggressive systemic mastocytosis.

Systemic mastocytosis (SM) is a rare myeloproliferative disease without curative therapy. Despite clinical variability, the majority of patients harbour a KIT-D816V mutation, but efforts to inhibit mutant KIT with tyrosine kinase inhibitors have been unsatisfactory, indicating a need for new preclinical approaches to identify alternative targets and novel therapies in this disease. Murine models to date have been limited and do not fully recapitulate the most aggressive forms of SM. We describe the generation of a transgenic zebrafish model expressing the human KIT-D816V mutation. Adult fish demonstrate a myeloproliferative disease phenotype, including features of aggressive SM in haematopoeitic tissues and high expression levels of endopeptidases, consistent with SM patients. Transgenic embryos demonstrate a cell-cycle phenotype with corresponding expression changes in genes associated with DNA maintenance and repair, such as reduced dnmt1. In addition, epcam was consistently downregulated in both transgenic adults and embryos. Decreased embryonic epcam expression was associated with reduced neuromast numbers, providing a robust in vivo phenotypic readout for chemical screening in KIT-D816V-induced disease. This study represents the first zebrafish model of a mast cell disease with an aggressive adult phenotype and embryonic markers that could be exploited to screen for novel agents in SM.

Carboxypeptidase A5 identifies a novel mast cell lineage in the zebrafish providing new insight into mast cell fate determination.

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 MC-specific 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 cepbalpha, identifying these early MCs as a distinct myeloid population arising from a common granulocyte/monocyte progenitor. Morpholino "knock-down" 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.

Comparative performance of different methods for circulating tumor cell enrichment in metastatic breast cancer patients.

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.

Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins.

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the Delta 1 and Delta 2 mutants retain their ability to bind substrate, other factor(s), such as decreased access to the substrate binding pocket, may be responsible for the loss of enzyme activity.

Identification of the epitope of a monoclonal antibody that disrupts binding of human transferrin to the human transferrin receptor.

The molecular basis of the transferrin (TF)-transferrin receptor (TFR) interaction is not known. The C-lobe of TF is required to facilitate binding to the TFR and both the N- and C-lobes are necessary for maximal binding. Several mAb have been raised against human transferrin (hTF). One of these, designated F11, is specific to the C-lobe of hTF and does not recognize mouse or pig TF. Furthermore, mAb F11 inhibits the binding of TF to TFR on HeLa cells. To map the epitope for mAb F11, constructs spanning various regions of hTF were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The recombinant fusion proteins were analysed in an iterative fashion by immunoblotting using mAb F11 as the probe. This process resulted in the localization of the F11 epitope to the C1 domain (residues 365-401) of hTF. Subsequent computer modelling suggested that the epitope is probably restricted to a surface patch of hTF consisting of residues 365-385. Mutagenesis of the F11 epitope of hTF to the sequence of either mouse or pig TF confirmed the identity of the epitope as immunoreactivity was diminished or lost. In agreement with other studies, these epitope mapping studies support a role for residues in the C1 domain of hTF in receptor binding.

Novel ubiquitin fusion proteins: ribosomal protein P1 and actin.

Ubiquitin is a small, highly conserved protein found in all eukaryotic cells. Through its covalent attachment to other proteins, ubiquitin regulates numerous important cellular processes including apoptosis, transcription, and the progression of the cell cycle. Ubiquitin expression is unusual: it is encoded and expressed as multimeric head-to-tail repeats (polyubiquitins) that are post-translationally cleaved into monomers, or fused with ribosomal proteins L40 and S27a. The ubiquitin moiety is removed from these fusion proteins, but is thought to act as a chaperone in ribosome biogenesis prior to cleavage. Here we show that the chlorarachniophyte algae express several novel ubiquitin fusion proteins. An expressed sequence tag (EST) survey revealed ubiquitin fusions with an unidentified open reading frame (ORF), ribosomal protein P1 and, most interestingly, actin. Actin is an essential component of the eukaryotic cytoskeleton and is involved in a variety of cellular processes. In other eukaryotes, actin genes only exist as stand-alone ORFs, but in all chlorarachniophytes examined, actin is always encoded as a ubiquitin fusion protein. The variety of ubiquitin fusion proteins in these organisms raises interesting questions about the evolutionary origins of ubiquitin fusions, as well as their possible biochemical functions in other processes, such as cytoskeletal regulation.

Zebrafish mast cells possess an FcɛRI-like receptor and participate in innate and adaptive immune responses.

We previously identified a zebrafish mast cell (MC) lineage and now aim to determine if these cells function analogously in innate and adaptive immunity like their mammalian counterparts. Intraperitoneal (IP) injection of compound 48/80 or live Aeromonas salmonicida resulted in significant MC degranulation evident histologically and by increased plasma tryptase compared with saline-injected controls (p=0.0006, 0.005, respectively). Pre-treatment with ketotifen abrogated these responses (p=0.0004, 0.005, respectively). Cross-reactivity was observed in zebrafish to anti-human high-affinity IgE receptor gamma (FcɛRIγ) and IgE heavy chain-directed antibodies. Whole mount in situ hybridization on 7-day embryos demonstrated co-localization of cpa5, a MC-specific marker, with myd88, a toll-like receptor adaptor, and zebrafish FcɛRI subunit homologs. Zebrafish injected IP with matched dinitrophenyl-sensitized mouse (anti-DNP) IgE and DNP-BSA or trinitrophenyl-sensitized mouse (anti-TNP) IgE and TNP-BSA demonstrated increased plasma tryptase compared with mismatched controls (p=0.03, 0.010, respectively). These results confirm functional conservation and validate the zebrafish model as an in vivo screening tool for novel MC modulating agents.