Mast Cells Granular Contents Are Crucial for Deep Vein Thrombosis in Mice

Rationale: Deep vein thrombosis (DVT) and its complication pulmonary embolism have high morbidity reducing quality of life and leading to death. Cellular mechanisms of DVT initiation remain poorly understood. Objective: We sought to determine the role of mast cells (MCs) in DVT initiation and validate MCs as a potential target for DVT prevention. Methods and Results: In a mouse model, DVT was induced by partial ligation (stenosis) of the inferior vena cava. We demonstrated that 2 strains of mice deficient for MCs were completely protected from DVT. Adoptive transfer of in vitro differentiated MCs restored thrombosis. MCs were present in the venous wall, and the number of granule-containing MCs decreased with thrombosis. Pharmacological depletion of MCs granules or prevention of MC degranulation also reduced DVT. Basal plasma levels of von Willebrand factor and recruitment of platelets to the inferior vena cava wall after DVT induction were reduced in MC-deficient mice. Stenosis application increased plasma levels of soluble P-selectin in wild-type but not in MC-deficient mice. MC releasate elevated ICAM-1 (intercellular adhesion molecule-1) expression on HUVEC (human umbilical vein endothelial cells) in vitro. Topical application of compound 48/80, an MC secretagogue, or histamine, a Weibel–Palade body secretagogue from MCs, potentiated DVT in wild-type mice, and histamine restored thrombosis in MC-deficient animals. Conclusions: MCs exacerbate DVT likely through endothelial activation and Weibel–Palade body release, which is, at least in part, mediated by histamine. Because MCs do not directly contribute to normal hemostasis, they can be considered potential targets for prevention of DVT in humans

Tissue factor/FVIIa activates Bcl-2 and prevents doxorubicin-induced apoptosis in neuroblastoma cells

<p>Abstract</p> <p>Background</p> <p>Tissue factor (TF) is a transmembrane protein that acts as a receptor for activated coagulation factor VII (FVIIa), initiating the coagulation cascade. Recent studies demonstrate that expression of tumor-derived TF also mediates intracellular signaling relevant to tumor growth and apoptosis. Our present study investigates the possible mechanism by which the interaction between TF and FVIIa regulates chemotherapy resistance in neuroblastoma cell lines.</p> <p>Methods</p> <p>Gene and siRNA transfection was used to enforce TF expression in a TF-negative neuroblastoma cell line and to silence endogenous TF expression in a TF-overexpressing neuroblastoma line, respectively. The expression of TF, Bcl-2, STAT5, and Akt as well as the phosphorylation of STAT5 and Akt in gene transfected cells or cells treated with JAK inhibitor and LY294002 were determined by Western blot assay. Tumor cell growth was determined by a clonogenic assay. Cytotoxic and apoptotic effect of doxorubicin on neuroblastoma cell lines was analyzed by WST assay and annexin-V staining (by flow cytometry) respectively.</p> <p>Results</p> <p>Enforced expression of TF in a TF-negative neuroblastoma cell line in the presence of FVIIa induced upregulation of Bcl-2, leading to resistance to doxorubicin. Conversely, inhibition of endogenous TF expression in a TF-overexpressing neuroblastoma cell line using siRNA resulted in down-regulation of Bcl-2 and sensitization to doxorubicin-induced apoptosis. Additionally, neuroblastoma cells expressing high levels of either endogenous or transfected TF treated with FVIIa readily phosphorylated STAT5 and Akt. Using selective pharmacologic inhibitors, we demonstrated that JAK inhibitor I, but not the PI3K inhibitor LY294002, blocked the TF/FVIIa-induced upregulation of Bcl-2.</p> <p>Conclusion</p> <p>This study shows that in neuroblastoma cell lines overexpressed TF ligated with FVIIa produced upregulation of Bcl-2 expression through the JAK/STAT5 signaling pathway, resulting in resistance to apoptosis. We surmise that this TF-FVIIa pathway may contribute, at least in part, to chemotherapy resistance in neuroblastoma.</p

Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis

<p>Abstract</p> <p>Background</p> <p>Nerve growth factor (NGF) is a potent growth factor that plays a key role in neuronal cell differentiation and may also play a role in hematopoietic differentiation. It has been shown that NGF induced synergistic action for the colony formation of CD34 positive hematopoietic progenitor cells treated with macrophage-colony stimulating factor (M-CSF or CSF-1), or stem cell factor (SCF). However, the exact role of NGF in hematopoietic system is unclear. It is also not clear whether NGF mediated signals in hematopoietic cells are identical to those in neuronal cells.</p> <p>Results</p> <p>To study the signal transduction pathways induced by NGF treatment in hematopoietic cells, we utilized the mastocytoma cell line HMC-1(V560G c-Kit) which expresses the NGF receptor, tropomyosin-receptor-kinase (Trk)A, as well as the constitutively activated SCF receptor, V560G c-Kit, which can be inhibited completely by treatment with the potent tyrosine kinase inhibitor imatinib mesylate (imatinib). NGF rescues HMC-1(V560G c-Kit) cells from imatinib mediated cell death and promotes proliferation. To examine the NGF mediated proliferation and survival in these cells, we compared the NGF mediated upregulated genes (30 and 120 min after stimulation) to the downregulated genes by imatinib treatment (downregulation of c-Kit activity for 4 h) by transcriptome analysis. The following conclusions can be drawn from the microarray data: Firstly, gene expression profiling reveals 50% overlap of genes induced by NGF-TrkA with genes expressed downstream of V560G c-Kit. Secondly, NGF treatment does not enhance expression of genes involved in immune related functions that were down regulated by imatinib treatment. Thirdly, more than 55% of common upregulated genes are involved in cell proliferation and survival. Fourthly, we found Kruppel-like factor (KLF) 2 and Smad family member 7 (SMAD7) as the NGF mediated novel downstream genes in hematopoietic cells. Finally, the downregulation of KLF2 gene enhanced imatinib induced apoptosis.</p> <p>Conclusion</p> <p>NGF does not induce genes which are involved in immune related functions, but induces proliferation and survival signals in HMC-1(V560G c-Kit) cells. Furthermore, the current data provide novel candidate genes, KLF2 and SMAD7 which are induced by NGF/TrkA activation in hematopoietic cells. Since the depletion of KLF2 causes enhanced apoptosis of HMC-1(V560G c-Kit), KLF2 may play a role in the NGF mediated survival signal.</p

Notes for genera: basal clades of Fungi (including Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota)

Compared to the higher fungi (Dikarya), taxonomic and evolutionary studies on the basal clades of fungi are fewer in number. Thus, the generic boundaries and higher ranks in the basal clades of fungi are poorly known. Recent DNA based taxonomic studies have provided reliable and accurate information. It is therefore necessary to compile all available information since basal clades genera lack updated checklists or outlines. Recently, Tedersoo et al. (MycoKeys 13:1--20, 2016) accepted Aphelidiomycota and Rozellomycota in Fungal clade. Thus, we regard both these phyla as members in Kingdom Fungi. We accept 16 phyla in basal clades viz. Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota. Thus, 611 genera in 153 families, 43 orders and 18 classes are provided with details of classification, synonyms, life modes, distribution, recent literature and genomic data. Moreover, Catenariaceae Couch is proposed to be conserved, Cladochytriales Mozl.-Standr. is emended and the family Nephridiophagaceae is introduced

Cre/loxP-based mouse models of mast cell deficiency and mast cell-specific gene inactivation

Over the past decades, research on in vivo functions of mast cells has largely relied on kit-mutant mouse strains. Recently, new mouse models for investigation of mast cell functions based on the Cre/loxP recombination system have been published and results in these new models challenged findings of previous studies in kit-mutant mice. Herein we describe procedures central to mast cell-specific gene inactivation and the generation of mast cell-deficient mice based on the mouse strain Mcpt5-Cre, which expresses Cre recombinase selectively in connective tissue mast cells

Enterococcal Rgg-Like Regulator ElrR Activates Expression of the elrA Operon

The Enterococcus faecalis leucine-rich protein ElrA promotes virulence by stimulating bacterial persistence in macrophages and production of the interleukin-6 (IL-6) cytokine. The ElrA protein is encoded within an operon that is poorly expressed under laboratory conditions but induced in vivo. In this study, we identify ef2687 (renamed elrR), which encodes a member of the Rgg (regulator gene for glucosyltransferase) family of putative regulatory proteins. Using quantitative reverse transcription-PCR, translational lacZ fusions, and electrophoretic mobility shift assays, we demonstrate that ElrR positively regulates expression of elrA. These results correlate with the attenuated virulence of the \u394elrR strain in a mouse peritonitis model. Virulence of simple and double elrR and elrA deletion mutants also suggests a remaining ElrR-independent expression of elrA in vivo and additional virulence-related genes controlled by ElrR