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Find video protocols related to scientific articles indexed in Pubmed.
I?B kinase 2 is essential for IgE-induced mast cell de novo cytokine production but not for degranulation.
Cell Rep
PUBLISHED: 08-28-2014
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The immunoglobulin E (IgE)-mediated mast cell (MC) response is central to the pathogenesis of type I allergy and asthma. I?B kinase 2 (IKK2) was reported to couple IgE-induced signals to MC degranulation by phosphorylating the SNARE protein SNAP23. We investigated MC responses in mice with MC-specific inactivation of IKK2 or NF-?B essential modulator (NEMO), or animals with MC-specific expression of a mutant, constitutively active IKK2. We show that the IgE-induced late-phase cytokine response is reduced in mice lacking IKK2 or NEMO in MCs. However, anaphylactic in vivo responses of these animals are not different from those of control mice, and in vitro IKK2-deficient MCs readily phosphorylate SNAP23 and degranulate similarly to control cells in response to allergen or calcium ionophore. Constitutive overactivation of the NF-?B pathway has only slight effects on allergen-triggered MC responses. Thus, IKK2 is dispensable for MC degranulation, and the important question how IgE-induced signals trigger MC vesicle fusion remains open.
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Mast cells are activated by Staphylococcus aureus in vitro but do not influence the outcome of intraperitoneal S. aureus infection in vivo.
Immunology
PUBLISHED: 03-24-2014
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Staphylococcus aureus is a major pathogen that can cause a broad spectrum of serious infections including skin infections, pneumonia and sepsis. Peritoneal mast cells have been implicated in the host response towards various bacterial insults and to provide mechanistic insight into the role of mast cells in intraperitoneal bacterial infection we here studied the global effects of S. aureus on mast cell gene expression. After co-culture of peritoneal mast cells with live S. aureus we found by gene array analysis that they up-regulate a number of genes. Many of these corresponded to pro-inflammatory cytokines, including interleukin-3, interleukin-13 and tumour necrosis factor-?. The cytokine induction in response to S. aureus was confirmed by ELISA. To study the role of peritoneal mast cells during in vivo infection with S. aureus we used newly developed Mcpt5-Cre(+) × R-DTA mice in which mast cell deficiency is independent of c-Kit. This is in contrast to previous studies in which an impact of mast cells on bacterial infection has been proposed based on the use of mice whose mast cell deficiency is a consequence of defective c-Kit signalling. Staphylococcus aureus was injected intraperitoneally into mast-cell-deficient Mcpt5-Cre(+) × R-DTA mice using littermate mast-cell-sufficient mice as controls. We did not observe any difference between mast-cell-deficient and control mice with regard to weight loss, bacterial clearance, inflammation or cytokine production. We conclude that, despite peritoneal mast cells being activated by S. aureus in vitro, they do not influence the in vivo manifestations of intraperitoneal S. aureus infection.
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Interleukin-31: a novel diagnostic marker of allergic diseases.
Curr Allergy Asthma Rep
PUBLISHED: 02-11-2014
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Interleukin-31 (IL-31) is a newly discovered cytokine associated with chronic skin inflammation and pruritus. Patients with atopic dermatitis, chronic spontaneous urticaria, allergic contact dermatitis, prurigo nodularis, primary cutaneous lymphoma and mastocytosis exhibit increased serum levels of IL-31 protein and elevated IL-31 mRNA in the skin. Interestingly, in some of these diseases, IL-31 serum levels correlate with disease activity. In the present review, we particularly focus on studies investigating IL-31 as a novel diagnostic biomarker indicating the severity of allergic diseases. We highlight a recent study on IL-31 in mastocytosis, which reports on elevated serum levels of IL-31 in adults correlating with the severity of disease categories, tryptase levels and percentage of bone marrow infiltration. We conclude that growing knowledge about IL-31, its receptors and signaling pathways serves to better understand the pathogenesis of allergic diseases and may lead to the development of novel treatment approaches.
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Genetic ablation of mast cells redefines the role of mast cells in skin wound healing and bleomycin-induced fibrosis.
J. Invest. Dermatol.
PUBLISHED: 01-09-2014
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Conclusive evidence for the impact of mast cells (MCs) in skin repair is still lacking. Studies in mice examining the role of MC function in the physiology and pathology of skin regenerative processes have obtained contradictory results. To clarify the specific role of MCs in regenerative conditions, here we used a recently developed genetic mouse model that allows conditional MC ablation to examine MC-specific functions in skin. This mouse model is based on the cell type-specific expression of Cre recombinase in connective tissue-type MCs under control of the Mcpt5 promoter and the Cre-inducible diphtheria toxin receptor-mediated cell lineage ablation by diphtheria toxin. In response to excisional skin injury, genetic ablation of MCs did not affect the kinetics of reepithelialization, the formation of vascularized granulation tissue, or scar formation. Furthermore, genetic ablation of MCs failed to prevent the development of skin fibrosis upon bleomycin challenge. The amount of deposited collagen and the biochemistry of collagen fibril crosslinks within fibrotic lesions were comparable in MC-depleted and control mice. Collectively, our findings strongly suggest that significant reduction of MC numbers does not affect skin wound healing and bleomycin-induced fibrosis in mice, and provide to our knowledge previously unreported insight in the long-debated contribution of MCs in skin regenerative processes.
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Mast cells are dispensable for normal and activin-promoted wound healing and skin carcinogenesis.
J. Immunol.
PUBLISHED: 11-13-2013
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The growth and differentiation factor activin A is a key regulator of tissue repair, inflammation, fibrosis, and tumorigenesis. However, the cellular targets, which mediate the different activin functions, are still largely unknown. In this study, we show that activin increases the number of mature mast cells in mouse skin in vivo. To determine the relevance of this finding for wound healing and skin carcinogenesis, we mated activin transgenic mice with CreMaster mice, which are characterized by Cre recombinase-mediated mast cell eradication. Using single- and double-mutant mice, we show that loss of mast cells neither affected the stimulatory effect of overexpressed activin on granulation tissue formation and reepithelialization of skin wounds nor its protumorigenic activity in a model of chemically induced skin carcinogenesis. Furthermore, mast cell deficiency did not alter wounding-induced inflammation and new tissue formation or chemically induced angiogenesis and tumorigenesis in mice with normal activin levels. These findings reveal that mast cells are not major targets of activin during wound healing and skin cancer development and also argue against nonredundant functions of mast cells in wound healing and skin carcinogenesis in general.
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The KIT D816V expressed allele burden for diagnosis and disease monitoring of systemic mastocytosis.
Ann. Hematol.
PUBLISHED: 10-15-2013
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The activating KIT D816V mutation plays a central role in the pathogenesis, diagnosis, and targeted treatment of systemic mastocytosis (SM). For improved and reliable identification of KIT D816V, we have developed an allele-specific quantitative real-time PCR (RQ-PCR) with an enhanced sensitivity of 0.01-0.1 %, which was superior to denaturing high-performance liquid chromatography (0.5-1 %) or conventional sequencing (10-20 %). Overall, KIT D816 mutations were identified in 146/147 (99 %) of patients (D816V, n?=?142; D816H, n?=?2; D816Y, n?=?2) with SM, including indolent SM (ISM, n?=?63, 43 %), smoldering SM (n?=?8, 5 %), SM with associated hematological non-mast cell lineage disease (SM-AHNMD, n?=?16, 11 %), and aggressive SM/mast cell leukemia?±?AHNMD (ASM/MCL, n?=?60, 41 %). If positive in BM, the KIT D816V mutation was found in PB of all patients with advanced SM (SM-AHNMD, ASM, and MCL) and in 46 % (23/50) of patients with ISM. There was a strong correlation between the KIT D816V expressed allele burden (KIT D816V EAB) with results obtained from DNA by genomic allele-specific PCR and also with disease activity (e.g., serum tryptase level), disease subtype (e.g., indolent vs. advanced SM) and survival. In terms of monitoring of residual disease, qualitative and quantitative assessment of KIT D816V and KIT D816V EAB was successfully used for sequential analysis after chemotherapy or allogeneic stem cell transplantation. We therefore conclude that RQ-PCR assays for KIT D816V are useful complimentary tools for diagnosis, disease monitoring, and evaluation of prognosis in patients with SM.
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Dimethylfumarate induces apoptosis in human mast cells.
Exp. Dermatol.
PUBLISHED: 09-17-2013
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Mast cells modulate autoimmune diseases such as psoriasis and multiple sclerosis. Fumaric acid esters (FAE) are widely used for the treatment of psoriasis and dimethylfumarate (DMF) has recently been approved for multiple sclerosis. In the present study, we analyzed the cytotoxic effect of FAE on human mast cells. Specifically, cell death was analyzed in the human mast cell line HMC-1 and in primary cord blood-derived mast cells (CBMC) after incubation with fumaric acid (FA), monomethylfumarate (MMF), DMF and calcium bis(monomethylfumarate) (Ca-MF). Our data show that only DMF potently induces apoptotic cell death in HMC-1 cells and CBMC. DMF-mediated apoptosis was associated with increased expression of Bax and Bak and activation of caspase-9 and caspase-6. Interestingly, DMF also enhanced the sensitivity of CBMC towards TRAIL- and dexamethasone-induced apoptosis. These findings demonstrate for the first time that DMF induces apoptosis of human mast cells, primarily via the mitochondrial apoptotic pathway. Our study contributes to the understanding of the beneficial effects of FAE in autoimmune diseases and provides a rationale for exploiting FAE for other diseases associated with mast cells. This article is protected by copyright. All rights reserved.
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Zebrafish as a novel model to assess Na(+)/K(+)-ATPase-related neurological disorders.
Neurosci Biobehav Rev
PUBLISHED: 05-30-2013
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Modeling neurological disorders using zebrafish increases rapidly as this model system allows easy access to all developmental stages and imaging of pathological processes. A surprising degree of functional conservation has been demonstrated between human genes implicated in neurodegenerative diseases and their zebrafish orthologues. Zebrafish offers rapid high throughput screening of therapeutic compounds and live imaging of pathogenic mechanisms in vivo. Several recent zebrafish studies functionally assessed the role of the sodium-potassium pump (Na(+)/K(+)-ATPase). The Na(+)/K(+)-ATPase maintains the electrochemical gradients across the plasma membrane, essential for e.g. signaling, secondary active transport, glutamate re-uptake and neuron excitability in animal cells. Na(+)/K(+)-ATPase mutations are associated with neurological disorders, where mutations in the Na(+)/K(+)-ATPase ?2 and ?3 isoforms cause Familial hemiplegic migraine type 2 (FHM2) and Rapid-onset dystonia-parkinsonism (RDP)/Alternating hemiplegic childhood (AHC), respectively. In zebrafish, knock-down of Na(+)/K(+)-ATPase isoforms included skeletal and heart muscle defects, impaired embryonic motility, depolarized Rohon-beard neurons and abrupt brain ventricle development. In this review, we discuss zebrafish as a model to assess Na(+)/K(+)-ATPase isoform functions. Furthermore, studies investigating proteomic changes in both ?2- and ?3-isoform deficient embryos and their potential connections to the Na(+)/K(+)-ATPase functions will be discussed.
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Selective ablation of mast cells or basophils reduces peanut-induced anaphylaxis in mice.
J. Allergy Clin. Immunol.
PUBLISHED: 05-16-2013
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Studies with c-kit mutant mast cell (MC)-deficient mice and antibody-mediated depletion of basophils suggest that both MCs and basophils can contribute to peanut-induced anaphylaxis (PIA). However, interpretation of data obtained by using such approaches is complicated because c-kit mutant mice have several phenotypic abnormalities in addition to MC deficiency and because basophil-depleting antibodies can also react with MCs.
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Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation.
Blood
PUBLISHED: 05-03-2013
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Neutrophil recruitment is an important early step in controlling tissue infections or injury. Here, we report that this influx depends on both tissue-resident mast cells and macrophages. Mice with mast cell deficiency recruit reduced numbers of neutrophils in the first few hours of intraperitoneal lipopolysaccharide (LPS) stimulation. Conversely, in mice with clodronate-ablated macrophages, neutrophils extravasate, but have limited ability to reach the peritoneal fluid. Tissue macrophages synthesize neutrophil chemoattractants CXCL1/CXCL2 (CXC chemokine ligands 1/2) in response to LPS. Mast cells also produce these chemokines of which a proportion are preformed in granules. Release of the granules and new CXCL1/CXCL2 synthesis is Toll-like receptor 4-dependent. Both in vivo studies with blocking monoclonal antibodies and in vitro chemotaxis experiments show the neutrophil response to mast cells and macrophages to be CXCL1/CXCL2-dependent. The data are in keeping with the model that mast cells, optimally positioned in close proximity to the vasculature, initiate an early phase of neutrophil recruitment by releasing the chemoattractants CXCL1/CXCL2. Having arrived within the stimulated tissue, neutrophils penetrate further in a macrophage-dependent manner. Therefore, we demonstrate a positive role for mast cells in tissue inflammation and define how this comes about with contribution from a second tissue cell, the macrophage.
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The dormant and the fully competent oocyte: comparing the transcriptome of human oocytes from primordial follicles and in metaphase II.
Mol. Hum. Reprod.
PUBLISHED: 04-18-2013
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Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human oocyte in the quiescent state and at the pinnacle of maturity at ovulation. In silico bioinformatic comparisons were made between the transcriptome of human oocytes from dormant primordial follicles and that of human metaphase II (MII) oocytes and granulosa cells and unique gene expression profiles were identified as well as functional and pathway enrichments associated with the oocytes from the two developmental hallmarks. A total of 729 genes were highly enriched in oocytes from primodial follicles and 1456 genes were highly enriched in MII oocytes (>10-fold, P < 0.001) representing functional categories such as cell cycle regulation, DNA protection and epigenetics, with representative genes validated by qPCR analysis. Dominating canonical pathways in the oocytes from primordial follicles were androgen, estrogen receptor, glucocorticoid receptor and PI3K/AKT signaling (P < 0.001). In the MII, mitotic roles of polo-like kinases, estrogen receptor, JAK/Stat signaling (P < 0.001) and the ERK/MAPK (P < 0.01) signaling were enriched. Some of the highly differentially expressed genes were completely new in human reproduction (CDR1, TLC1A, UHRF2) while other genes [ABO, FOLR1 (folate receptor), CHRNA3 (nicotine receptor)] may relate to clinical observations as diverse as premature ovarian failure, folic acid deficiency and smoking affecting female fertility. The in silico analysis identified novel reproduction-associated genes and highlighted molecular mechanisms and pathways associated with the unique functions of the human oocyte in its two extremes during folliculogenesis. The data provides a fundamental basis for future functional studies in regulation of human oogenesis.
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?3Na+/K+-ATPase deficiency causes brain ventricle dilation and abrupt embryonic motility in zebrafish.
J. Biol. Chem.
PUBLISHED: 02-11-2013
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Na(+)/K(+)-ATPases are transmembrane ion pumps that maintain ion gradients across the basolateral plasma membrane in all animal cells to facilitate essential biological functions. Mutations in the Na(+)/K(+)-ATPase ?3 subunit gene (ATP1A3) cause rapid-onset dystonia-parkinsonism, a rare movement disorder characterized by sudden onset of dystonic spasms and slow movements. In the brain, ATP1A3 is principally expressed in neurons. In zebrafish, the transcripts of the two ATP1A3 orthologs, Atp1a3a and Atp1a3b, show distinct expression in the brain. Surprisingly, targeted knockdown of either Atp1a3a or Atp1a3b leads to brain ventricle dilation, a likely consequence of ion imbalances across the plasma membrane that cause accumulation of cerebrospinal fluid in the ventricle. The brain ventricle dilation is accompanied by a depolarization of spinal Rohon-Beard neurons in Atp1a3a knockdown embryos, suggesting impaired neuronal excitability. This is further supported by Atp1a3a or Atp1a3b knockdown results where altered responses to tactile stimuli as well as abnormal motility were observed. Finally, proteomic analysis identified several protein candidates highlighting proteome changes associated with the knockdown of Atp1a3a or Atp1a3b. Our data thus strongly support the role of ?3Na(+)/K(+)-ATPase in zebrafish motility and brain development, associating for the first time the ?3Na(+)/K(+)-ATPase deficiency with brain ventricle dilation.
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International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) & European Competence Network on Mastocytosis (ECNM) consensus response criteria in advanced systemic mastocytosis.
Blood
PUBLISHED: 01-16-2013
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Systemic mastocytosis (SM) is characterized by accumulation of neoplastic mast cells and is classified into indolent and aggressive forms. The latter include aggressive SM (ASM), mast cell leukemia (MCL), and SM associated with a myeloid neoplasm wherein 1 or both disease compartments exhibit advanced features. These variants, henceforth collectively referred to as advanced SM for the purposes of this report, are typically characterized by organ damage and shortened survival duration. In contrast to indolent SM, in which symptoms are usually managed by noncytotoxic antimediator therapy, cytoreduction is usually necessary for disease control in advanced SM. Unfortunately, current drug treatment of these patients rarely results in complete clinical and histopathologic remissions or improved survival time. Previously defined response criteria were adapted to the heterogeneous presentations of advanced SM and the limited effects of available drugs. However, recent advances in understanding the molecular pathogenesis of SM and the corresponding prospect in targeted therapy make it a priority to modify these criteria. Our current study is the product of an international group of experts and summarizes the challenges in accomplishing this task and forwards a new proposal for response criteria, which builds on prior proposals and should facilitate response evaluation in clinical trials.
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Perivascular mast cells dynamically probe cutaneous blood vessels to capture immunoglobulin E.
Immunity
PUBLISHED: 01-03-2013
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Mast cells are tissue-resident immune cells that play a central role in allergic disease. These contributions are largely dependent on the acquisition of antigen-specific immunoglobulin E (IgE). Despite this requirement, studies of mast cell and IgE interactions have overlooked the mechanism by which mast cells acquire IgE from the blood. To address this gap, we developed reporter IgE molecules and employed imaging techniques to study mast cell function in situ. Our data demonstrate that skin mast cells exhibit selective uptake of IgE based on perivascular positioning. Furthermore, perivascular mast cells acquire IgE by extending cell processes across the vessel wall to capture luminal IgE. These data demonstrate how tissue mast cells acquire IgE and reveal a strategy by which extravascular cells monitor blood contents to capture molecules central to cellular function.
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Migraine- and dystonia-related disease-mutations of Na+/K+-ATPases: relevance of behavioral studies in mice to disease symptoms and neurological manifestations in humans.
Neurosci Biobehav Rev
PUBLISHED: 05-13-2011
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The two autosomal dominantly inherited neurological diseases: familial hemiplegic migraine type 2 (FHM2) and familial rapid-onset of dystonia-parkinsonism (Familial RDP) are caused by in vivo mutations of specific alpha subunits of the sodium-potassium pump (Na(+)/K(+)-ATPase). Intriguingly, patients with classical FHM2 and RDP symptoms additionally suffer from other manifestations, such as epilepsy/seizures and developmental disabilities. Recent studies of FHM2 and RDP mouse models provide valuable tools for dissecting the vital roles of the Na(+)/K(+)-ATPases, and we discuss their relevance to the complex patient symptoms and manifestations. Thus, it is interesting that mouse models targeting a specific ?-isoform cause different, although still comparable, phenotypes consistent with classical symptoms and other manifestations observed in FHM2 and RDP patients. This review highlights that use of mouse models have broad potentials for future research concerning migraine and dystonia-related diseases, which will contribute towards understanding the, yet unknown, pathophysiologies.
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Distribution of Na/K-ATPase alpha 3 isoform, a sodium-potassium P-type pump associated with rapid-onset of dystonia parkinsonism (RDP) in the adult mouse brain.
J. Comp. Neurol.
PUBLISHED: 03-19-2011
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The Na(+)/K(+)-ATPase1 alpha subunit 3 (ATP1?(3)) is one of many essential components that maintain the sodium and potassium gradients across the plasma membrane in animal cells. Mutations in the ATP1A3 gene cause rapid-onset of dystonia parkinsonism (RDP), a rare movement disorder characterized by sudden onset of dystonic spasms and slowness of movement. To achieve a better understanding of the pathophysiology of the disease, we used immunohistochemical approaches to describe the regional and cellular distribution of ATP1?(3) in the adult mouse brain. Our results show that localization of ATP1?(3) is restricted to neurons, and it is expressed mostly in projections (fibers and punctuates), but cell body expression is also observed. We found high expression of ATP1?(3) in GABAergic neurons in all nuclei of the basal ganglia (striatum, globus pallidus, subthalamic nucleus, and substantia nigra), which is a key circuitry in the fine movement control. Several thalamic nuclei structures harboring connections to and from the cortex expressed high levels of the ATP1?(3) isoform. Other structures with high expression of ATP1?(3) included cerebellum, red nucleus, and several areas of the pons (reticulotegmental nucleus of pons). We also found high expression of ATP1?(3) in projections and cell bodies in hippocampus; most of these ATP1?(3)-positive cell bodies showed colocalization to GABAergic neurons. ATP1?(3) expression was not significant in the dopaminergic cells of substantia nigra. In conclusion, and based on our data, ATP1?(3) is widely expressed in neuronal populations but mainly in GABAergic neurons in areas and nuclei related to movement control, in agreement with RDP symptoms.
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Mild recessive dystrophic epidermolysis bullosa associated with two compound heterozygous COL7A1 mutations.
Eur J Dermatol
PUBLISHED: 03-09-2011
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Dystrophic epidermolysis bullosa is a group of inherited skin blistering disorders caused by mutations in the COL7A1 gene coding for type VII collagen. More than 500 different COL7A1 mutations have been detected in dystrophic epidermolysis bullosa to date. Clarification of genotype-phenotype correlations is of particular importance for the development of novel therapeutic approaches. Here we report a female patient with mild dystrophic epidermolysis bullosa harbouring two compound heterozygous COL7A1 mutations, namely the intronic splice site mutation c.3832-2A?>?G and the glycine substitution p.G1347W. Our data extend the current knowledge on genotype-phenotype correlations in dystrophic epidermolysis bullosa.
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Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.
Immunity
PUBLISHED: 02-03-2011
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A prominent feature of sensitizing environmental compounds that cause allergic contact dermatitis is the rapid induction of an innate inflammatory response that seems to provide danger signals for efficient T cell priming. We generated mouse models of mast cell deficiency, mast cell-specific gene inactivation, and mast cell reporter mice for intravital imaging and showed that these adjuvant effects of contact allergens are mediated by mast cells and histamine. Mast cell deficiency resulted in impaired emigration of skin DCs to the lymph node and contact hypersensitivity was dramatically reduced in the absence of mast cells. In addition, mast cell-specific inactivation of the Il10 gene did not reveal any role for mast cell-derived IL-10 in the regulation of contact allergy. Collectively, we demonstrate that mast cells are essential promoters of contact hypersensitivity, thereby highlighting their potential to promote immune responses to antigens entering via the skin.
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Definitions, criteria and global classification of mast cell disorders with special reference to mast cell activation syndromes: a consensus proposal.
Int. Arch. Allergy Immunol.
PUBLISHED: 01-24-2011
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Activation of tissue mast cells (MCs) and their abnormal growth and accumulation in various organs are typically found in primary MC disorders also referred to as mastocytosis. However, increasing numbers of patients are now being informed that their clinical findings are due to MC activation (MCA) that is neither associated with mastocytosis nor with a defined allergic or inflammatory reaction. In other patients with MCA, MCs appear to be clonal cells, but criteria for diagnosing mastocytosis are not met. A working conference was organized in 2010 with the aim to define criteria for diagnosing MCA and related disorders, and to propose a global unifying classification of all MC disorders and pathologic MC reactions. This classification includes three types of MCA syndromes (MCASs), namely primary MCAS, secondary MCAS and idiopathic MCAS. MCA is now defined by robust and generally applicable criteria, including (1) typical clinical symptoms, (2) a substantial transient increase in serum total tryptase level or an increase in other MC-derived mediators, such as histamine or prostaglandin D(2), or their urinary metabolites, and (3) a response of clinical symptoms to agents that attenuate the production or activities of MC mediators. These criteria should assist in the identification and diagnosis of patients with MCAS, and in avoiding misdiagnoses or overinterpretation of clinical symptoms in daily practice. Moreover, the MCAS concept should stimulate research in order to identify and exploit new molecular mechanisms and therapeutic targets.
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Mast cell hyperplasia, B-cell malignancy, and intestinal inflammation in mice with conditional expression of a constitutively active kit.
Blood
PUBLISHED: 12-09-2010
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Signaling through the receptor tyrosine kinase kit controls proliferation and differentiation of hematopoietic precursor cells and mast cells. Somatic point mutations of the receptor that constitutively activate kit signaling are associated with mastocytosis and various hematopoietic malignancies. We generated a Cre/loxP-based bacterial artificial chromosome transgenic mouse model that allows conditional expression of a kit gene carrying the kitD814V mutation (the murine homolog of the most common mutation in human mastocytosis, kitD816V) driven by the kit promoter. Expression of the mutant kit in cells of adult mice, including hematopoietic precursors, caused severe mastocytosis with 100% penetrance at young age frequently associated with additional hematopoietic (mostly B lineage-derived) neoplasms and focal colitis. Restriction of transgene expression to mature mast cells resulted in a similar mast cell disease developing with slower kinetics. Embryonic expression led to a hyperproliferative dysregulation of the erythroid lineage with a high rate of perinatal lethality. In addition, most adult animals developed colitis associated with mucosal mast cell accumulation. Our findings demonstrate that the effects of constitutive kit signaling critically depend on the developmental stage and the state of differentiation of the cell hit by the gain-of-function mutation.
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The chromosome passenger complex is required for fidelity of chromosome transmission and cytokinesis in meiosis of mouse oocytes.
J. Cell. Sci.
PUBLISHED: 12-03-2010
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The existence of two forms of the chromosome passenger complex (CPC) in the mammalian oocyte has meant that its role in female meiosis has remained unclear. Here we use loss- and gain-of function approaches to assess the meiotic functions of one of the shared components of these complexes, INCENP, and of the variable kinase subunits, Aurora B or Aurora C. We show that either the depletion of INCENP or the combined inhibition of Aurora kinases B and C activates the anaphase-promoting complex or cyclosome (APC/C) before chromosomes have properly congressed in meiosis I and also prevents cytokinesis and hence extrusion of the first polar body. Overexpression of Aurora C also advances APC/C activation and results in cytokinesis failure in a high proportion of oocytes, indicative of a dominant effect on CPC function. Together, this points to roles for the meiotic CPC in functions similar to the mitotic roles of the complex: correcting chromosome attachment to microtubules, facilitating the spindle-assembly checkpoint (SAC) function and enabling cytokinesis. Surprisingly, overexpression of Aurora B leads to a failure of APC/C activation, stabilization of securin and consequently a failure of chiasmate chromosomes to resolve - a dominant phenotype that is completely suppressed by depletion of INCENP. Taken together with the differential distribution of Aurora proteins B and C on chiasmate chromosomes, this points to differential functions of the two forms of CPC in regulating the separation of homologous chromosomes in meiosis I.
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Early developmental expression of Mus musculus zinc finger RNA-binding protein compared to orthologs in Caenorhabditis elegans and Danio rerio and subcellular localization of Mus musculus and Caenorhabditis elegans zinc finger RNA-binding protein in 2-cel
DNA Cell Biol.
PUBLISHED: 11-22-2010
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In mouse, knock-out of the Zfr gene encoding the zinc finger RNA-binding protein (ZFR) is associated with early lethality during gastrulation, suggesting that a pool of maternally contributed Zfr mRNA might compensate to allow development. ZFR is an ancient and highly conserved chromosome-associated protein from nematodes to mammals. We characterized expression of the Zfr transcript during early development in Mus musculus, Danio rerio, and Caenorhabditis elegans by quantitative real-time polymerase chain reaction. Mouse Zfr mRNA was detected in all stages tested during mouse preimplantation, with higher levels at the 1-cell stage that includes the maternal contribution of Zfr mRNA. In D. rerio, Zfr mRNA expression was highest in unfertilized eggs and declines throughout development. In C. elegans, Zfr mRNA expression was barely detectable in the fertilized egg and the L1 stage, but increased in the adult organism. Microinjections of green fluorescent protein (GFP)-tagged versions of in vitro-transcribed mouse and C. elegans Zfr mRNAs into early mouse embryos allowed analysis of the intracellular localization of the protein. Mouse ZFR-GFP was localized in the nucleus in 2-cell stage embryos although absent from nucleoli. Deletion studies revealed that this nuclear localization required the C-terminal part of ZFR, as deletion of the C-terminal resulted in the localization to the nuclear membrane. Despite the lack of a conserved nuclear localization signal, the C. elegans ZFR-GFP fusion protein also displayed an intranuclear localization in the 2-cell mouse embryo.
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Mastocytosis - an update.
J Dtsch Dermatol Ges
PUBLISHED: 07-29-2010
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Mastocytosis (MC) encompasses a range of disorders characterized by a clonal, pathological accumulation of mast cells having a somatic activating mutation of the tyrosine kinase receptor Kit (exon 17, codon 816; D816V) in more than 90 % of adult patients. The mutation is much less common in children. Skin and bone marrow are most often affected. Symptoms and clinical course are very heterogeneous due to a variable degree of local or systemic mediator release or organ dysfunction as a result of mast cell infiltrates. Pruritus, wheals, flushing and gastrointestinal symptoms are often reported. The majority of pediatric patients experience spontaneous remission of MC. Adults usually have chronic disease, rarely transforming into an aggressive or lethal type. Indolent systemic MC with involvement of skin and bone is the most common type. In MC the risk for anaphylactic reactions following an insect sting (and other causes of mast cell activation) is increased significantly. Diagnostic hallmarks are biopsies from skin and bone marrow using tryptase antibodies for staining as well as serum tryptase levels. At present a curative treatment for MC is not available. Systemic histamine H(1) receptor antagonists are widely used. Aggressive types of MC respond partially to IFN-alpha or cladribine. A variety of receptor tyrosine kinase inhibitors is still under critical evaluation for systemic treatment of MC. After introduction of the WHO classification for MC and the development a German MC guideline, as well as the foundation of national and international competence networks for MC, a significantly improved quality of medical care for MC patients can be expected for the future.
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Localization and differential expression of the Krüppel-associated box zinc finger proteins 1 and 54 in early mouse development.
DNA Cell Biol.
PUBLISHED: 07-14-2010
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Upon fertilization, the zygotic genome is activated. To ensure the transcription of specific genes and avoid promiscuous gene expression, a chromatin-mediated repressive state is established. To characterize potential heterochromatin factors present during the first cleavage, two putative transcriptional repressors, zinc finger protein (ZFP1) and ZFP54, belonging to the Krüppel-associated box (KRAB) zinc finger family, were isolated. ZFP1 and ZFP54 contain an N-terminally located KRAB repressor domain followed by 8 and 12 repeats of Krüppel zinc-finger motifs, respectively. Reverse transcription (RT) and quantitative (q) PCR show that maternally contributed Zfp1 and Zfp54 mRNA are detected throughout preimplantation development. α-Amanitin-treated zygotes revealed that maternal Zfp1 and Zfp54 are fully degraded at the two-cell stage. Microinjections of in vitro-transcribed mRNA encoding a gfp-fused reporter gene into zygotes demonstrated the intracellular distribution of ZFP1-green fluorescent protein (GFP) and ZFP54-GFP colocalized with a DNA marker in the two-cell embryo. The KRAB domain was essential to colocalize with DNA, and deletion of the KRAB domain in ZFP1-GFP and ZFP54-GFP localized in nucleoli and in a ubiquitously manner, respectively. Taken together, this suggests a role for ZFP1 and ZFP54 in transcriptional regulation in early development.
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The receptor tyrosine kinase c-Kit controls IL-33 receptor signaling in mast cells.
Blood
PUBLISHED: 03-03-2010
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Members of the Toll/interleukin-1 receptor (TIR) family are of importance for host defense and inflammation. Here we report that the TIR-family member interleukin-33R (IL-33R) cross-activates the receptor tyrosine kinase c-Kit in human and murine mast cells. The IL-33R-induced activation of signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase B (PKB), and Jun NH(2)-terminal kinase 1 (JNK1) depends on c-Kit and is required to elicit optimal effector functions. Costimulation with the c-Kit ligand stem cell factor (SCF) is necessary for IL-33-induced cytokine production in primary mast cells. The structural basis for this cross-activation is the complex formation between c-Kit, IL-33R, and IL-1R accessory protein (IL-1RAcP). We found that c-Kit and IL-1RAcP interact constitutively and that IL-33R joins this complex upon ligand binding. Our findings support a model in which signals from seemingly disparate receptors are integrated for full cellular responses.
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European Competence Network on Mastocytosis (ECNM): 10-year jubilee, update, and future perspectives.
Wien. Klin. Wochenschr.
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The European Competence Network on Mastocytosis (ECNM) was initiated in 2002 as a multidisciplinary and multinational cooperative approach to increase awareness and to improve diagnosis and therapy of mastocytosis. The network is composed of local centers, physicians, and scientists who have dedicated their work to patients with mastocytosis. A strategic goal of the ECNM is to provide the best available information about the disease to patients and physicians. During the past 10 years, the ECNM has expanded to various countries and contributed successfully to the development of markers, definitions, and standards in the field of mastocytosis. Members of the ECNM organized Annual Meetings in Europe and two Working Conferences on Mastocytosis in Vienna (in 2005 and 2010), and initiated and supported several preclinical and clinical trials. In all these activities, representatives of the ECNM cooperate closely with their US colleagues, with patient-organizations in Europe and in the USA, and with other scientific networks. The ECNM also launched a mastocytosis registry that has been activated in 2012. Using the central database of this registry, cooperative multicenter studies, which should include sufficient numbers of patients and robust evaluations, will be conducted. These studies will increase our knowledge about optimal management and therapy of patients with mastocytosis in the future.
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The ?2Na+/K+-ATPase is critical for skeletal and heart muscle function in zebrafish.
J. Cell. Sci.
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The Na(+)/K(+)-ATPase generates ion gradients across the plasma membrane, essential for multiple cellular functions. In mammals, four different Na(+)/K(+)-ATPase ?-subunit isoforms are associated with characteristic cell-type expression profiles and kinetics. We found the zebrafish ?2Na(+)/K(+)-ATPase associated with striated muscles and that knockdown causes a significant depolarization of the resting membrane potential in slow-twitch fibers of skeletal muscles. Abrupt mechanosensory responses were observed in ?2Na(+)/K(+)-ATPase-deficient embryos, possibly linked to a postsynaptic defect. The ?2Na(+)/K(+)-ATPase deficiency reduced the heart rate and caused a loss of left-right asymmetry in the heart tube. Similar phenotypes from knockdown of the Na(+)/Ca(2+) exchanger indicated a role for the interplay between these two proteins in the observed phenotypes. Furthermore, proteomics identified up- and downregulation of specific phenotype-related proteins, such as parvalbumin, CaM, GFAP and multiple kinases, thus highlighting a potential proteome change associated with the dynamics of ?2Na(+)/K(+)-ATPase. Taken together, our findings show that zebrafish ?2Na(+)/K(+)-ATPase is important for skeletal and heart muscle functions.
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Anti-Fas/CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) differentially regulate apoptosis in normal and neoplastic human basophils.
Leuk. Lymphoma
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Basophilia is associated with allergic and parasitic diseases and advanced chronic myeloid leukemia. In the present study, we characterized the expression and function of the death receptors Fas/CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors in basophils from healthy donors compared to neoplastic basophils. Peripheral blood basophils obtained from healthy donors (HD-PBB) and from patients with chronic myeloid leukemia (CML-PBB) were found to express high levels of Fas/CD95 and low levels of TRAIL-R2, whereas the basophil-like chronic myeloid leukemia cell line KU-812 expressed significant levels of TRAIL-R1 and TRAIL-R2. HD-PBB underwent apoptosis in response to anti-Fas/CD95, but showed resistance to TRAIL, unless they were co-treated with actinomycin D. Interestingly, CML-PBB and KU-812 cells exhibited the opposite response pattern with resistance to anti-Fas/CD95, but significant susceptibility to TRAIL-induced apoptosis. Our data show that anti-Fas/CD95 and TRAIL differentially regulate apoptosis of normal and neoplastic human basophils, which may direct the development of novel therapeutic strategies.
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Mast cells play a protumorigenic role in primary cutaneous lymphoma.
Blood
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Primary cutaneous lymphomas (PCLs) are clonal T- or B-cell neoplasms, which originate in the skin. In recent years, mast cells were described as regulators of the tumor microenvironment in different human malignancies. Here, we investigated the role of mast cells in the tumor microenvironment of PCL. We found significantly increased numbers of mast cells in skin biopsies from patients with cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma (CBCL). Mast cell infiltration was particularly prominent in the periphery, at lymphoma rims. Interestingly, CTCL and CBCL patients with a progressive course showed higher mast cell counts than stable patients, and mast cell numbers in different stages of CTCL correlated positively with disease progression. In addition, mast cell numbers positively correlated with microvessel density. Incubating primary CTCL cells with mast cell supernatant, we observed enhanced proliferation and production of cytokines. In line with our in vitro experiments, in a mouse model of cutaneous lymphoma, tumor growth in mast cell-deficient transgenic mice was significantly decreased. Taken together, these experiments show that mast cells play a protumorigenic role in CTCL and CBCL. Our data provide a rationale for exploiting tumor-associated mast cells as a prognostic marker and therapeutic target in PCL.
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SH2 domain-containing phosphatase 2 is a critical regulator of connective tissue mast cell survival and homeostasis in mice.
Mol. Cell. Biol.
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Mast cells require KIT receptor tyrosine kinase signaling for development and survival. Here, we report that SH2 domain-containing phosphatase 2 (SHP2) signaling downstream of KIT is essential for mast cell survival and homeostasis in mice. Using a novel mouse model with shp2 deletion within mature mast cells (MC-shp2 knockout [KO]), we find that SHP2 is required for the homeostasis of connective tissue mast cells. Consistently with the loss of skin mast cells, MC-shp2 KO mice fail to mount a passive late-phase cutaneous anaphylaxis response. To better define the phenotype of shp2-deficient mast cells, we used an inducible shp2 knockout approach in bone marrow-derived mast cells (BMMCs) or cultured peritoneal mast cells and found that SHP2 promotes mast cell survival. We show that SHP2 promotes KIT signaling to extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase and downregulation of the proapoptotic protein Bim in BMMCs. Also, SHP2-deficient BMMCs failed to repopulate mast cells in mast cell-deficient mice. Silencing of Bim partially rescued survival defects in shp2-deficient BMMCs, consistent with the importance of a KIT ? SHP2 ? Ras/ERK pathway in suppressing Bim and promoting mast cell survival. Thus, SHP2 is a key node in a mast cell survival pathway and a new potential therapeutic target in diseases involving mast cells.
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Transcription factor E3, a major regulator of mast cell-mediated allergic response.
J. Allergy Clin. Immunol.
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Microphthalmia transcription factor, an MiT transcription family member closely related to transcription factor E3 (TFE3), is essential for mast cell development and survival. TFE3 was previously reported to play a role in the functions of B and T cells; however, its role in mast cells has not yet been explored.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.