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Dean D Metcalfe

Publications and source records attributed to Dean D Metcalfe.

At least 55 records · Page 3Linked to original sources
PubMed2003

Identification of Fyn-binding proteins in MC/9 mast cells using mass spectrometry.

Fyn is a Src kinase known to have an essential role in mast cell degranulation induced following aggregation of the high affinity IgE-receptor. Although Fyn possesses SH2 and SH3 protein binding domains, the molecules that interact with Fyn have not been characterized in mast cells. We thus analyzed Fyn-binding proteins in MC/9 mast cells to explore the Fyn-mediated signaling pathway. On mass spectrometric analysis of proteins binding to the SH2 and SH3 domains of Fyn, we identified six proteins that bind to Fyn including vimentin, pyruvate kinase, p62 ras-GAP associated phosphoprotein, SLP-76, HS-1, and FYB. Among these proteins, vimentin and pyruvate kinase have not been shown to bind to Fyn. After IgE-receptor mediated stimulation, binding of vimentin to Fyn was increased; and this interaction was via binding to the SH2, but not the SH3, domain of Fyn. Mast cells from vimentin-deficient mice showed enhanced mediator release and tyrosine phosphorylation of intracellular proteins including NTAL and LAT. The observation that vimentin and pyruvate kinase bind to Fyn provides additional insight into Fyn-mediated signaling pathways, and suggests a critical role for Fyn in mast cell degranulation in interacting with both cytosolic and structural proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2003

17-Allylamino-17-demethoxygeldanamycin (17-AAG) is effective in down-regulating mutated, constitutively activated KIT protein in human mast cells.

Mutations in the proto-oncogene c-kit cause constitutive kinase activity of its product, KIT protein, and are associated with human mastocytosis and gastrointestinal stromal tumors (GISTs). Although currently available tyrosine kinase inhibitors are effective in the treatment of GISTs, there has been limited success in the treatment of mastocytosis. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a benzoquinoid ansamycin antibiotic, which binds to heat shock protein 90 (hsp90) causes destabilization of various hsp90-dependent kinases important in oncogenesis. Treatment with 17-AAG of the mast cell line HMC-1.2, harboring the Asp816Val and Val560Gly KIT mutations, and the cell line HMC-1.1, harboring a single Val560Gly mutation, causes both the level and activity of KIT and downstream signaling molecules AKT and STAT3 to be down-regulated following drug exposure. These data were validated using Cos-7 cells transfected with wild-type and mutated KIT. 17-AAG promotes cell death of both HMC mast cell lines. In addition, neoplastic mast cells isolated from patients with mastocytosis, incubated with 17-AAG ex vivo, are selectively sensitive to the drug compared to the mononuclear fraction. These data provide compelling evidence that 17-AAG may be effective in the treatment of c-kit-related diseases including mastocytosis, GISTs, mast cell leukemia, subtypes of acute myelogenous leukemia, and testicular cancer.

Animals↗
PubMed2003

Molecular remission and reversal of myelofibrosis in response to imatinib mesylate treatment in patients with the myeloproliferative variant of hypereosinophilic syndrome.

We recently described a subset of patients with a myeloproliferative variant of hypereosinophilic syndrome (MHES) characterized by elevated serum tryptase levels, increased atypical mast cells in the bone marrow, tissue fibrosis, and the presence of the fusion tyrosine kinase, FIP1L1-PDGFRalpha, which is a therapeutic target of imatinib mesylate. Seven patients with MHES were treated with imatinib mesylate (300-400 mg daily). Clinical improvement and resolution of eosinophilia was observed in all patients, although cardiac dysfunction, when present, was not altered by therapy. Reversal of bone marrow pathology, including increased cellularity, the presence of spindle-shaped mast cells, and myelofibrosis, was evident in all patients at 4 to 8 weeks following initiation of therapy. This was accompanied by a decrease in activated eosinophils and mast cells in the peripheral blood and bone marrow, respectively. Serum tryptase levels declined rapidly to normal levels in all patients and remained in the normal range throughout therapy. Molecular remission, with disappearance of detectable FIP1L1/PDGFRA (F/P) transcripts, was achieved in 5 of 6 patients tested. The lack of reversal of cardiac abnormalities and persistence of the F/P mutation in some patients suggests that early intervention with higher doses of imatinib mesylate may be desirable in the treatment of patients with MHES.

Adult↗
PubMed2003

The phospholipase C gamma 1-dependent pathway of Fc epsilon RI-mediated mast cell activation is regulated independently of phosphatidylinositol 3-kinase.

Mast cell degranulation following Fc epsilon RI aggregation is generally believed to be dependent on phosphatidylinositide 3-kinase (PI 3-kinase)-mediated phospholipase C (PLC)gamma activation. Here we report evidence that the PLC gamma 1-dependent pathway of Fc epsilon RI-mediated activation of mast cells is independent of PI 3-kinase activation. In primary cultures of human mast cells, Fc epsilon RI aggregation induced a rapid translocation and phosphorylation of PLC gamma 1, and subsequent inositol trisphosphate (IP3) production, which preceded PI 3-kinase-related signals. In addition, although PI 3-kinase-mediated responses were completely inhibited by wortmannin, even at high concentrations, this PI 3-kinase inhibitor had no effect on parameters of Fc epsilon RI-mediated PLC gamma activation, and had little effect on the initial increase in intracellular calcium levels that correlated with PLC gamma activation. Wortmannin, however, did produce a partial (approximately 50%) concentration-dependent inhibition of Fc epsilon RI-mediated degranulation in human mast cells and a partial inhibition of the later calcium response at higher concentrations. Further studies, conducted in mast cells derived from the bone marrow of mice deficient in the p85 alpha and p85 beta subunits of PI 3-kinase, also revealed no defects in Fc epsilon RI-mediated PLC gamma 1 activation. These data are consistent with the conclusion that the PLC gamma-dependent component of Fc epsilon RI-mediated calcium flux leading to degranulation of mast cells is independent of PI 3-kinase. However, PI 3-kinase may contribute to the later phase of Fc epsilon RI-mediated degranulation in human mast cells.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Elevated serum tryptase levels identify a subset of patients with a myeloproliferative variant of idiopathic hypereosinophilic syndrome associated with tissue fibrosis, poor prognosis, and imatinib responsiveness.

Since serum tryptase levels are elevated in some patients with myeloproliferative disorders, we examined their utility in identifying a subset of patients with hypereosinophilic syndrome (HES) and an underlying myeloproliferative disorder. Elevated serum tryptase levels (> 11.5 ng/mL) were present in 9 of 15 patients with HES and were associated with other markers of myeloproliferation, including elevated B12 levels and splenomegaly. Although bone marrow biopsies in these patients showed increased numbers of CD25+ mast cells and atypical spindle-shaped mast cells, patients with HES and elevated serum tryptase could be distinguished from patients with systemic mastocytosis and eosinophilia by their clinical manifestations, the absence of mast cell aggregates, the lack of a somatic KIT mutation, and the presence of the recently described fusion of the Fip1-like 1 (FIP1L1) gene to the platelet-derived growth factor receptor alpha gene (PDGFRA). Patients with HES and elevated serum tryptase were more likely to develop fibroproliferative end organ damage, and 3 of 9 died within 5 years of diagnosis in contrast to 0 of 6 patients with normal serum tryptase levels. All 6 patients with HES and elevated tryptase treated with imatinib demonstrated a clinical and hematologic response. In summary, elevated serum tryptase appears to be a sensitive marker of a myeloproliferative variant of HES that is characterized by tissue fibrosis, poor prognosis, and imatinib responsiveness.

Adolescent↗
PubMed2003

F(ab)'2-mediated neutralization of C3a and C5a anaphylatoxins: a novel effector function of immunoglobulins.

High-dose intravenous immunoglobulin (IVIG) prevents immune damage by scavenging complement fragments C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)2-IVIG and irrelevant human monoclonal antibody. C3a- and C5a-induced thromboxane (TXB2) generation and histamine release from HMC-1 cells and whole-blood basophils were also suppressed by exogenous immunoglobulins. In a mouse model of asthma, immunoglobulin treatment reduced cellular migration to the lung. Lethal C5a-mediated circulatory collapse in pigs was prevented by pretreatment with F(ab)2-IVIG. Molecular modeling, surface plasmon resonance (SPR) and western blot analyses suggested a physical association between anaphylatoxins and the constant region of F(ab)2. This binding could interfere with the role of C3a and C5a in inflammation.

Animals↗
PubMed2003

Comparison of Fc epsilon RI- and Fc gamma RI-mediated degranulation and TNF-alpha synthesis in human mast cells: selective utilization of phosphatidylinositol-3-kinase for Fc gamma RI-induced degranulation.

We have demonstrated that CD34(+) IFN-gamma-treated human mast cells (HuMC) express functional Fc gamma RI and that aggregation of these receptors leads to mediator release. As the signaling pathways linking Fc gamma RI aggregation to mediator release are unknown, we examined Fc gamma RI-dependent activation of specific signal transduction molecules and determined the relative involvement of these events in HuMC degranulation and TNF-alpha production following both Fc gamma RI and Fc epsilon RI aggregation. Fc gamma RI aggregation resulted in the phosphorylation/activation of src kinases and p72(syk) and subsequent tyrosine phosphorylation of multiple substrates. Inhibitor studies revealed that these responses were required for degranulation and TNF-alpha synthesis. Both Fc gamma RI and Fc epsilon RI aggregation also activated the MAP kinases ERK 1/2, JNK and p38 and this was necessary for TNF-alpha synthesis, but not degranulation for both receptors. Thus, signaling events in HuMC following aggregation of Fc gamma RI were generally similar to those observed following Fc epsilon RI aggregation. The one exception was that, although phosphatidylinositol-3-kinase was activated after both Fc epsilon RI and Fc gamma RI aggregation, only the Fc gamma RI appeared to require this molecule for degranulation.

Animals↗
PubMed2003

Gene expression analysis in mastocytosis reveals a highly consistent profile with candidate molecular markers.

BACKGROUND: Mastocytosis is a rare clonal disorder that might be accompanied by non-mast-cell clonal hematologic disorders, such as myeloproliferative or myelodysplastic syndromes. OBJECTIVE: Our aim was to further understand the pathologic basis of mastocytosis and to identify novel molecular markers of disease. METHODS: Microarray analysis was performed on RNA preparations obtained from bone marrow mononuclear cells of patients with mastocytosis. Results were compared with gene expression profiles performed on bone marrow mononuclear cells of healthy subjects. RESULTS: Analysis of gene expression in neoplastic bone marrow tissues revealed highly consistent profiles. One hundred four genes were significantly upregulated, and 64 genes were significantly downregulated in the bone marrow of patients with mastocytosis. The most prominent differentially expressed gene was alpha-tryptase (44.6-fold increase). Also upregulated were genes involved in cell proliferation, neoplastic transformation, and apoptosis. Both hierarchical and K-means clustering analyses identified an identical group of 10 genes highly coordinately overexpressed in patients with mastocytosis, including genes for the mast-cell-associated enzymes alpha- and beta-tryptase and carboxypeptidase A. The expression level of 3 of these 10 genes (alpha-tryptase, the activating transcription factor type 3, and the muscle aponeurotic fibrosarcoma type F oncogene) was significantly correlated with serum tryptase levels, a surrogate marker of disease. CONCLUSION: The data presented in this study reveal significant differences in gene expression in the bone marrow of patients with mastocytosis compared with healthy subjects, demonstrate highly coordinated genes that might contribute to pathology, and identify 3 genes as candidate molecular markers for systemic disease.

Activating Transcription Factor 3↗
PubMed2003

Human dendritic cell 1 and dendritic cell 2 subsets express FcepsilonRI: correlation with serum IgE and allergic asthma.

BACKGROUND: Type 1 dendritic cells (DC1) express the high-affinity IgE receptor (FcepsilonRI); however, the regulation of FcepsilonRI expression by DCs is not well understood. Type 2 DC (DC2) expression of FcepsilonRI has not been demonstrated. OBJECTIVE: We hypothesized that DC2 cellsalso express FcepsilonRI and that expression of FcepsilonRI by the DC1 and DC2 subsets correlates with serum IgE and allergic asthma disease status. METHODS: To test these hypotheses, we quantitated FcepsilonRI alpha chain expression by the peripheral blood precursor DC1 (pDC1) and pDC2 subsets by using flow cytometry. RESULTS: FcepsilonRI was expressed by the pDC1 and pDC2 subsets, as well as tissue DCs from tonsils. Relative FcepsilonRI expression by basophil, pDC1, and pDC2 subsets was 12:6.5:1, respectively. In both pDC subsets, FcepsilonRI expression was significantly greater in allergic asthmatic subjects than in nonatopic control subjects. pDC1 and pDC2 expression of FcepsilonRI was highly correlated to serum IgE concentration. The pDC1, pDC2, and basophil subsets demonstrated a similar magnitude of increase in FcepsilonRI expression relative to changes in serum IgE. CONCLUSIONS: FcepsilonRI expression is characteristic of both the DC1 and DC2 subsets. Furthermore, FcepsilonRI expression by these cells is highly correlated to serum IgE and to basophil FcepsilonRI expression and is greater in subjects with allergic asthma. These data support the concept that novel therapeutic approaches directly targeted at FcepsilonRI expression would affect both the sensitization and the effector phases of the allergen-specific immune response.

Asthma↗
PubMed2003

Aggressive systemic mastocytosis and related mast cell disorders: current treatment options and proposed response criteria.

Aggressive systemic mastocytosis (ASM) is a clonal mast cell disease characterized by progressive growth of neoplastic cells in diverse organs leading to organopathy. The organ-systems most frequently affected are the bone marrow, skeletal system, liver, spleen, and the gastrointestinal tract. Respective clinical findings (so called C-Findings) include cytopenias, osteolysis (or osteoporosis) with pathologic fractures, hepatosplenomegaly with impaired liver function and ascites, and malabsorption. During the past decade several treatment strategies for ASM have been proposed. One promising approach may be combination treatment with interferon-alpha (IFN-alpha) and glucocorticoids. This concept has been based on the notion that systemic mastocytosis involves multilineage hematopoietic progenitors indicating a relationship with myeloproliferative disorders. However, relatively little is known about the quality of responses to IFN-alpha in ASM and the actual response rates. This may be due in part to the fact that disease criteria for ASM have only recently been established, and no response criteria are available. In the current article, we propose surrogate markers and treatment response criteria for patients with ASM. In addition, we have applied these criteria retrospectively to ASM patients described in the available literature. In these analyses, the calculated rate of major response (=complete resolution of C-Findings) in patients treated with IFN-alpha (with or without additional glucocorticoids) amounts to approximately 21%. This confirms clinical activity in some patients for this drug-combination, but also points to the need to search for more effective strategies in the treatment of patients with aggressive mast cell disorders.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2003

Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcepsilonRI or FcgammaRI.

Two novel stem cell factor (SCF) dependent human mast cell lines, designated LAD 1 and 2, were established from bone marrow aspirates from a patient with mast cell sarcoma/leukemia. LAD 1 and 2 cells have the ultrastructural features of human mast cells, and express FcepsilonRI, CD4, 9, 13, 14, 22, 31, 32, 45, 64, 71, 103, 117, 132, CXCR4 (CD184), CCR5 (CD195); and intracytoplasmic histamine, tryptase and chymase. LAD 1 and 2 do not exhibit activating mutations at codon 816 of c-kit. Both LAD 1 and 2 release beta-hexosaminidase following FcepsilonRI or FcgammaRI aggregation. The availability of these cell lines offers an unparalleled circumstance to examine the biology of human mast cells.

Adult↗
PubMed2003

Effects of tyrosine kinase inhibitor STI571 on human mast cells bearing wild-type or mutated c-kit.

OBJECTIVE: STI571 is a tyrosine kinase inhibitor which inhibits the kinase activity of kit, the receptor for stem cell factor (SCF). Because activating mutations of c-kit affecting codon 816 are associated with human mast cell neoplasms, we determined whether STI571 exerted a similar cytotoxic effect on neoplastic and normal human mast cells. METHODS: We investigated the effect of addition of STI571 in increasing concentrations (0.01 to 10 micromolar) to two HMC-1 human mast cell leukemia cell lines carrying two different activating c-kit mutations in codons 816 or 560, as well as the effect of the drug on short-term bone marrow cultures obtained from patients who carry a mutated codon 816 or wild-type c-kit. RESULTS: STI571 failed to inhibit the growth of HMC-1(560,816) cells bearing a codon 816 mutation but effectively suppressed the proliferation of HMC-1(560) carrying c-kit with the wild-type codon 816. STI571 did not induce preferential killing of neoplastic bone marrow mast cells in short-term cultures from patients bearing a codon 816 c-kit mutation. In contrast, STI571 caused a dramatic reduction in mast cells in patients without codon 816 c-kit mutations. CONCLUSION: These results suggest that STI571, while effectively killing mast cells with wild-type c-kit, did not show preferential cytotoxicity to neoplastic human mast cells and thus may not be effective in the treatment of human systemic mastocytosis associated with codon 816 c-kit mutations.

Adult↗
PubMed2003

Mast cell proliferative disorders: current view on variants recognized by the World Health Organization.

The term mastocytosis covers a heterogeneous group of disorders characterized by the abnormal growth and accumulation of MCs in one or more organ systems. Clinical symptoms occur from the release of chemical mediators or pathologic infiltration of MCs. CM typically presents as UP and is a benign disease confined to the skin. In many cases, particularly in children, this disease regresses spontaneously. By contrast, SM is a clonal persistent disease of MC-committed or precommitted hematopoietic progenitors. In most of these patients, the transforming c-kit mutation Asp-816-Val is detectable. The clinical course in SM is variable. Many cases remain in an indolent stage over decades. In a few patients, significant organopathy is found, and this may lead to the diagnosis of aggressive SM. In other patients, AHNMD is diagnosed. MCL is a rare form of SM characterized by leukemic spread of MCs in the marrow and blood and rapid progression. In contrast to indolent SM, patients with MCL or aggressive SM often present without UP-like skin lesions. Patients with indolent SM should be treated with mediator-targeting drugs but not with cytoreductive drugs. By contrast, patients with aggressive SM or MCL are candidates for cytoreductive therapy. Some patients with aggressive SM may benefit from IFNalpha2b. Patients with rapid progression or MCL are candidates for more aggressive treatment, including cladribine, chemotherapy, and stem cell transplantation. In patients with SM-AHNMD, the SM should be treated as if no AHNMD is present, and the AHNMD should be treated as if no SM had been diagnosed.

Bone Marrow↗
PubMed2003

Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-kit proto-oncogene.

The human mast cell line (HMC)-1 cell line is growth-factor independent because of a constitutive activity of the receptor tyrosine kinase Kit. Such deregulated Kit activity has also been suggested causative in gastrointestinal stromal tumours (GISTs) and mastocytosis. HMC-1 is the only established continuously growing human mast cell line and has therefore been widely employed for in vitro studies of human mast cell biology. In this paper we describe two sublines of HMC-1, named HMC-1(560 ) and HMC-1(560,816 ), with different phenotypes and designated by the locations of specific mutations in the c-kit proto-oncogene. Activating mutations in the Kit receptor were characterized using the pyrosequencing trade mark method. Both sublines have a heterozygous T to G mutation at codon 560 in the juxtamembrane region of the c-kit gene causing an amino acid substitution of Gly-560 for Val. In contrast, only HMC-1(560,816) cells have the c-kitV816 mutation found in mast cell neoplasms causing an Asp-->Val substitution in the intracellular kinase domain. Kit was constitutively phosphorylated on tyrosine residues and associated with phosphatidylinositol 3'-kinase (PI 3-kinase) in both variants of HMC-1, but this did not lead to a constitutive phosphorylation of Akt or extracellular regulated protein kinase (ERK), which are signalling molecules normally activated by the interaction of stem cell factor (SCF) with Kit. The documentation and characterization of two sublines of HMC-1 cells provides both information on the biological consequences of mutations in Kit and recognition of the availability of what in reality are two distinct cultured human mast cell lines.

Cell Culture Techniques↗
PubMed2003

4. IgE, mast cells, basophils, and eosinophils.

IgE, mast cells, basophils, and eosinophils constitute essential elements in allergic inflammation. Allergen-specific IgE, synthesized in response to allergens in the environment and in susceptible individuals, becomes fixed to high-affinity receptors on cellular membranes, especially of mast cells and basophils. If these receptor-bound IgE molecules are aggregated on reexposure to specific allergen, these mast cells and basophils produce mediators that result in the allergic response. Principal among the cells drawn to sites of mediator release is the eosinophil.

Animals↗
PubMed2003

Assessment of the extent of cutaneous involvement in children and adults with mastocytosis: relationship to symptomatology, tryptase levels, and bone marrow pathology.

BACKGROUND: Cutaneous involvement occurs in most patients with systemic mastocytosis. OBJECTIVE: We sought to determine whether the extent of cutaneous involvement is predictive of systemic disease. METHODS: In a prospective survey of 48 adults and 19 children, the extent and density of cutaneous lesions were compared with patient history, symptoms, internal organ involvement, serum total mast cell tryptase level, and bone marrow pathology. RESULTS: Cutaneous lesions in children were of a greater mean and maximum diameter, but similar in extent and density compared with lesions in adults. In adults with skin lesions, the extent of lesions correlated to disease duration. Adults with extensive cutaneous disease experienced more pruritus and flushing. Fatigue, splenomegaly, and hepatomegaly were more frequent in adults without cutaneous involvement; and in those with a greater density of lesions and disease duration. Increased tryptase levels were found in children and adults with systemic disease and correlated to skin lesion density and bone marrow pathology. CONCLUSION: An examination of the extent and density of cutaneous lesions in adults helps identify those with more extensive extracutaneous disease and, thus, requiring a more thorough evaluation.

Adolescent↗
PubMed2003

Introduction: what are the issues in addressing the allergenic potential of genetically modified foods?

There is growing concern among the general public and the scientific community regarding the potential toxicity of genetically modified organisms (GMOs). The use of biotechnology to enhance pest resistance or nutritional value has raised a number of fundamental questions including the consequences of insertion of reporter genes, the spread of resistance genes to surrounding plants, and the use of suicide genes to prohibit reuse of seed from engineered plants. Of particular interest is the ability of proteins from GMOs to elicit potentially harmful immunologic responses, including allergic hypersensitivity. The lack of information of the potential toxicity of these products suggests a need to identify the critical issues and research needs regarding these materials and to develop testing strategies to examine the allergenicity of these compounds.

Animals↗