Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity.

PubMed

Feyerabend, Thorsten B; Weiser, Anne; Tietz, Annette; Stassen, Michael; Harris, Nicola; Kopf, Manfred; Radermacher, Peter; Möller, Peter; Benoist, Christophe; Mathis, Diane; Fehling, Hans Jörg; Rodewald, Hans-Reimer

2011-11-23

Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy. Copyright © 2011 Elsevier Inc. All rights reserved.

C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways

PubMed Central

Al-Maqtari, Tareq; Cao, Pengxiao; Keith, Matthew C. L.; Wysoczynski, Marcin; Zhao, John; Moore IV, Joseph B.; Bolli, Roberto

2015-01-01

A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit. PMID:26474484

Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers

PubMed Central

Roberts, Kathryn G.; Smith, Amanda M.; McDougall, Fiona; Carpenter, Helen; Horan, Martin; Neviani, Paolo; Powell, Jason A.; Thomas, Daniel; Guthridge, Mark A.; Perrotti, Danilo; Sim, Alistair T.R.; Ashman, Leonie K.; Verrills, Nicole M.

2010-01-01

Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors (GIST), systemic mastocytosis, and some acute myeloid leukemias (AML). Whilst juxtamembrane mutations commonly detected in GIST are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study we show that myeloid cells expressing activated c-KIT mutants that are imatinib-sensitive (V560G) or –resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with reduced expression of PP2A structural (A) and regulatory subunits (B55α; B56α; B56γ and B56δ). Overexpression of PP2A-Aα in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacological activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential and induced apoptosis of mutant c-KIT+ cells, whilst having no effect on WT c-KIT cells or empty vector controls. FTY720 treatment caused dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5 and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT-mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT+ cancers. PMID:20551067

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

PubMed

Reith, A D; Ellis, C; Lyman, S D; Anderson, D M; Williams, D E; Bernstein, A; Pawson, T

1991-09-01

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.

A change in structural integrity of c-Kit mutant D816V causes constitutive signaling.

PubMed

Raghav, Pawan Kumar; Singh, Ajay Kumar; Gangenahalli, Gurudutta

2018-03-01

Several signaling pathways, ligands, and genes that regulate proliferative and self-renewal properties of the Hematopoietic Stem Cells (HSCs) have been studied meticulously. One of the signaling pathways that play a crucial role in the process of hematopoiesis is the Stem Cell Factor (SCF) mediated c-Kit pathway. The c-Kit is a Receptor Tyrosine Kinase (RTK), which is expressed in the cells including HSCs. It undergoes dimerization upon binding with its cognate ligand SCF. As a result, phosphorylation of the Juxtamembrane (JM) domain of c-Kit takes place at Tyr568 and Tyr570 residues. These phosphorylated residues become the docking sites for protein tyrosine phosphatases (PTPs) namely SHP-1 and SHP-2, which in turn cause dephosphorylation and negative regulation of the downstream signaling responsible for the cell proliferation. Interestingly, it has been reported that the mutation of c-Kit at D816V makes it independent of SCF stimulation and SHP-1/SHP-2 inhibition, thereby, causing its constitutive activation. The present study was commenced to elucidate the structural behavior of this mutation in the JM and A-loop region of c-Kit using Molecular Dynamics (MD) simulations of the wild-type and mutant c-Kit in unphosphorylated and phosphorylated states. The energy difference computed between the wild type and mutant (D816V) c-Kit, and protein-protein docking and complex analysis revealed the impact of this single residue mutation on the integrity dynamics of c-Kit that makes it independent of SHP-1/SHP-2 negative regulation. Copyright © 2018 Elsevier B.V. All rights reserved.

Resveratrol Improved the Progression of Chronic Prostatitis via the Downregulation of c-kit/SCF by Activating Sirt1.

PubMed

He, Yi; Zeng, Huizhi; Yu, Yang; Zhang, Jiashu; Zeng, Xiaona; Gong, Fengtao; Duan, Xingping; Liu, Qi; Yang, Bo

2017-07-19

The regulation mechanism of inflammation inducing prostate carcinogenesis remains largely unknown. Therefore, we investigated the role of the c-kit/SCF pathway, which has been associated with the control of prostate carcinogenesis, in chronic prostatitis (CP) rats and evaluated the anti-prostatitis effect of resveratrol. We performed hemolysin and eosin staining to evaluate the histopathological changes in prostates. Multiple approaches evaluated the expression levels of c-kit, stem cell factor (SCF), Sirt1, and carcinogenesis-associated proteins. The CP group exhibited severe diffuse chronic inflammation. Meanwhile, the prostate cells appeared atypia; the activity of c-kit/SCF was upregulated, and carcinogenesis-associated proteins are dysregulated significantly in CP rats. Resveratrol treatment significantly improved these factors by Sirt1 activation. In summary, CP could further cause prostate carcinogenesis, which may be associated with activated c-kit/SCF signaling. Resveratrol treatment could improve the progression of CP via the downregulation of c-kit/SCF by activating Sirt1.

GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells

PubMed Central

Barroeta Seijas, Amairelys Belen; Simonetti, Sonia; Vitale, Sara; Runci, Daniele; Quinci, Angela Caterina; Soriani, Alessandra; Criscuoli, Mattia; Filippi, Irene; Naldini, Antonella; Sacchetti, Federico Maria; Tarantino, Umberto; Oliva, Francesco; Piccirilli, Eleonora; Santoni, Angela; Di Rosa, Francesca

2017-01-01

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity. PMID:28261209

Growth control of genetically modified cells using an antibody/c-Kit chimera.

PubMed

Kaneko, Etsuji; Kawahara, Masahiro; Ueda, Hiroshi; Nagamune, Teruyuki

2012-05-01

Gene therapy has been regarded as an innovative potential treatment against serious congenital diseases. However, applications of gene therapy remain limited, partly because its clinical success depends on therapeutic gene-transduced cells acquiring a proliferative advantage. To address this problem, we have developed the antigen-mediated genetically modified cell amplification (AMEGA) system, which uses chimeric receptors to enable the selective proliferation of gene-transduced cells. In this report, we describe mimicry of c-Kit signaling and its application to the AMEGA system. We created an antibody/c-Kit chimera in which the extracellular domain of c-Kit is replaced with an anti-fluorescein single-chain Fv antibody fragment and the extracellular D2 domain of the erythropoietin receptor. A genetically modified mouse pro-B cell line carrying this chimera showed selective expansion in the presence of fluorescein-conjugated BSA (BSA-FL) as a growth inducer. By further engineering the transmembrane domain of the chimera to reduce interchain interaction we attained stricter ligand-dependency. Since c-Kit is an important molecule in the expansion of hematopoietic stem cells (HSCs), this antibody/c-Kit chimera could be a promising tool for gene therapy targeting HSCs. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

AMD3100 treatment attenuates pulmonary angiogenesis by reducing the c-kit (+) cells and its pro-angiogenic activity in CBDL rat lungs.

PubMed

Shen, Cheng-Cheng; Chen, Bing; Gu, Jian-Teng; Ning, Jiao-Lin; Zeng, Jing; Yi, Bin; Lu, Kai-Zhi

2018-03-01

Recent studies have shown that pulmonary angiogenesis is an important pathological process in the development of hepatopulmonary syndrome (HPS), and growing evidence has indicated that Stromal cell-derived factor 1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4) axis is involved in pulmonary vascular disease by mediating the accumulation of c-kit+ cells. This study aimed to test the effect of AMD3100, an antagonist of CXCR4, in HPS pulmonary angiogenesis. Common bile duct ligation (CBDL) rats were used as experimental HPS model and were treated with AMD3100 (1.25mg/kg/day, i.p.) or 0.9% saline for 3weeks. The sham rats underwent common bile duct exposure without ligation. The c-kit+ cells accounts and its angiogenic-related functions, prosurvival signals, pulmonary angiogenesis and arterial oxygenation were analysed in these groups. Our results showed that pulmonary SDF-1/CXCR4, Akt, Erk and VEGF/VEGFR2 were significantly activated in CBDL rats, and the numbers of circulating and pulmonary c-kit+ cells were increased in CBDL rats compared with control rats. Additionally, the angiogenic-related functions of c-kit+ cells and pulmonary microvessel counts were also elevated in CBDL rats. CXCR4 inhibition reduced pulmonary c-kit+ cells and microvessel counts and improved arterial oxygenation within 3weeks in CBDL rats. The pulmonary prosurvival signals and pro-angiogenic activity of c-kit+ cells were also down-regulated in AMD3100-treated rats. In conclusion, AMD3100 treatment attenuated pulmonary angiogenesis in CBDL rats and prevented the development of HPS via reductions in pulmonary c-kit+ cells and inhibition of the prosurvival signals. Our study provides new insights in HPS treatment. Copyright © 2017. Published by Elsevier B.V.

Selective tyrosine kinase inhibition by imatinib mesylate for the treatment of autoimmune arthritis

PubMed Central

Paniagua, Ricardo T.; Sharpe, Orr; Ho, Peggy P.; Chan, Steven M.; Chang, Anna; Higgins, John P.; Tomooka, Beren H.; Thomas, Fiona M.; Song, Jason J.; Goodman, Stuart B.; Lee, David M.; Genovese, Mark C.; Utz, Paul J.; Steinman, Lawrence; Robinson, William H.

2006-01-01

Tyrosine kinases play a central role in the activation of signal transduction pathways and cellular responses that mediate the pathogenesis of rheumatoid arthritis. Imatinib mesylate (imatinib) is a tyrosine kinase inhibitor developed to treat Bcr/Abl-expressing leukemias and subsequently found to treat c-Kit–expressing gastrointestinal stromal tumors. We demonstrate that imatinib potently prevents and treats murine collagen-induced arthritis (CIA). We further show that micromolar concentrations of imatinib abrogate multiple signal transduction pathways implicated in RA pathogenesis, including mast cell c-Kit signaling and TNF-α release, macrophage c-Fms activation and cytokine production, and fibroblast PDGFR signaling and proliferation. In our studies, imatinib attenuated PDGFR signaling in fibroblast-like synoviocytes (FLSs) and TNF-α production in synovial fluid mononuclear cells (SFMCs) derived from human RA patients. Imatinib-mediated inhibition of a spectrum of signal transduction pathways and the downstream pathogenic cellular responses may provide a powerful approach to treat RA and other inflammatory diseases. PMID:16981009

FUNCTIONAL DEREGULATION OF KIT: LINK TO MAST CELL PROLIFERATIVE DISEASES AND OTHER NEOPLASMS

PubMed Central

Cruse, Glenn; Metcalfe, Dean D.; Olivera, Ana

2014-01-01

SYNOPSIS Signaling through the receptor tyrosine kinase KIT mediates differentiation, proliferation and survival of hematopoietic precursor cells and mast cells. Constitutive KIT signaling due to somatic point mutations in c-Kit is an important occurrence in the development of mast cell proliferation disorders and other hematological malignancies. In this review, we discuss the common gain-of-function mutations found in these malignancies, particularly in mast cell proliferation disorders, and summarize the current understanding of the molecular mechanisms by which transforming point mutations in KIT may affect KIT structure and function and lead to altered downstream signaling and cellular transformation. Drugs targeting KIT have shown mixed success in the treatment of these diseases. A brief overview of the most common KIT inhibitors currently used, the reasons for the varied clinical results of such inhibitors and a discussion of potential new strategies are provided. PMID:24745671

Distinct tissue site-specific requirements of mast cells and complement components C3/C5a receptor in IgG immune complex-induced injury of skin and lung.

PubMed

Baumann, U; Chouchakova, N; Gewecke, B; Köhl, J; Carroll, M C; Schmidt, R E; Gessner, J E

2001-07-15

We induced the passive reverse Arthus reaction to IgG immune complexes (IC) at different tissue sites in mice lacking C3 treated or not with a C5aR-specific antagonist, or in mice lacking mast cells (Kit(W)/Kit(W-v) mice), and compared the inflammatory responses with those in the corresponding wild-type mice. We confirmed that IC inflammation of skin can be mediated largely by mast cells expressing C5aR and FcgammaRIII. In addition, we provided evidence for C3-independent C5aR triggering, which may explain why the cutaneous Arthus reaction develops normally in C3(-/-) mice. Furthermore, some, but not all, of the acute changes associated with the Arthus response in the lung were significantly more intense in normal mice than in C3(-/-) or Kit(W)/Kit(W-v) mice, indicating for C3- and mast cell-dependent and -independent components. Finally, we demonstrated that C3 contributed to the elicitation of neutrophils to alveoli, which corresponded to an increased synthesis of TNF-alpha, macrophage-inflammatory protein-2, and cytokine-induced neutrophil chemoattractant. While mast cells similarly influenced alveolar polymorphonuclear leukocyte influx, the levels of these cytokines remained largely unaffected in mast cell deficiency. Together, the phenotypes of C3(-/-) mice and Kit(W)/Kit(W-v) mice suggest that complement and mast cells have distinct tissue site-specific requirements acting by apparently distinct mechanisms in the initiation of IC inflammation.

Characterization of various types of mast cells derived from model mice of familial gastrointestinal stromal tumors with KIT-Asp818Tyr mutation

PubMed Central

Kajimoto, Noriko; Nakai, Norihiro; Ohkouchi, Mizuka; Hashikura, Yuka; Liu-Kimura, Ning-Ning; Isozaki, Koji; Hirota, Seiichi

2015-01-01

Sporadic mast cell neoplasms and gastrointestinal stromal tumors (GISTs) often have various types of somatic gain-of-function mutations of the c-kit gene which encodes a receptor tyrosine kinase, KIT. Several types of germline gain-of-function mutations of the c-kit gene have been detected in families with multiple GISTs. All three types of model mice for the familial GISTs with germline c-kit gene mutations at exon 11, 13 or 17 show development of GIST, while they are different from each other in skin mast cell number. Skin mast cell number in the model mice with exon 17 mutation was unchanged compared to the corresponding wild-type mice. In the present study, we characterized various types of mast cells derived from the model mice with exon 17 mutation (KIT-Asp818Tyr) corresponding to human familial GIST case with human KIT-Asp820Tyr to clarify the role of the c-kit gene mutation in mast cells. Bone marrow-derived cultured mast cells (BMMCs) derived from wild-type mice, heterozygotes and homozygotes were used for the experiments. Immortalized BMMCs, designated as IMC-G4 cells, derived from BMMCs of a homozygote during long-term culture were also used. Ultrastructure, histamine contents, proliferation profiles and phosphorylation of various signaling molecules in those cells were examined. In IMC-G4 cells, presence of additional mutation(s) of the c-kit gene and effect of KIT inhibitors on both KIT autophosphorylation and cell proliferation were also analyzed. We demonstrated that KIT-Asp818Tyr did not affect ultrastructure and proliferation profiles but did histamine contents in BMMCs. IMC-G4 cells had an additional novel c-kit gene mutation of KIT-Tyr421Cys which is considered to induce neoplastic transformation of mouse mast cells and the mutation appeared to be resistant to a KIT inhibitor of imatinib but sensitive to another KIT inhibitor of nilotinib. IMC-G4 cells might be a useful mast cell line to investigate mast cell biology. PMID:26722383

Proliferation of murine c-kit(pos) cardiac stem cells stimulated with IGF-1 is associated with Akt-1 mediated phosphorylation and nuclear export of FoxO3a and its effect on downstream cell cycle regulators.

PubMed

Johnson, Ann Mary; Kartha, C C

2014-04-01

Insulin-like growth factor-1 (IGF-1) is known to promote proliferation in many cell types including c-kit(pos) cardiac stem cells (CSCs). Downstream signaling pathways of IGF-1 induced CSC proliferation have not been investigated. An important downstream target of IGF-1/Akt-1 signaling is FoxO3a, a key negative regulator of cell-cycle progression. We studied the effect of IGF-1 on proliferation of c-kit(pos) murine CSCs and found that IGF-1-mediated cell proliferation is associated with FoxO3a phosphorylation and inactivation of its transcriptional activity. PI3 inhibitors LY294002 and Wortmannin abolished the effect of IGF-1 on FoxO3a phosphorylation indicating that FoxO3a phosphorylation is mediated by PI3/Akt-1 pathway. In cells with FoxO3a translocation to the cytoplasm, there is decreased expression of cell-cycle inhibitors such as p27(kip1) and p57(kip2) and increased expression of CyclinD1. Our study provides evidence that IGF-1 induced CSC proliferation could be the result of FoxO3a inactivation and its downstream effect on cell-cycle regulators.

A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets

PubMed Central

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-01-01

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways. PMID:25475013

A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

PubMed

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-12-05

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

Molecular Characterization of PDGFR-α/PDGF-A and c-KIT/SCF in Gliosarcomas

PubMed Central

Reis, Rui M.; Martins, Albino; Ribeiro, Susana A.; Basto, Diana; Longatto-Filho, Adhemar; Schmitt, Fernando C.; Lopes, José M.

2005-01-01

Gliosarcomas are rare and poorly characterized malignant brain tumors that exhibit a biphasic tissue pattern with areas of gliomatous and sarcomatous differentiation. These tumors are histological variants of glioblastoma, displaying a similar genetic profile and dismal prognosis. Up-regulation of PDGFR subfamily of tyrosine kinase members, PDGFR-α and c-Kit, and their intracellular effectors RAS/RAF/MAPK has a crucial role in the cancer development. In addition, signal transduction mediated by activating mutations of c-Kit and PDGFR can be effectively blocked by specific tyrosine kinase inhibitors, such as Imatinib mesylate. The aim of this study was to characterize the molecular alterations of PDGFR signaling in gliosarcomas. Six cases were analyzed by immunohistochemistry for the expression of PDGFR-α, c-Kit and their ligands PDGF-A and SCF, respectively. The cases were further evaluated for the presence of activating mutations of PDGFR-α (exons 12 and 18) and c-kit (exons 9, 11, 13, and 17), as well as B-RAF (exons 11 and 15). Expression of PDGF-A was found in all cases and co-expression of PDGFR-α was observed in three cases. Four cases showed expression of SCF, and c-Kit was observed only in one case that also expressed SCF. Generally, immunoreaction predominates in the glial component. The mutational analysis of PDGFR-α showed the presence of an IVS17-50insT intronic insertion in two cases, one of them also with a 2472C > T silent mutation; this silent mutation was also found in another case. Glioma cell line analysis of IVS17-50insT insertion showed no influence on PDGFR-α gene splicing. No mutations were detected in c-kit and B-RAF oncogenes. Our Results indicate that activating mutations of PDGFR-α, c-kit and B-RAF are absent in gliosarcomas. Nevertheless, the presence of a PDGFR-a/PDGFA and c-Kit/SCF autocrine/paracrine stimulation loop in a proportion of cases, supports the potential role of specific tyrosine kinase inhibitors in the treatment of gliosarcomas. PMID:16373964

SLAM family member 8 is involved in oncogenic KIT-mediated signaling in human mastocytosis.

PubMed

Sugimoto, Akihiko; Kataoka, Tatsuki R; Ueshima, Chiyuki; Takei, Yusuke; Kitamura, Kyohei; Hirata, Masahiro; Nomura, Takashi; Haga, Hironori

2018-03-02

The signaling lymphocytic activation molecule family member 8 (SLAMF8)/CD353 is a member of the CD2 family of proteins. Its ligand has not been identified. SLAMF8 is expressed by macrophages and suppresses cellular functions. No study has yet explored SLAMF8 expression or function in human mastocytosis, which features oncogenic KIT-mediated proliferation of human mast cells. SLAMF8 protein was expressed in human mastocytosis cells, immunohistochemically. SLAMF8 expression was also evident in the human mast cell lines, HMC1.2 (expressing oncogenic KIT) and LAD2 (expressing wild-type KIT) cells. SLAMF8-knockdown significantly reduced the KIT-mediated growth of HMC1.2 cells but not that of LAD2 cells. SLAMF8-knockdown HMC1.2 cells exhibited significant attenuation of SHP-2 activation and oncogenic KIT-mediated RAS-RAF-ERK signaling. An interaction between SLAMF8 and SHP-2 was confirmed in HMC1.2 cells and all pathological mastocytosis specimens examined (19 of 19 cases, 100%). Thus, SLAMF8 is involved in oncogenic KIT-mediated RAS-RAF-ERK signaling and the subsequent growth of human neoplastic mast cells mediated by SHP-2. SLAMF8 is a possible therapeutic target in human mastocytosis patients. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Megakaryocytes compensate for Kit insufficiency in murine arthritis.

PubMed

Cunin, Pierre; Penke, Loka R; Thon, Jonathan N; Monach, Paul A; Jones, Tatiana; Chang, Margaret H; Chen, Mary M; Melki, Imene; Lacroix, Steve; Iwakura, Yoichiro; Ware, Jerry; Gurish, Michael F; Italiano, Joseph E; Boilard, Eric; Nigrovic, Peter A

2017-05-01

The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell-deficient murine lines: KitWsh/Wsh, which develops robust arthritis, and KitW/Wv, which does not. Reciprocal bone marrow transplantation between KitW/Wv and KitWsh/Wsh mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In KitW/Wv mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/megakaryocyte markers NF-E2 and glycoprotein VI. In KitW/Wv mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in KitW/Wv mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1-dependent manner. Transfer of WT but not IL-1-deficient megakaryocytes restored arthritis susceptibility to KitW/Wv mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1-driven systemic inflammatory disease.

Allelic Heterogeneity at the Equine KIT Locus in Dominant White (W) Horses

PubMed Central

Haase, Bianca; Brooks, Samantha A; Schlumbaum, Angela; Azor, Pedro J; Bailey, Ernest; Alaeddine, Ferial; Mevissen, Meike; Burger, Dominik; Poncet, Pierre-André; Rieder, Stefan; Leeb, Tosso

2007-01-01

White coat color has been a highly valued trait in horses for at least 2,000 years. Dominant white (W) is one of several known depigmentation phenotypes in horses. It shows considerable phenotypic variation, ranging from ∼50% depigmented areas up to a completely white coat. In the horse, the four depigmentation phenotypes roan, sabino, tobiano, and dominant white were independently mapped to a chromosomal region on ECA 3 harboring the KIT gene. KIT plays an important role in melanoblast survival during embryonic development. We determined the sequence and genomic organization of the ∼82 kb equine KIT gene. A mutation analysis of all 21 KIT exons in white Franches-Montagnes Horses revealed a nonsense mutation in exon 15 (c.2151C>G, p.Y717X). We analyzed the KIT exons in horses characterized as dominant white from other populations and found three additional candidate causative mutations. Three almost completely white Arabians carried a different nonsense mutation in exon 4 (c.706A>T, p.K236X). Six Camarillo White Horses had a missense mutation in exon 12 (c.1805C>T, p.A602V), and five white Thoroughbreds had yet another missense mutation in exon 13 (c.1960G>A, p.G654R). Our results indicate that the dominant white color in Franches-Montagnes Horses is caused by a nonsense mutation in the KIT gene and that multiple independent mutations within this gene appear to be responsible for dominant white in several other modern horse populations. PMID:17997609

A new human mast cell line expressing a functional IgE receptor converts to tumorigenic growth by KIT D816V transfection.

PubMed

Saleh, Rosine; Wedeh, Ghaith; Herrmann, Harald; Bibi, Siham; Cerny-Reiterer, Sabine; Sadovnik, Irina; Blatt, Katharina; Hadzijusufovic, Emir; Jeanningros, Sylvie; Blanc, Catherine; Legarff-Tavernier, Magali; Chapiro, Elise; Nguyen-Khac, Florence; Subra, Frédéric; Bonnemye, Patrick; Dubreuil, Patrice; Desplat, Vanessa; Merle-Béral, Hélène; Willmann, Michael; Rülicke, Thomas; Valent, Peter; Arock, Michel

2014-07-03

In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF) -dependent human MC line, ROSA(KIT WT), expressing a fully functional immunoglobulin E (IgE) receptor. Transfection with KIT D816V converted ROSA(KIT WT) cells into an SCF-independent clone, ROSA(KIT D816V), which produced a mastocytosis-like disease in NSG mice. Although several signaling pathways were activated, ROSA(KIT D816V) did not exhibit an increased, but did exhibit a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSA(KIT D816V)-derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSA(KIT D816V) may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients. © 2014 by The American Society of Hematology.

SRC-like adaptor protein 2 (SLAP2) is a negative regulator of KIT-D816V-mediated oncogenic transformation.

PubMed

Rupar, Kaja; Moharram, Sausan A; Kazi, Julhash U; Rönnstrand, Lars

2018-04-23

KIT is a receptor tyrosine kinase (RTK) involved in several cellular processes such as regulation of proliferation, survival and differentiation of early hematopoietic cells, germ cells and melanocytes. Activation of KIT results in phosphorylation of tyrosine residues in the receptor, and recruitment of proteins that mediate downstream signaling and also modulate receptor signaling. Here we show that the SRC-like adaptor protein 2 (SLAP2) binds to wild-type KIT in a ligand-dependent manner and is furthermore found constitutively associated with the oncogenic mutant KIT-D816V. Peptide fishing analysis mapped pY568 and pY570 as potential SLAP2 association sites in KIT, which overlaps with the SRC binding sites in KIT. Expression of SLAP2 in cells expressing the transforming mutant KIT-D816V led to reduced cell viability and reduced colony formation. SLAP2 also partially blocked phosphorylation of several signal transduction molecules downstream of KIT such as AKT, ERK, p38 and STAT3. Finally, SLAP2 expression enhanced ubiquitination of KIT and its subsequent degradation. Taken together, our data demonstrate that SLAP2 negatively modulates KIT-D816V-mediated transformation by enhancing degradation of the receptor.

Broadening the Notion of Text: An Exploration of an Artistic Composing Process.

ERIC Educational Resources Information Center

Smagorinsky, Peter; Coppock, John

In language arts classes a "composition" generally refers to a written text. Semiotic theory based on C. S. Peirce's work suggests that writing is only one of many forms of composition available for mediating thought and activity. According to J. V. Wertsch (1991), writing should be one tool in a tool kit of mediational means available…

Data on quantification of signaling pathways activated by KIT and PDGFRA mutants.

PubMed

Bahlawane, Christelle; Schmitz, Martine; Letellier, Elisabeth; Arumugam, Karthik; Nicot, Nathalie; Nazarov, Petr V; Haan, Serge

2016-12-01

The present data are related to the article entitled "Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling" (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot, P.V. Nazarov, S. Haan, 2016) [1]. Constitutive and ligand-derived signaling pathways mediated by KIT and PDGFRA mutated proteins found in gastrointestinal stromal tumors (GIST) were compared. Expression of mutant proteins was induced by doxycycline in an isogenic background (Hek293 cells). Kit was identified by FACS at the cell surface and found to be quickly degraded or internalized upon SCF stimulation for both Kit Wild type and Kit mutant counterparts. Investigation of the main activated pathways in GIST unraveled a new feature specific for oncogenic KIT mutants, namely their ability to be further activated by Kit ligand, the stem cell factor (scf). We were also able to identify the MAPK pathway as the most prominent target for a common inhibition of PDGFRA and KIT oncogenic signaling. Western blotting and micro-array analysis were applied to analyze the capacities of the mutant to induce an effective STATs response. Among all Kit mutants, only Kit Ex11 deletion mutant was able to elicit an effective STATs response whereas all PDGFRA were able to do so.

XK-related protein 5 (XKR5) is a novel negative regulator of KIT/D816V-mediated transformation.

PubMed

Sun, Jianmin; Thingholm, Tine; Højrup, Peter; Rönnstrand, Lars

2018-06-18

In order to investigate the molecular mechanisms by which the oncogenic mutant KIT/D816V causes transformation of cells, we investigated proteins that selectively bind KIT/D816V, but not wild-type KIT, as potential mediators of transformation. By mass spectrometry several proteins were identified, among them a previously uncharacterized protein denoted XKR5 (XK-related protein 5), which is related to the X Kell blood group proteins. We could demonstrate that interaction between XKR5 and KIT/D816V leads to phosphorylation of XKR5 at Tyr 369, Tyr487, and Tyr 543. Tyrosine phosphorylated XKR5 acts as a negative regulator of KIT signaling, which leads to downregulation of phosphorylation of ERK, AKT, and p38. This led to reduced proliferation and colony forming capacity in semi-solid medium. Taken together, our data demonstrate that XKR5 is a novel type of negative regulator of KIT-mediated transformation.

Diagnosis of Clostridium difficile-associated disease: examination of multiple algorithms using toxin EIA, glutamate dehydrogenase EIA and loop-mediated isothermal amplification.

PubMed

Bamber, A I; Fitzsimmons, K; Cunniffe, J G; Beasor, C C; Mackintosh, C A; Hobbs, G

2012-01-01

The laboratory diagnosis of Clostridium difficile infection (CDI) needs to be accurate and timely to ensure optimal patient management, infection control and reliable surveillance. Three methods are evaluated using 810 consecutive stool samples against toxigenic culture: CDT TOX A/B Premier enzyme immunoassay (EIA) kit (Meridian Bioscience, Europe), Premier EIA for C. difficile glutamate dehydrogenase (GDH) (Meridian Bioscience, Europe) and the Illumigene kit (Meridian Bioscience, Europe), both individually and within combined testing algorithms. The study revealed that the CDT TOX A/B Premier EIA gave rise to false-positive and false-negative results and demonstrated poor sensitivity (56.47%), compared to Premier EIA for C. difficile GDH (97.65%), suggesting this GDH EIA can be a useful negative screening method. Results for the Illumigene assay alone showed sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of 91.57%, 98.07%, 99.03% and 84.44%, respectively. A two-stage algorithm using Premier EIA for C. difficile GDH/Illumigene assay yielded superior results compared with other testing algorithms (91.57%, 98.07%, 99.03% and 84.44%, respectively), mirroring the Illumigene performance. However, Illumigene is approximately half the cost of current polymerase chain reaction (PCR) methods, has a rapid turnaround time and requires no specialised skill base, making it an attractive alternative to assays such as the Xpert C. difficile assay (Cepheid, Sunnyvale, CA). A three-stage algorithm offered no improvement and would hamper workflow.

Clinicopathological characteristics of KIT and protein kinase C-δ expression in adenoid cystic carcinoma: comparison with chromophobe renal cell carcinoma and gastrointestinal stromal tumour.

PubMed

Park, Cheol Keun; Kim, Won Kyu; Kim, Hoguen

2017-10-01

KIT overexpression is frequently observed in adenoid cystic carcinomas (AdCCs), chromophobe renal cell carcinomas (ChRCCs), and gastrointestinal stromal tumours (GISTs). Persistent KIT activation has been reported to be mediated by protein kinase C (PKC)-δ in a subset of colon cancers with wild-type KIT overexpression, and by PKC-θ in GISTs with mutant KIT overexpression. To elucidate the clinical implications of PKC-δ and PKC-θ expression in KIT-expressing tumours, we investigated the expression of KIT, PKC-δ and PKC-θ in AdCCs and ChRCCs in comparison with GISTs. KIT expression, PKC-δ expression and PKC-θ expression were analysed in whole sections from 41 AdCCs, 40 ChRCCs and 56 GISTs by immunohistochemistry. Membranous expression of KIT was found in 34 AdCCs and all ChRCCs, whereas cytoplasmic expression of KIT was found in 46 GISTs. In AdCCs, PKC-δ expression was associated with histological grade (P = 0.049), lymphovascular invasion (P = 0.004), perineural invasion (P = 0.002), and KIT positivity (P = 0.002). PKC-δ positivity was associated with shorter relapse-free survival (RFS) (P = 0.017) and a tendency for there to be shorter overall survival (OS) (P = 0.090) in patients with AdCCs. No clinicopathological associations were observed between PKC-δ and KIT expression in ChRCCs. In GISTs, PKC-θ expression was associated with higher mitotic count (P = 0.011) and high grade according to the modified National Institutes of Health criteria (P < 0.001). PKC-θ positivity was associated with shorter RFS (P = 0.016) and a tendency for there to be shorter OS (P = 0.051) in patients with GISTs. PKC-δ expression is associated with KIT expression and the prognosis of patients with AdCCs, suggesting that PKC-δ may be a potential therapeutic target for AdCCs. © 2017 The Authors. Histopathology published by John Wiley & Sons Ltd.

Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling

PubMed Central

Ho, Chia Chi M.; Chhabra, Akanksha; Starkl, Philipp; Schnorr, Peter-John; Wilmes, Stephan; Moraga, Ignacio; Kwon, Hye-Sook; Gaudenzio, Nicolas; Sibilano, Riccardo; Wehrman, Tom S.; Gakovic, Milica; Sockolosky, Jonathan T.; Tiffany, Matthew R.; Ring, Aaron M.; Piehler, Jacob; Weissman, Irving L.; Galli, Stephen J.; Shizuru, Judith A.; Garcia, K. Christopher

2017-01-01

SUMMARY Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion, but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems. PMID:28283060

Inhibition of MicroRNA-23 Contributes to the Isoflurane-Mediated Cardioprotection Against Oxidative Stress.

PubMed

Liu, Hai-Jian; Liu, Bin

2018-04-07

Isoflurane is one of the most frequently used volatile anesthetics in clinical practice for inhalational anesthesia. It is widely studied that isoflurane mediates cardioprotection during multiple pathological processes. However, the precise mechanisms have not been fully elucidated. Neonatal cardiomyocytes were isolated and cultured, followed by treatments with isoflurane at 0, 50, 100 or 200 µM. Rat cardiomyoblast cell line, H9c2, was treated with H 2 O 2 . Expression of miR-23 was measured by qRT-PCR. The cell survival rate of H9c2 in response to H 2 O 2 treatments was evaluated by MTT assay. The ROS and GSH/GSSG levels were measured using Superoxide Detection Kit and GSH/GSSG Ratio Detection Assay Kit. In this study, we report an isoflurane-miR-23-antioxidant axis in cardiomyocyte. We observed that miR-23 was suppressed by isoflurane treatments at 50, 100 or 200 µM. Moreover, cardiomyocyte with isoflurane exposure was insensitive to H 2 O 2 treatment in vitro. Inhibition of miR-23 protected cardiomyocyte against oxidative stress induced by H 2 O 2 treatments at 30, 60, 90 or 120 µM. In addition, overexpression of miR-23 induced ROS generation over twofolds and rendered cardiomyocyte sensitive to H 2 O 2 treatments. We demonstrate that miR-23 inhibited intracellular GSH, an antioxidant against oxidative stress. Our results reveal that with isoflurane exposure, overexpression of miR-23 rendered cardiomyocyte sensitive to H 2 O 2 treatments at 20, 30, 40, 50 µM. Pretreatments with GSH in miR-23 overexpressing cells rescued the cell death under oxidative stress. In summary, our results illustrate that the isoflurane-mediated protection of cardiomyocytes under oxidative stress is through inhibition of miR-23. This study provides an aspect for the miRNAs-modulated cardiomyocyte sensitivity to oxidative stress, contributing to the development of therapeutic agents.

Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers.

PubMed

Heidari, Banafsheh; Rahmati-Ahmadabadi, Maryam; Akhondi, Mohammad Mehdi; Zarnani, Amir Hassan; Jeddi-Tehrani, Mahmood; Shirazi, Abolfazl; Naderi, Mohammad Mehdi; Behzadi, Bahareh

2012-10-01

Presently the techniques for making transgenic animals are cumbersome, required costly instruments and trained man-power. The ability of spermatogonial stem cells (SSCs) to integrate foreign genes has provided the opportunity for developing alternate methods for generation of transgenic animals. One of the big challenges in this field is development of the methods to identify and purify donor SSCs by antibody mediated cell sorting. The present study was aimed to identify goat subpopulations of SSCs using polyclonal antibodies against PGP9.5 and c-kit molecular markers as well as the growth characteristics of SSCs during short term culture. One month old goats' testicular samples were subjected for immunohistochemical and immunocytochemical evaluations. The enzymatically isolated SSCs were cultured in DMEM plus FCS supplemented with (treatment) or without (control) growth factors (GDNF, LIF, FGF, and EGF) for 2 weeks. At the end of culture the morphological characteristics of SSCs colonies and immunocytochemical staining were evaluated. The number and size of colonies in treatment groups were significantly (P < 0.01) higher than corresponding values in controls. The presence of PGP 9.5 and c-kit antigens was confirmed in immunocytochemical evaluation. In immunocytochemical evaluation, the proportion of c-kit and PGP9.5 positive cells were significantly (P < 0.001) higher in control and treatment groups, respectively. The presence of PGP9.5 and c-kit antigens was confirmed in goat SSCs. Moreover, culture medium supplementation with growth factors could effectively retain the undifferentiation status of SSCs, reflected as a higher population of PGP9.5 positive cells, after short term culture.

A de novo mutation in KIT causes white spotting in a subpopulation of German Shepherd dogs.

PubMed

Wong, A K; Ruhe, A L; Robertson, K R; Loew, E R; Williams, D C; Neff, M W

2013-06-01

Although variation in the KIT gene is a common cause of white spotting among domesticated animals, KIT has not been implicated in the diverse white spotting observed in the dog. Here, we show that a loss-of-function mutation in KIT recapitulates the coat color phenotypes observed in other species. A spontaneous white spotting observed in a pedigree of German Shepherd dogs was mapped by linkage analysis to a single locus on CFA13 containing KIT (pairwise LOD = 15). DNA sequence analysis identified a novel 1-bp insertion in the second exon that co-segregated with the phenotype. The expected frameshift and resulting premature stop codons predicted a severely truncated c-Kit receptor with presumably abolished activity. No dogs homozygous for the mutation were recovered from multiple intercrosses (P = 0.01), suggesting the mutation is recessively embryonic lethal. These observations are consistent with the effects of null alleles of KIT in other species. © 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.

Cadmium exposure in newborn rats ovary induces developmental disorders of primordial follicles and the differential expression of SCF/c-kit gene.

PubMed

Zhang, Wenchang; Wu, Tingting; Zhang, Chenyun; Luo, Lingfeng; Xie, Meimei; Huang, Huiling

2017-10-05

Since the 1990s, the rising problem that gonad reproductive toxicity on adult female after exposing to cadmium (Cd), an environmental endocrine disruptor, has attracted high attention at home and abroad,and was systematically studied. Our research focuses on a further problem is that early cadmium exposure (during birth to before puberty) impact on development and function of ovarian cells and its possible mechanism. Our research focuses on the changes of ovarian cells growth and development after the newborn rat ovaries with cadmium exposure in vitro, and different expression of ovarian cells development-related factors, SCF/c-kit and changes of their DNA methylation status. We obtained ovaries from 4-day-old SD rats and cultured them in DMEM/F12 mixed with α-MEM media in vitro. Different doses of cadmium were designed as control, 0.5, 5, 10 and 50μM, and then the constituent ratio of ovarian follicle and follicular oocytes diameter were observed with microscope after 4-h exposure. We found that the increased constituent ratio of original follicle and decreased diameter of all levels of follicular oocytes(compared with control, with statistically significant differences, P<0.01).After the measurement of expression of SCF/c-kit by qRT-PCR and Western Blotting, the mRNA and protein expression of SCF/c-kit in ovarian were both decreased. We further found that the increased constituent ratio of growth follicle and increased diameter of oocytes under the treatment of adding SCF in cell culture media. Finally, MALDI-TOF-MS method showed DNA-low methylation status of SCF/c-kit promoter region after Cd exposure. Overall, we concluded that the exposure of cadimium (5-50μM) on newborn rats ovaries could inhibit follicle development.SCF/c-kit system might mediate follicle development damage caused by cadmium, which is associated with DNA hypomethylation of SCF/c-kit promoter region may be worthy of further study. Copyright © 2017. Published by Elsevier B.V.

Tumor Necrosis Factor-α Is Required for Mast Cell-Mediated Host Immunity Against Cutaneous Staphylococcus aureus Infection.

PubMed

Liu, Chao; Ouyang, Wei; Xia, Jingyan; Sun, Xiaoru; Zhao, Liying; Xu, Feng

2018-06-05

Mast cells (MCs) play a key role in immune process response to invading pathogens. This study assessed the involvement of MCs in controlling Staphylococcus aureus infection in a cutaneous infection model of MC-deficient (KitW-sh/W-sh) mice. KitW-sh/W-sh mice developed significantly larger skin lesions after the cutaneous S. aureus challenge, when compared to wild-type (WT) mice, while MC dysfunction reduced the inflammation response to S. aureus. The levels of tumor necrosis factor (TNF)-α in skin tissues were significantly decreased in KitW-sh/W-sh mice upon infection. Moreover, the exogenous administration of MCs or recombinant TNF-α effectively restored the immune response against S. aureus in KitW-sh/W-sh mice via the recruitment of neutrophils to the infected site. These results indicate that the effects of MC deficiency are largely attributed to the decrease in production of TNF-α in cutaneous S. aureus infection. In addition, S. aureus-induced MC activation was dependent on the c-kit receptor-activated phosphoinositide 3-kinase (PI3K)/AKT/P65-nuclear factor (NF-κB) pathway, which was confirmed by treatment with Masitinib (a c-kit receptor inhibitor), Wortmannin (a PI3K inhibitor), and pyrrolidine dithiocarbamate (a NF-κB inhibitor), respectively. The present study identifies the critical role of MCs in the host defense against S. aureus infection.

Ischemia-reperfusion injury and pregnancy initiate time-dependent and robust signs of up-regulation of cardiac progenitor cells.

PubMed

Genead, Rami; Fischer, Helene; Hussain, Alamdar; Jaksch, Marie; Andersson, Agneta B; Ljung, Karin; Bulatovic, Ivana; Franco-Cereceda, Anders; Elsheikh, Elzafir; Corbascio, Matthias; Smith, C I Edvard; Sylvén, Christer; Grinnemo, Karl-Henrik

2012-01-01

To explore how cardiac regeneration and cell turnover adapts to disease, different forms of stress were studied for their effects on the cardiac progenitor cell markers c-Kit and Isl1, the early cardiomyocyte marker Nkx2.5, and mast cells. Adult female rats were examined during pregnancy, after myocardial infarction and ischemia-reperfusion injury with/out insulin like growth factor-1(IGF-1) and hepatocyte growth factor (HGF). Different cardiac sub-domains were analyzed at one and two weeks post-intervention, both at the mRNA and protein levels. While pregnancy and myocardial infarction up-regulated Nkx2.5 and c-Kit (adjusted for mast cell activation), ischemia-reperfusion injury induced the strongest up-regulation which occurred globally throughout the entire heart and not just around the site of injury. This response seems to be partly mediated by increased endogenous production of IGF-1 and HGF. Contrary to c-Kit, Isl1 was not up-regulated by pregnancy or myocardial infarction while ischemia-reperfusion injury induced not a global but a focal up-regulation in the outflow tract and also in the peri-ischemic region, correlating with the up-regulation of endogenous IGF-1. The addition of IGF-1 and HGF did boost the endogenous expression of IGF and HGF correlating to focal up-regulation of Isl1. c-Kit expression was not further influenced by the exogenous growth factors. This indicates that there is a spatial mismatch between on one hand c-Kit and Nkx2.5 expression and on the other hand Isl1 expression. In conclusion, ischemia-reperfusion injury was the strongest stimulus with both global and focal cardiomyocyte progenitor cell marker up-regulations, correlating to the endogenous up-regulation of the growth factors IGF-1 and HGF. Also pregnancy induced a general up-regulation of c-Kit and early Nkx2.5+ cardiomyocytes throughout the heart. Utilization of these pathways could provide new strategies for the treatment of cardiac disease.

Radotinib induces high cytotoxicity in c-KIT positive acute myeloid leukemia cells.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-06-05

Previously, we reported that radotinib, a BCR-ABL1 tyrosine kinase inhibitor, induced cytotoxicity in acute myeloid leukemia (AML) cells. However, the effects of radotinib in the subpopulation of c-KIT-positive AML cells were unclear. We observed that low-concentration radotinib had more potent cytotoxicity in c-KIT-positive cells than c-KIT-negative cells from AML patients. To address this issue, cell lines with high c-KIT expression, HEL92.1.7, and moderate c-KIT expression, H209, were selected. HEL92.1.7 cells were grouped into intermediate and high c-KIT expression populations. The cytotoxicity of radotinib against the HEL92.1.7 cell population with intermediate c-KIT expression was not different from that of the population with high c-KIT expression. When H209 cells were grouped into c-KIT expression-negative and c-KIT expression-positive populations, radotinib induced cytotoxicity in the c-KIT-positive population, but not the c-KIT-negative population. Thus, radotinib induces cytotoxicity in c-KIT-positive cells, regardless of the c-KIT expression intensity. Therefore, radotinib induces significant cytotoxicity in c-KIT-positive AML cells, suggesting that radotinib is a potential target agent for the treatment of c-KIT-positive malignancies including AML. Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amagai, Yosuke; Tanaka, Akane; Ohmori, Keitaro

Much is known regarding participations of mast cells with innate and acquired immunity by secreting various cytokines and chemical mediators. However, details of mast cell biology still remain unclear. In this study, we successfully established a novel growth factor-independent mast cell line (MPT-1) derived from canine mast cell tumor. MPT-1 cells manifested factor-independent proliferation as floating cells containing a large amount of histamine, as well as chymase-like dog mast cell protease 3, in cytosolic granules. Particularly, MPT-1 cells expressed high-affinity IgE receptors (Fc{epsilon}RI) and wild-type c-kit receptors. Degranulation of MPT-1 cells was induced not only by stimulation with calcium ionophoremore » but also by cross-linkage of the surface IgE. Given that MPT-1 is the first mast cell line with Fc{epsilon}RI which has no c-kit mutations, MPT-1 cells may provide great contribution for investigation of IgE-mediated activation mechanisms of mast cells, leading to development of effective treatment for allergic disorders.« less

The pivotal role of fibrocytes and mast cells in mediating fibrotic reactions to biomaterials

PubMed Central

Thevenot, Paul T.; Baker, David W.; Weng, Hong; Sun, Man-Wu; Tang, Liping

2011-01-01

Almost all biomaterial implants are surrounded by a fibrotic capsule, although the mechanism of biomaterial-mediated fibrotic reactions is mostly unclear. To search for the types of cells responsible for triggering the tissue responses, we used poly-L glycolic acid polymers capable of releasing various reagents. We first identified that CD45+ /Collagen 1+ fibrocytes are recruited and resided within the fibrotic capsule at the implant interface. Interestingly, we found that the recruitment of fibrocytes and the extent of fibrotic tissue formation (collagen type I production) were substantially enhanced and reduced by the localized release of compound 48/80 and cromolyn, respectively. Since it is well established that compound 48/80 and cromolyn alter mast cell reactions, we hypothesized that mast cells are responsible for triggering fibrocyte recruitment and subsequent fibrotic capsule formation surrounding biomaterial implants. To directly test this hypothesis, similar studies were carried out using mast cell deficient mice, WBB6F1/J-KitW/KitW-v/, and their congenic controls. Indeed, mast cell deficient mice prompted substantially less fibrocyte and myofibroblast responses in comparison to C-57 wild type mice controls. Most interestingly, subcutaneous mast cell reconstitution of WBB6F1/J-KitW/KitW-v/J mice almost completely restored the fibrocyte response in comparison to the C-57 wild type response. These results indicate that the initial biomaterial interaction resulting in the stimulation of mast cells and degranulation byproducts not only stimulates the inflammatory cascade but significantly alters the downstream fibrocyte response and degree of fibrosis. PMID:21864899

A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells.

PubMed

Kobayashi, Masato; Kuroki, Shiori; Kurita, Sena; Miyamoto, Ryo; Tani, Hiroyuki; Tamura, Kyoichi; Bonkobara, Makoto

2017-10-01

Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re‑treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.

Detection of enterotoxigenic Clostridium perfringens in meat samples by using molecular methods.

PubMed

Kaneko, Ikuko; Miyamoto, Kazuaki; Mimura, Kanako; Yumine, Natsuko; Utsunomiya, Hirotoshi; Akimoto, Shigeru; McClane, Bruce A

2011-11-01

To prevent food-borne bacterial diseases and to trace bacterial contamination events to foods, microbial source tracking (MST) methods provide important epidemiological information. To apply molecular methods to MST, it is necessary not only to amplify bacterial cells to detection limit levels but also to prepare DNA with reduced inhibitory compounds and contamination. Isolates carrying the Clostridium perfringens enterotoxin gene (cpe) on the chromosome or a plasmid rank among the most important food-borne pathogens. Previous surveys indicated that cpe-positive C. perfringens isolates are present in only ∼5% of nonoutbreak food samples and then only at low numbers, usually less than 3 cells/g. In this study, four molecular assays for the detection of cpe-positive C. perfringens isolates, i.e., ordinary PCR, nested PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP), were developed and evaluated for their reliability using purified DNA. For use in the artificial contamination of meat samples, DNA templates were prepared by three different commercial DNA preparation kits. The four molecular assays always detected cpe when >10³ cells/g of cpe-positive C. perfringens were present, using any kit. Of three tested commercial DNA preparation kits, the InstaGene matrix kit appeared to be most suitable for the testing of a large number of samples. By using the InstaGene matrix kit, the four molecular assays efficiently detected cpe using DNA prepared from enrichment culture specimens of meat samples contaminated with low numbers of cpe-positive C. perfringens vegetative cells or spores. Overall, the current study developed molecular assay protocols for MST to detect the contamination of foods with low numbers of cells, and at a low frequency, of cpe-positive C. perfringens isolates.

Hematopoietic Kit Deficiency, rather than Lack of Mast Cells, Protects Mice from Obesity and Insulin Resistance.

PubMed

Gutierrez, Dario A; Muralidhar, Sathya; Feyerabend, Thorsten B; Herzig, Stephan; Rodewald, Hans-Reimer

2015-05-05

Obesity, insulin resistance, and related pathologies are associated with immune-mediated chronic inflammation. Kit mutant mice are protected from diet-induced obesity and associated co-morbidities, and this phenotype has previously been attributed to their lack of mast cells. We performed a comprehensive metabolic analysis of Kit-dependent Kit(W/Wv) and Kit-independent Cpa3(Cre/+) mast-cell-deficient mouse strains, employing diet-induced or genetic (Lep(Ob/Ob) background) models of obesity. Our results show that mast cell deficiency, in the absence of Kit mutations, plays no role in the regulation of weight gain or insulin resistance. Moreover, we provide evidence that the metabolic phenotype observed in Kit mutant mice, while independent of mast cells, is immune regulated. Our data underscore the value of definitive mast cell deficiency models to conclusively test the involvement of this enigmatic cell in immune-mediated pathologies and identify Kit as a key hematopoietic factor in the pathogenesis of metabolic syndrome. Copyright © 2015 Elsevier Inc. All rights reserved.

Immunohistochemical expression of c-KIT protein in feline soft tissue fibrosarcomas.

PubMed

Smith, A J; Njaa, B L; Lamm, C G

2009-09-01

C-KIT is the cellular homolog of the feline sarcoma viral oncogene v-KIT, which encodes the tyrosine kinase receptor protein KIT. Mutations and varied expression of this gene have been demonstrated within multiple neoplasms in people and domestic animals. The purpose of this study was to determine if KIT protein is expressed in feline soft tissue fibrosarcomas (ST FSA) using immunohistochemistry (IHC). The computer database at the Oklahoma Animal Disease Diagnostic Laboratory was searched from January 1, 2006, to December 31, 2007, for any domestic cat with an ST FSA. Routinely stained slides from 46 feline ST FSAs were reviewed and graded based on the scale outlined by Kuntz et al. Immunohistochemistry for KIT protein was performed on one representative section from each cat. There were a total of 12/46 (26%) cats that were immunoreactive for KIT. Immunoreactivity was detected in greater than 80% of the neoplastic cells in 4/46 (9%) cats. Immunoreactivity was detected in less than 10% of the neoplastic cells in 8/46 (17%) cats. Immunoreactivity was characterized by evenly distributed cytoplasmic stippling within the neoplastic spindle-shaped cells and/or multinucleated giant cells. Based on these results, KIT immunoreactivity can be detected within feline ST FSAs using IHC. The results of this study also indicate that KIT immunoreactivity in feline ST FSA does not correlate with the histologic grade (P = .141, X(2) = 2.166), survivability (P = .241, X(2) = 1.373), or whether the neoplasm was a spontaneous or an injection site FSA (P = .074, X(2) = 3.184).

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

PubMed

Vicinanza, Carla; Aquila, Iolanda; Scalise, Mariangela; Cristiano, Francesca; Marino, Fabiola; Cianflone, Eleonora; Mancuso, Teresa; Marotta, Pina; Sacco, Walter; Lewis, Fiona C; Couch, Liam; Shone, Victoria; Gritti, Giulia; Torella, Annalaura; Smith, Andrew J; Terracciano, Cesare Mn; Britti, Domenico; Veltri, Pierangelo; Indolfi, Ciro; Nadal-Ginard, Bernardo; Ellison-Hughes, Georgina M; Torella, Daniele

2017-12-01

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kit pos ) cells. The adult heart indeed contains a heterogeneous mixture of c-kit pos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit pos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kit pos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit pos sorting. The blood/endothelial lineage-committed (Lineage pos ) CD45 pos c-kit pos cardiac cells were compared to CD45 neg (Lineage neg /Lin neg ) c-kit pos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit pos cardiac cells are blood/endothelial lineage-committed CD45 pos CD31 pos c-kit pos cells. In contrast, the Lin neg CD45 neg c-kit pos cardiac cell cohort, which represents ⩽10% of the total c-kit pos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit neg and the blood/endothelial lineage-committed c-kit pos cardiac cells. Single Lin neg c-kit pos cell-derived clones, which represent only 1-2% of total c-kit pos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Lin neg c-kit pos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kit pos cardiac cells were injected. Thus, among the cardiac c-kit pos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

Pathologic Stimulus Determines Lineage Commitment of Cardiac C-kit+ Cells.

PubMed

Chen, Zhongming; Zhu, Wuqiang; Bender, Ingrid; Gong, Wuming; Kwak, Il-Youp; Yellamilli, Amritha; Hodges, Thomas J; Nemoto, Natsumi; Zhang, Jianyi; Garry, Daniel J; van Berlo, Jop H

2017-12-12

Although cardiac c-kit + cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit + cells in vivo are unknown. Recent findings suggest that endogenous cardiac c-kit + cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit + cells. We used single-cell sequencing and genetic lineage tracing of c-kit + cells to determine whether various pathological stimuli would result in different fates of c-kit + cells. Single-cell sequencing of cardiac CD45 - c-kit + cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit + cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit + cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation. These results demonstrate that different pathological stimuli induce different cell fates of c-kit + cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit + cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit + cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit + cells. © 2017 American Heart Association, Inc.

Hemogenic endothelial cell specification requires c-kit, notch signaling, and p27-mediated cell-cycle control

USDA-ARS?s Scientific Manuscript database

Delineating the mechanism or mechanisms that regulate the specification of hemogenic endothelial cells from primordial endothelium is critical for optimizing their derivation from human stem cells for clinical therapies. We previously determined that retinoic acid (RA) is required for hemogenic spec...

Loss of Asxl1 leads to myelodysplastic syndrome-like disease in mice.

PubMed

Wang, Jiapeng; Li, Zhaomin; He, Yongzheng; Pan, Feng; Chen, Shi; Rhodes, Steven; Nguyen, Lihn; Yuan, Jin; Jiang, Li; Yang, Xianlin; Weeks, Ophelia; Liu, Ziyue; Zhou, Jiehao; Ni, Hongyu; Cai, Chen-Leng; Xu, Mingjiang; Yang, Feng-Chun

2014-01-23

ASXL1 is mutated/deleted with high frequencies in multiple forms of myeloid malignancies, and its alterations are associated with poor prognosis. De novo ASXL1 mutations cause Bohring-Opitz syndrome characterized by multiple congenital malformations. We show that Asxl1 deletion in mice led to developmental abnormalities including dwarfism, anophthalmia, and 80% embryonic lethality. Surviving Asxl1(-/-) mice lived for up to 42 days and developed features of myelodysplastic syndrome (MDS), including dysplastic neutrophils and multiple lineage cytopenia. Asxl1(-/-) mice had a reduced hematopoietic stem cell (HSC) pool, and Asxl1(-/-) HSCs exhibited decreased hematopoietic repopulating capacity, with skewed cell differentiation favoring granulocytic lineage. Asxl1(+/-) mice also developed mild MDS-like disease, which could progress to MDS/myeloproliferative neoplasm, demonstrating a haploinsufficient effect of Asxl1 in the pathogenesis of myeloid malignancies. Asxl1 loss led to an increased apoptosis and mitosis in Lineage(-)c-Kit(+) (Lin(-)c-Kit(+)) cells, consistent with human MDS. Furthermore, Asxl1(-/-) Lin(-)c-Kit(+) cells exhibited decreased global levels of H3K27me3 and H3K4me3 and altered expression of genes regulating apoptosis (Bcl2, Bcl2l12, Bcl2l13). Collectively, we report a novel ASXL1 murine model that recapitulates human myeloid malignancies, implying that Asxl1 functions as a tumor suppressor to maintain hematopoietic cell homeostasis. Future work is necessary to clarify the contribution of microenvironment to the hematopoietic phenotypes observed in the constitutional Asxl1(-/-) mice.

Grafted c-kit+/SSEA1- eye-wall progenitor cells delay retinal degeneration in mice by regulating neural plasticity and forming new graft-to-host synapses.

PubMed

Chen, Xi; Chen, Zehua; Li, Zhengya; Zhao, Chen; Zeng, Yuxiao; Zou, Ting; Fu, Caiyun; Liu, Xiaoli; Xu, Haiwei; Yin, Zheng Qin

2016-12-30

Despite diverse pathogenesis, the common pathological change observed in age-related macular degeneration and in most hereditary retinal degeneration (RD) diseases is photoreceptor loss. Photoreceptor replacement by cell transplantation may be a feasible treatment for RD. The major obstacles to clinical translation of stem cell-based cell therapy in RD remain the difficulty of obtaining sufficient quantities of appropriate and safe donor cells and the poor integration of grafted stem cell-derived photoreceptors into the remaining retinal circuitry. Eye-wall c-kit + /stage-specific embryonic antigen 1 (SSEA1) - cells were isolated via fluorescence-activated cell sorting, and their self-renewal and differentiation potential were detected by immunochemistry and flow cytometry in vitro. After labeling with quantum nanocrystal dots and transplantation into the subretinal space of rd1 RD mice, differentiation and synapse formation by daughter cells of the eye-wall c-kit + /SSEA1 - cells were evaluated by immunochemistry and western blotting. Morphological changes of the inner retina of rd1 mice after cell transplantation were demonstrated by immunochemistry. Retinal function of rd1 mice that received cell grafts was tested via flash electroretinograms and the light/dark transition test. Eye-wall c-kit + /SSEA1 - cells were self-renewing and clonogenic, and they retained their proliferative potential through more than 20 passages. Additionally, eye-wall c-kit + /SSEA1 - cells were capable of differentiating into multiple retinal cell types including photoreceptors, bipolar cells, horizontal cells, amacrine cells, Müller cells, and retinal pigment epithelium cells and of transdifferentiating into smooth muscle cells and endothelial cells in vitro. The levels of synaptophysin and postsynaptic density-95 in the retinas of eye-wall c-kit + /SSEA1 - cell-transplanted rd1 mice were significantly increased at 4 weeks post transplantation. The c-kit + /SSEA1 - cells were capable of differentiating into functional photoreceptors that formed new synaptic connections with recipient retinas in rd1 mice. Transplantation also partially corrected the abnormalities of inner retina of rd1 mice. At 4 and 8 weeks post transplantation, the rd1 mice that received c-kit + /SSEA1 - cells showed significant increases in a-wave and b-wave amplitude and the percentage of time spent in the dark area. Grafted c-kit + /SSEA1 - cells restored the retinal function of rd1 mice via regulating neural plasticity and forming new graft-to-host synapses.

'W' mutant forms of the Fms receptor tyrosine kinase act in a dominant manner to suppress CSF-1 dependent cellular transformation.

PubMed

Reith, A D; Ellis, C; Maroc, N; Pawson, T; Bernstein, A; Dubreuil, P

1993-01-01

Point mutations in highly conserved amino acid residues in the catalytic domain of the Kit receptor tyrosine kinase (RTK) are responsible for the coat color, fertility and hematopoietic defects of mice bearing mutant alleles at the dominant white-spotting (W) locus. The dominant nature of structural Kit mutations suggests that expression of other kinase-defective RTKs might also specifically interfere with signal transduction by normal receptors. To test this possibility, we have investigated the functional consequences of introducing analogous mutations into the RTK encoded by the c-fms proto-oncogene. Both Fms37 (glu582-->lys) and Fms42 (asp776-->asn) mutant proteins, corresponding to the strongly dominant-negative W37 and W42 mutant c-kit alleles, had undetectable in vitro kinase activity and were unable to transform Rat-2 fibroblasts in the presence of exogenous CSF-1. Moreover, expression of Fms37 or Fms42 proteins in Rat-2 cells specifically inhibited anchorage-independent growth mediated by the normal Fms receptor in the presence of exogenous CSF-1 and conferred a dominant loss of Fms-associated PI3-kinase activity on CSF-1 stimulation. Mutant RTKs, bearing point substitutions identical to those present in mild or severe W mutants, may provide a generally applicable strategy for inducing dominant loss of function defects in RTK-mediated signalling pathways.

Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.

PubMed Central

Furitsu, T; Tsujimura, T; Tono, T; Ikeda, H; Kitayama, H; Koshimizu, U; Sugahara, H; Butterfield, J H; Ashman, L K; Kanayama, Y

1993-01-01

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells. Images PMID:7691885

Evaluation of the kinase domain of c-KIT in canine cutaneous mast cell tumors

PubMed Central

Webster, Joshua D; Kiupel, Matti; Yuzbasiyan-Gurkan, Vilma

2006-01-01

Background Mutations in the c-KIT proto-oncogene have been implicated in the progression of several neoplastic diseases, including gastrointestinal stromal tumors and mastocytosis in humans, and cutaneous mast cell tumors (MCTs) in canines. Mutations in human mastocytosis patients primarily occur in c-KIT exon 17, which encodes a portion of its kinase domain. In contrast, deletions and internal tandem duplication (ITD) mutations are found in the juxtamembrane domain of c-KIT in approximately 15% of canine MCTs. In addition, ITD c-KIT mutations are significantly associated with aberrant KIT protein localization in canine MCTs. However, some canine MCTs have aberrant KIT localization but lack ITD c-KIT mutations, suggesting that other mutations or other factors may be responsible for aberrant KIT localization in these tumors. Methods In order to characterize the prevalence of mutations in the phospho-transferase portion of c-KIT's kinase domain in canine MCTs exons 16–20 of 33 canine MCTs from 33 dogs were amplified and sequenced. Additionally, in order to determine if mutations in c-KIT exon 17 are responsible for aberrant KIT localization in MCTs that lack juxtamembrane domain c-KIT mutations, c-KIT exon 17 was amplified and sequenced from 18 canine MCTs that showed an aberrant KIT localization pattern but did not have ITD c-KIT mutations. Results No mutations or polymorphisms were identified in exons 16–20 of any of the MCTs examined. Conclusion In conclusion, mutations in the phospho-transferase portion of c-KIT's kinase domain do not play an important role in the progression of canine cutaneous MCTs, or in the aberrant localization of KIT in canine MCTs. PMID:16579858

Tranilast prevents renal interstitial fibrosis by blocking mast cell infiltration in a rat model of diabetic kidney disease.

PubMed

Yin, Dan-Dan; Luo, Jun-Hui; Zhao, Zhu-Ye; Liao, Ying-Jun; Li, Ying

2018-05-01

Renal interstitial fibrosis is a final pathway that is observed in various types of kidney diseases, including diabetic kidney disease (DKD). The present study investigated the effect of tranilast on renal interstitial fibrosis and the association between its role and mast cell infiltration in a rat model of DKD. A total of 30 healthy 6‑week‑old male Sprague‑Dawley rats were randomly divided into the following four groups: Normal control group; DKD model group; low‑dose tranilast group (200 mg/kg/day); and high‑dose tranilast group (400 mg/kg/day). The morphological alterations of tubulointerstitial fibrosis were evaluated by Masson's trichrome staining, while mast cell infiltration into the renal tubular interstitium was measured by toluidine blue staining and complement C3a receptor 1 (C3aR) immunohistochemical staining (IHC). The expression of fibronectin (FN), collagen I (Col‑I), stem cell factor (SCF) and proto‑oncogene c‑kit (c‑kit) was detected by IHC, western blotting and reverse transcription‑quantitative‑polymerase chain reaction. The results demonstrated that tubulointerstitial fibrosis and mast cell infiltration were observed in DKD model rats, and this was improved dose‑dependently in the tranilast treatment groups. The expression of FN, Col‑I, SCF and c‑kit mRNA and protein was upregulated in the tubulointerstitium of DKD model rats compared with the normal control rats, and tranilast inhibited the upregulated expression of these markers. Furthermore, the degree of SCF and c‑kit expression demonstrated a significant positive correlation with C3aR‑positive mast cells and the markers of renal interstitial fibrosis. The results of the present study indicate that mast cell infiltration may promote renal interstitial fibrosis via the SCF/c‑kit signaling pathway. Tranilast may prevent renal interstitial fibrosis through inhibition of mast cell infiltration mediated through the SCF/c-kit signaling pathway.

No GIST-type c-kit gain of function mutations in neuroblastic tumours

PubMed Central

Korja, M; Finne, J; Salmi, T T; Haapasalo, H; Tanner, M; Isola, J

2005-01-01

Aims: Neuroblastic tumours (NTs) have been shown to respond to imatinib treatment in vivo and in vitro, possibly via inactivating the c-kit receptor. The purpose of this study was to identify gastrointestinal stromal tumour (GIST)-type c-kit gene associated mutations in exons 9, 11, 13, and 17 in NTs to recognise a subset of tumours that would probably respond to imatinib treatment. Methods: Expression of the c-kit protein was detected immunohistochemically in a total of 37 archival paraffin wax embedded NTs using polyclonal rabbit antihuman c-kit antibody. After immunohistochemistry, c-kit gene associated chromosomal mutations in all cases of NT were detected with denaturing high performance liquid chromatography (HPLC). Results: Denaturing HLPC analysis did not reveal GIST-type mutations in four immunohistochemically detected c-kit positive or in 33 c-kit negative NTs. Conclusions: c-kit receptor expression and GIST-type c-kit gene mutations are rare events in NTs. Oncogenic activation of c-kit in NTs presumably differs from that of GISTs, which may influence their responsiveness to imatinib treatment. Whether c-kit has an essential role in the pathogenesis of NTs remains to be investigated. PMID:15976348

Pim1 kinase regulates c-Kit gene translation.

PubMed

An, Ningfei; Cen, Bo; Cai, Houjian; Song, Jin H; Kraft, Andrew; Kang, Yubin

2016-01-01

Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1 -/- mice and Pim1 -/- 2 -/- 3 -/- triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1 -/- and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit 35 S methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

Intratumoral CD3+ T-Lymphocytes Immunoexpression and Its Association with c-Kit, Angiogenesis, and Overall Survival in Malignant Canine Mammary Tumors

PubMed Central

Carvalho, Maria Isabel; Pires, Isabel; Dias, Marlene; Prada, Justina; Gregório, Hugo; Lobo, Luis; Queiroga, Felisbina

2015-01-01

In this study 80 malignant CMT were submitted to immunohistochemical detection of CD3, c-kit, VEGF, and CD31, together with clinicopathological parameters of tumor aggressiveness. CD3+ T-cells and c-kit overexpression revealed a positive correlation with VEGF (r = 0.503, P < 0.0001; r = 0.284, P = 0.023 for CD3 and c-kit, resp.) and CD31 (r = 0.654, P < 0.0001; r = 0.365, P = 0.003 for CD3 and c-kit, resp.). A significant association (P = 0.039) and a positive correlation (r = 0.263, P = 0.039) between CD3 and c-kit were also observed. High CD3/VEGF, c-kit/VEGF, and CD3/c-kit tumors were associated with elevated grade of malignancy (P < 0.0001 for all groups), presence of intravascular emboli (P < 0.0001 for CD3/VEGF and CD3/c-kit; P = 0.002 for c-kit/VEGF), and presence of lymph node metastasis (P < 0.0001 for all groups). Tumors with high CD3/VEGF (P = 0.006), c-kit/VEGF (P < 0.0001), and CD3/c-kit (P = 0.002) were associated with poor prognosis. Interestingly high c-kit/VEGF tumors retained their significance by multivariate analysis arising as independent prognostic factor. PMID:26346272

A quantitative analysis of whether elementary teachers' science kit usage and beliefs can predict state science assessment scores

NASA Astrophysics Data System (ADS)

Rice, Tony E.

The purpose of this survey was to describe and analyze the perceptions of elementary school teachers' in a Midwestern state concerning their use of a science kit program, including to what extent a school's state science assessment scores can be predicated from the level of science kit usage. Prior research indicates that elementary school teachers lack the confidence in teaching science primarily because of their weak undergraduate training in inquiry-based instruction and the lack of a strong science background. Authors such as Dickerson et al. (2006) and Riggs and Enochs (2006) argued that science kits and the materials included in them are valuable in increasing teacher confidence. The teacher perceptions I collected matched the literature quite closely as far as what the teachers found to be of the most value and use. Teachers perceptions of the science kits were positive including: (a) student engagement in using the science kits, (b) use of most of the instructional items included in the kits, (c) the amount of teacher confidence in using them, (d) the support from the math and science center for using them, (e) and the professional development provided. Teachers liked using many components of the kits, especially the experiments. Their main complaint concerned time: time to teach science and time to complete the kit lessons. I used multiple regression to understand the components of the kit program that had a significant correlation to the state test scores. The following variables could explain a high proportion of the variance (.796): (a) teacher confidence, (b) student science learning success, (c) teacher beliefs about science education and (d) the percentage of students eligible for the National School Lunch Program. These findings might lead to school principals and teachers increasing their 5th grade state science exam scores by using the findings to identify which components of the kit program are most important in this endeavor.

The clinical significance of c-Kit mutations in metastatic oral mucosal melanoma in China.

PubMed

Ma, Xuhui; Wu, Yunteng; Zhang, Tian; Song, Hao; Jv, Houyu; Guo, Wei; Ren, Guoxin

2017-10-10

c-Kit mutations are frequently detected in mucosal melanomas, but their clinical significance in metastatic oral mucosal melanomas (OMM) remains unclear. The main purpose of this study was to investigate the clinical and pathological features of metastatic OMMs with c-Kit mutations and the efficiency of the tyrosine kinase inhibitor imatinib in treating metastatic OMMs. We found thatresidual primary lesion and neck lymph nodes could act as independent prognostic factors in metastatic OMM patients. c-Kit mutations were detected in 22 out of 139 (15.8%) metastatic OMM patients. Under chemotherapy, the overall survival (OS) of c-Kit mutant patients was significantly shorter than that of wild-type patients. The Ki67 expression was significantly higher in c-Kit mutant patients than in wild-type patients. In distant metastatic OMM patients with c-Kit mutations, the treatment with c-Kit inhibitor resulted in a better OS. In conclusion, residual primary lesion, cervical lymph nodes and c-Kit mutations act as adverse prognostic factors of metastatic OMMs. The Kit inhibitor imatinib could benefit metastatic OMM patients with c-Kit mutations.

Targeting c-KIT (CD117) by dasatinib and radotinib promotes acute myeloid leukemia cell death.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jeong, Yoo Kyung; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-11-10

Dasatinib and radotinib are oral BCR-ABL tyrosine kinase inhibitors that were developed as drugs for the treatment of chronic myeloid leukemia. We report here that the c-KIT (CD117) targeting with dasatinib and radotinib promotes acute myeloid leukemia (AML) cell death, and c-KIT endocytosis is essential for triggering c-KIT-positive AML cell death by dasatinib and radotinib during the early stages. In addition, dasatinib and radotinib reduce heat shock protein 90β (HSP90β) expression and release Apaf-1 in c-KIT-positive AML cells. Finally, this activates a caspase-dependent apoptotic pathway in c-KIT-positive AML cells. Moreover, the inhibition of c-KIT endocytosis by dynamin inhibitor (DY) reversed cell viability and c-KIT expression by dasatinib and radotinib. HSP90β expression was recovered by DY in c-KIT-positive AML cells as well. Furthermore, the effect of radotinib on c-KIT and HSP90β showed the same pattern in a xenograft animal model using HEL92.1.7 cells. Therefore, dasatinib and radotinib promote AML cell death by targeting c-KIT. Taken together, these results indicate that dasatinib and radotinib treatment have a potential role in anti-leukemic therapy on c-KIT-positive AML cells.

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells

PubMed Central

Singh, Satyendra; Klarmann, Kimberly D.; Coppola, Vincenzo; Keller, Jonathan R.; Tessarollo, Lino

2016-01-01

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies. PMID:27835883

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells.

PubMed

Puverel, Sandrine; Kiris, Erkan; Singh, Satyendra; Klarmann, Kimberly D; Coppola, Vincenzo; Keller, Jonathan R; Tessarollo, Lino

2016-12-20

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.

Changes in c-Kit expression levels during the course of radiation therapy for nasopharyngeal carcinoma.

PubMed

Jiang, Feng; Hu, Wei; Zhang, Bicheng; Xu, Jing; Shui, Yongjie; Zhou, Xiaofeng; Ren, Xiaoqiu; Chen, Xiaozhong; Shen, Li; Wei, Qichun

2016-10-01

In the era of intensity-modulated radiotherapy, distant metastasis is currently the main cause of treatment failure for nasopharyngeal carcinoma (NPC). Additional therapeutic strategies are required to control the metastasis and improve survival. One strategy is targeted therapy, for example against c-Kit. In the current study, the frequency of c-Kit expression was determined immunohistochemically in 106 NPC patients. c-Kit expression changes during the course of radiation therapy were detected in 41 cases via weekly biopsy. Twelve cases (11.3%) had c-Kit expression scores of 3 + and 16 (15.1%) had scores of 2 + . Thus, c-Kit overexpression (2 + or 3 + ) was observed in 28 (26.4%) patients. There were 35 (33.0%) and 43 (40.6%) patients with c-Kit expression scores of 1 + and 0, respectively. Furthermore, a trend of decreased c-Kit expression was observed after commencing radiotherapy according to the 41 NPC patients who were biopsied weekly. Therefore, c-Kit overexpression was identified to be common in NPC, and evaluating c-Kit as a therapeutic target for metastatic NPC via c-Kit overexpression subsequent to first line treatment may be of interest. To the best of our knowledge, the present study is the first to demonstrate a trend of decreased c-Kit expression during the course of radiotherapy.

Exacerbated experimental autoimmune encephalomyelitis in mast-cell-deficient Kit W-sh/W-sh mice.

PubMed

Piconese, Silvia; Costanza, Massimo; Musio, Silvia; Tripodo, Claudio; Poliani, Pietro L; Gri, Giorgia; Burocchi, Alessia; Pittoni, Paola; Gorzanelli, Andrea; Colombo, Mario P; Pedotti, Rosetta

2011-04-01

Mast cell (MC)-deficient c-Kit mutant Kit(W/W-v) mice are protected against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, suggesting a detrimental role for MCs in this disease. To further investigate the role of MCs in EAE, we took advantage of a recently characterized model of MC deficiency, Kit(W-sh/W-sh). Surprisingly, we observed that myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced chronic EAE was exacerbated in Kit(W-sh/W-sh) compared with Kit(+/+) mice. Kit(W-sh/W-sh) mice showed more inflammatory foci in the central nervous system (CNS) and increased T-cell response against myelin. To understand whether the discrepant results obtained in Kit(W-sh/W-sh) and in Kit(W/W-v) mice were because of the different immunization protocols, we induced EAE in these two strains with varying doses of MOG(35-55) and adjuvants. Although Kit(W-sh/W-sh) mice exhibited exacerbated EAE under all immunization protocols, Kit(W/W-v) mice were protected from EAE only when immunized with high, but not low, doses of antigen and adjuvants. Kit(W-sh/W-sh) mice reconstituted systemically, but not in the CNS, with bone marrow-derived MCs still developed exacerbated EAE, indicating that protection from disease could be exerted by MCs mainly in the CNS, and/or by other cells possibly dysregulated in Kit(W-sh/W-sh) mice. In summary, these data suggest to reconsider MC contribution to EAE, taking into account the variables of using different experimental models and immunization protocols.

Anti-cancer effect of ursolic acid activates apoptosis through ROCK/PTEN mediated mitochondrial translocation of cofilin-1 in prostate cancer

PubMed Central

Gai, Wen-Tao; Yu, Da-Peng; Wang, Xin-Sheng; Wang, Pei-Tao

2016-01-01

Ursolic acid is a type of pentacyclic triterpene compound with multiple pharmacological activities including cancer resistance, protection from liver injury, antisepsis, anti-inflammation and antiviral activity. The present study aimed to investigate the anticancer effect of ursolic acid. Ursolic acid activates cell apoptosis and its pro-apoptotic mechanism remains to be fully elucidated. Cell Counting kit-8 assays, flow cytometric analysis and analysis of caspase-3 and caspase-9 activity were used to estimate the anticancer effect of ursolic acid on DU145 prostate cancer cells. The protein expression of cytochrome c, rho-associated protein kinase (ROCK), phosphatase and tensin homolog (PTEN) and cofilin-1 were examined using western blot analysis. In the present study, ursolic acid significantly suppressed cell growth and induced apoptosis, as well as increasing caspase-3 and caspase-9 activities of DU145 cells. Furthermore, cytoplasmic and mitochondrial cytochrome c protein expression was significantly activated and suppressed, respectively, by ursolic acid. Ursolic acid significantly suppressed the ROCK/PTEN signaling pathway and inhibited cofilin-1 protein expression in DU145 cells. The results of the present study indicate that the anticancer effect of ursolic acid activates cell apoptosis through ROCK/PTEN mediated mitochondrial translocation of cofilin-1 in prostate cancer. PMID:27698874

Single-cell analysis of the fate of c-kit-positive bone marrow cells

NASA Astrophysics Data System (ADS)

Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D.; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa

2017-10-01

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

Single-cell analysis of the fate of c-kit-positive bone marrow cells.

PubMed

Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa

2017-01-01

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

CD117/c-kit in Cancer Stem Cell-Mediated Progression and Therapeutic Resistance

PubMed Central

Young, Tyler R.; Mobley, Mary E.

2018-01-01

Metastasis is the primary cause of cancer patient morbidity and mortality, but due to persisting gaps in our knowledge, it remains untreatable. Metastases often occur as patient tumors progress or recur after initial therapy. Tumor recurrence at the primary site may be driven by a cancer stem-like cell or tumor progenitor cell, while recurrence at a secondary site is driven by metastatic cancer stem cells or metastasis-initiating cells. Ongoing efforts are aimed at identifying and characterizing these stem-like cells driving recurrence and metastasis. One potential marker for the cancer stem-like cell subpopulation is CD117/c-kit, a tyrosine kinase receptor associated with cancer progression and normal stem cell maintenance. Further, activation of CD117 by its ligand stem cell factor (SCF; kit ligand) in the progenitor cell niche stimulates several signaling pathways driving proliferation, survival, and migration. This review examines evidence that the SCF/CD117 signaling axis may contribute to the control of cancer progression through the regulation of stemness and resistance to tyrosine kinase inhibitors. PMID:29518044

Discovery of a highly selective KIT kinase primary V559D mutant inhibitor for gastrointestinal stromal tumors (GISTs).

PubMed

Yu, Kailin; Liu, Xuesong; Jiang, Zongru; Hu, Chen; Zou, Fengming; Chen, Cheng; Ge, Juan; Wu, Jiaxin; Liu, Xiaochuan; Wang, Aoli; Wang, Wenliang; Wang, Wenchao; Qi, Ziping; Wang, Beilei; Wang, Li; Yan, Hezhong; Wang, Jiaoxue; Ren, Tao; Tang, Jun; Liu, Qingsong; Liu, Jing

2017-12-19

KIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors (GISTs). Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031, which displayed about 10-20 fold selectivity over KIT wt in the biochemical assay (IC 50 : 28 nM over 168 nM; Kd: 266 nM versus 6640 nM) and in cell (EC 50 : 176 nM versus 2000 nM for pY703) examination. It also displayed 15∼400-fold selectivity over other primary mutants such as L576P and secondary mutants including T670I, V654A (ATP binding pocket) as well as N822K and D816V (activation loop). In addition, it exhibited a selectivity S score (1) of 0.01 among 468 kinases/mutants in the KINOMEScan ™ assay. CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo (Tumor Growth Inhibition: 68.5%). Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GISTs.

Discovery of a highly selective KIT kinase primary V559D mutant inhibitor for gastrointestinal stromal tumors (GISTs)

PubMed Central

Yu, Kailin; Liu, Xuesong; Jiang, Zongru; Hu, Chen; Zou, Fengming; Chen, Cheng; Ge, Juan; Wu, Jiaxin; Liu, Xiaochuan; Wang, Aoli; Wang, Wenliang; Wang, Wenchao; Qi, Ziping; Wang, Beilei; Wang, Li; Yan, Hezhong; Wang, Jiaoxue; Ren, Tao; Tang, Jun; Liu, Qingsong; Liu, Jing

2017-01-01

KIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors (GISTs). Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031, which displayed about 10-20 fold selectivity over KIT wt in the biochemical assay (IC50: 28 nM over 168 nM; Kd: 266 nM versus 6640 nM) and in cell (EC50: 176 nM versus 2000 nM for pY703) examination. It also displayed 15∼400-fold selectivity over other primary mutants such as L576P and secondary mutants including T670I, V654A (ATP binding pocket) as well as N822K and D816V (activation loop). In addition, it exhibited a selectivity S score (1) of 0.01 among 468 kinases/mutants in the KINOMEScan™ assay. CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo (Tumor Growth Inhibition: 68.5%). Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GISTs. PMID:29340041

Germline mutations of KIT in gastrointestinal stromal tumor (GIST) and mastocytosis.

PubMed

Ke, Hengning; Kazi, Julhash U; Zhao, Hui; Sun, Jianmin

2016-01-01

Somatic mutations of KIT are frequently found in mastocytosis and gastrointestinal stromal tumor (GIST), while germline mutations of KIT are rare, and only found in few cases of familial GIST and mastocytosis. Although ligand-independent activation is the common feature of KIT mutations, the phenotypes mediated by various germline KIT mutations are different. Germline KIT mutations affect different tissues such as interstitial cells of Cajal (ICC), mast cells or melanocytes, and thereby lead to GIST, mastocytosis, or abnormal pigmentation. In this review, we summarize germline KIT mutations in familial mastocytosis and GIST and discuss the possible cellular context dependent transforming activity of KIT mutations.

Survivin is a novel transcription regulator of KIT and is downregulated by miRNA-494 in gastrointestinal stromal tumors.

PubMed

Yun, SeongJu; Kim, Won Kyu; Kwon, Yujin; Jang, Mi; Bauer, Sebastian; Kim, Hoguen

2018-05-15

Gain-of-function mutations of KIT are pathognomonic in sporadic gastrointestinal stromal tumors (GISTs). Several microRNAs have been shown to be dysregulated in GISTs and impact KIT expression. Little is known though on KIT-independent targets of KIT-regulating mRNAs. We sought to investigate how miR-494 inhibits GIST proliferation and to identify novel target gene. We used microarray-based gene expression analyses to identify pathways and target genes affected by miR-494. The expressional relationship between survivin and miR-494 was determined in 35 GIST tissues. Cell proliferation assay, FACS analysis, colony formation assay, promoter assays and chromatin immunoprecipitation (ChiP) were performed to clarify the roles of survivin in GIST progression. Gene expression microarray analysis revealed that miR-494 inhibited GISTs by affecting multiple genes in the cell cycle pathway. Survivin (BIRC5) was a key target of miR-494, and its expression showed an inverse correlation with miR-494 expression in 35 GIST tissues (Pearson's correlation coefficient, r = -0.418, p = 0.012). Downregulation of survivin inhibited proliferation and colony formation, and resulted in cell cycle alteration. Induced survivin overexpression relieved miR-494-mediated inhibition of GIST progression. Targeting PI3K effectively suppressed proliferation of GISTs with downregulation of survivin. Survivin also regulated KIT expression at the transcription level. Immunohistochemical analysis using 113 GISTs revealed that survivin expression was significantly correlated with overall survival of GIST patients (p = 0.004). Our findings indicated that miR-494 synergistically suppressed GISTs by concomitantly targeting survivin and KIT. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

Investigation of c-KIT and Ki67 expression in normal, preneoplastic and neoplastic canine prostate.

PubMed

Fonseca-Alves, Carlos Eduardo; Kobayashi, Priscilla Emiko; Palmieri, Chiara; Laufer-Amorim, Renée

2017-12-06

c-KIT expression has been related to bone metastasis in human prostate cancer, but whether c-KIT expression can be similarly classified in canine prostatic tissue is unknown. This study assessed c-KIT and Ki67 expression in canine prostate cancer (PC). c-KIT gene and protein expression and Ki67 expression were evaluated in forty-four canine prostatic tissues by immunohistochemistry, RT-qPCR and western blot. Additionally, we have investigated c-KIT protein expression by immunoblotting in two primary canine prostate cancer cell lines. Eleven normal prostates, 12 proliferative inflammatory atrophy (PIA) prostates, 18 PC, 3 metastatic lesions and two prostate cancer cell cultures (PC1 and PC2) were analysed. The prostatic tissue exhibited varying degrees of membranous, cytoplasmic or membranous/cytoplasmic c-KIT staining. Four normal prostates, 4 PIA and 5 prostatic carcinomas showed positive c-KIT expression. No c-KIT immunoexpression was observed in metastases. Canine prostate cancer and PIA samples contained a higher number of Ki67-positive cells compared to normal samples. The median relative quantification (RQ) for c-KIT expression in normal, PIA and prostate cancer and metastatic samples were 0.6 (0.1-2.5), 0.7 (0.09-2.1), 0.7 (0.09-5.1) and 0.1 (0.07-0.6), respectively. A positive correlation between the number of Ki67-positive cells and c-KIT transcript levels was observed in prostate cancer samples. In the cell line, PC1 was negative for c-KIT protein expression, while PC2 was weakly positive. The present study identified a strong correlation between c-KIT expression and proliferative index, suggesting that c-KIT may influence cell proliferation. Therefore, c-KIT heterogeneous protein expression among the samples (five positive and thirteen negative prostate cancer samples) indicates a personalized approach for canine prostate cancer.

Discovery of amido-benzisoxazoles as potent c-Kit inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kunz, Roxanne K.; Rumfelt, Shannon; Chen, Ning

2010-01-12

Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.

Ultrastructural study of relationships between c-kit immunoreactive interstitial cells and other cellular elements in the human colon.

PubMed

Mazzia, C; Porcher, C; Julé, Y; Christen, M O; Henry, M

2000-05-01

C-kit immunocytochemistry was performed on ultrathin sections of human distal colon. Our attention was focused on relationships between c-kit immunoreactive interstitial cells (c-kit ICs) and muscular cells and nervous elements located in the external muscular layers of the colonic wall. C-kit ICs established membrane apposition with both nerve fibers and smooth muscle cells of, respectively, the longitudinal and circular muscle layers, the myenteric area, and the extremus submucosus plexus. C-kit ICs also surrounded the external submucosus plexus and established membrane appositions with nerve elements located inside the myenteric ganglia. These membrane appositions were observed either at the level of the c-kit IC bodies or at that of their cytoplasmic processes. In some cases, membrane appositions were observed concomitantly between the c-kit ICs, nerve fibers, and smooth muscle cells. In all the regions studied, the c-kit ICs were also found to be located in the close vicinity of blood vessels and to have established close contacts with non-immunoreactive fibroblast-like cells. The results of the present study shed essential light on the relationships of c-kit ICs with the neighboring muscle cells and nerve elements, and confirm that the intercalated c-kit ICs well fit with the so-called "interstitial cells of Cajal".

Expression of Fas and Fas-ligand in donor hematopoietic stem and progenitor cells is dissociated from the sensitivity to apoptosis.

PubMed

Pearl-Yafe, Michal; Yolcu, Esma S; Stein, Jerry; Kaplan, Ofer; Shirwan, Haval; Yaniv, Isaac; Askenasy, Nadir

2007-10-01

The interaction between the Fas receptor and its cognate ligand (FasL) has been implicated in the mutual suppression of donor and host hematopoietic cells after transplantation. Following the observation of deficient early engraftment of Fas and FasL-defective donor cells and recipients, we determined the role of the Fas-FasL interaction. Donor cells were recovered after syngeneic (CD45.1-->CD45.2) transplants from various organs and assessed for expression of Fas/FasL in reference to lineage markers, carboxyfluorescein succinimidyl ester dilution, Sca-1 and c-kit expression. Naïve and bone marrow-homed cells were challenged for apoptosis ex vivo. The Fas receptor and ligand were markedly upregulated to 40% to 60% (p < 0.001 vs 5-10% in naïve cells) within 2 days after syngeneic transplantation, while residual host cells displayed modest and delayed upregulation of these molecules ( approximately 10%). All lin(-)Sca(+)c-kit(+) cells were Fas(+)FasL(+), including 95% of Sca-1(+) and 30% of c-kit(+) cells. Fas and FasL expression varied in donor cells that homed to bone marrow, spleen, liver and lung, and was induced by interaction with the stroma, irradiation, cell cycling, and differentiation. Bone marrow-homed donor cells challenged with supralethal doses of FasL were insensitive to apoptosis (3.2% +/- 1% vs 38% +/- 5% in naïve bone marrow cells), and engraftment was not affected by pretransplantation exposure of donor cells to an apoptotic challenge with FasL. There was no evidence of Fas-mediated suppression of donor and host cell activity after transplantation. Resistance to Fas-mediated apoptosis evolves as a functional characteristic of hematopoietic reconstituting stem and progenitor cells, providing them competitive engraftment advantage over committed progenitors.

Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nobuhisa, Ikuo; Ohtsu, Naoki; Okada, Seiji

The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45{sup low} c-Kit{sup +} cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45{sup low} c-Kit{sup -} cells that showed a granulocyte morphology;more » CD45{sup high} c-Kit{sup low/-} that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45{sup low} c-Kit{sup +} cells from the AGM culture had the abilities to reproduce CD45{sup low} c-Kit{sup +} cells and differentiate into CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} cells, whereas CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} did not produce CD45{sup low} c-Kit{sup +} cells. Furthermore, CD45{sup low} c-Kit{sup +} cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45{sup low} c-Kit{sup +} cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.« less

Aberrant expressions of c-KIT and DOG-1 in mucinous and nonmucinous colorectal carcinomas and relation to clinicopathologic features and prognosis.

PubMed

Foda, Abd Al-Rahman Mohammad; Mohamed, Mie Ali

2015-10-01

c-KIT and DOG-1 are 2 highly expressed proteins in gastrointestinal stromal tumors. Few studies had investigated c-KIT, but not DOG-1, expression in colorectal carcinoma (CRC). This study aims to investigate expressions of c-KIT and DOG-1 in colorectal mucinous carcinoma and nonmucinous carcinoma using manual tissue microarray technique. In this work, we studied tumor tissue specimens from 150 patients with colorectal mucinous (MA) and nonmucinous adenocarcinoma (NMA). High-density manual tissue microarrays were constructed using modified mechanical pencil tip technique, and immunohistochemistry for c-KIT and DOG-1 was done. We found that aberrant c-KIT expression was detected in 12 cases (8%); 6 cases (4%) showed strong expression. Aberrant DOG-1 expression was detected in 15 cases (10%); among them, only 4 cases (2.7%) showed strong expression. Nonmucinous adenocarcinoma showed a significantly high expression of c-KIT, but not DOG-1, than MA. Aberrant c-KIT and DOG-1 expressions were significantly unrelated but were associated with excessive microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. In conclusion, aberrant c-KIT and DOG-1 expressions in CRC are rare events, either in NMA or MA. Nonmucinous adenocarcinoma showed a significantly higher expression of c-KIT, but not DOG-1, than MA. The expressions of both in CRC are significantly unrelated but are associated with microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. So, c-KIT and DOG-1 immunostaining is not a cost-effective method of identifying patients with CRC who may benefit from treatment with tyrosine kinase inhibitors. Copyright © 2015 Elsevier Inc. All rights reserved.

Disruption of c-Kit Signaling in Kit(W-sh/W-sh) Growing Mice Increases Bone Turnover.

PubMed

Lotinun, Sutada; Krishnamra, Nateetip

2016-08-16

c-Kit tyrosine kinase receptor has been identified as a regulator of bone homeostasis. The c-Kit loss-of-function mutations in WBB6F1/J-Kit(W/W-v) mice result in low bone mass. However, these mice are sterile and it is unclear whether the observed skeletal phenotype is secondary to a sex hormone deficiency. In contrast, C57BL/6J-Kit(W-sh)/(W-sh) (W(sh)/W(sh)) mice, which carry an inversion mutation affecting the transcriptional regulatory elements of the c-Kit gene, are fertile. Here, we showed that W(sh)/W(sh) mice exhibited osteopenia with elevated bone resorption and bone formation at 6- and 9-week-old. The c-Kit W(sh) mutation increased osteoclast differentiation, the number of committed osteoprogenitors, alkaline phosphatase activity and mineralization. c-Kit was expressed in both osteoclasts and osteoblasts, and c-Kit expression was decreased in W(sh)/W(sh)osteoclasts, but not osteoblasts, suggesting an indirect effect of c-Kit on bone formation. Furthermore, the osteoclast-derived coupling factor Wnt10b mRNA was increased in W(sh)/W(sh) osteoclasts. Conditioned medium from W(sh)/W(sh) osteoclasts had elevated Wnt10b protein levels and induced increased alkaline phosphatase activity and mineralization in osteoblast cultures. Antagonizing Wnt10b signaling with DKK1 or Wnt10b antibody inhibited these effects. Our data suggest that c-Kit negatively regulates bone turnover, and disrupted c-Kit signaling couples increased bone resorption with bone formation through osteoclast-derived Wnt 10 b.

CD45{sup low}c-Kit{sup high} cells have hematopoietic properties in the mouse aorta-gonad-mesonephros region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nobuhisa, Ikuo, E-mail: nobuhisa.scr@mri.tmd.ac.jp; Department of Cell Fate Modulation, Institute of Molecular Embryology and Genetics/Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, 860-0811; Yamasaki, Shoutarou

Long-term reconstituting hematopoietic stem cells first arise from the aorta of the aorta-gonad-mesonephros (AGM) region in a mouse embryo. We have previously reported that in cultures of the dispersed AGM region, CD45{sup low}c-Kit{sup +} cells possess the ability to reconstitute multilineage hematopoietic cells, but investigations are needed to show that this is not a cultured artifact and to clarify when and how this population is present. Based on the expression profile of CD45 and c-Kit in freshly dissociated AGM cells from embryonic day 9.5 (E9.5) to E12.5 and aorta cells in the AGM from E13.5 to E15.5, we defined sixmore » cell populations (CD45{sup -}c-Kit{sup -}, CD45{sup -}c-Kit{sup low}, CD45{sup -}c-Kit{sup high}, CD45{sup low}c-Kit{sup high}, CD45{sup high}c-Kit{sup high}, and CD45{sup high}c-Kit{sup very} {sup low}). Among these six populations, CD45{sup low}c-Kit{sup high} cells were most able to form hematopoietic cell colonies, but their ability decreased after E11.5 and was undetectable at E13.5 and later. The CD45{sup low}c-Kit{sup high} cells showed multipotency in vitro. We demonstrated further enrichment of hematopoietic activity in the Hoechst dye-effluxing side population among the CD45{sup low}c-Kit{sup high} cells. Here, we determined that CD45{sup low}c-Kit{sup high} cells arise from the lateral plate mesoderm using embryonic stem cell-derived differentiation system. In conclusion, CD45{sup low}c-Kit{sup high} cells are the major hematopoietic cells of mouse AGM.« less

Bone-induced c-kit expression in prostate cancer: a driver of intraosseous tumor growth

PubMed Central

Mainetti, Leandro E.; Zhe, Xiaoning; Diedrich, Jonathan; Saliganan, Allen D.; Cho, Won Jin; Cher, Michael L.; Heath, Elisabeth; Fridman, Rafael; Kim, Hyeong-Reh Choi; Bonfil, R. Daniel

2014-01-01

Loss of BRCA2 function stimulates prostate cancer (PCa) cell invasion and is associated with more aggressive and metastatic tumors in PCa patients. Concurrently, the receptor tyrosine kinase c-kit is highly expressed in skeletal metastases of PCa patients and induced in PCa cells placed into the bone microenvironment in experimental models. However, the precise requirement of c-kit for intraosseous growth of PCa and its relation to BRCA2 expression remain unexplored. Here, we show that c-kit expression promotes migration and invasion of PCa cells. Alongside, we found that c-kit expression in PCa cells parallels BRCA2 downregulation. Gene rescue experiments with human BRCA2 transgene in c-kit-transfected PCa cells resulted in reduction of c-kit protein expression and migration and invasion, suggesting a functional significance of BRCA2 downregulation by c-kit. The inverse association between c-kit and BRCA2 gene expressions in PCa cells was confirmed using laser capture microdissection in experimental intraosseous tumors and bone metastases of PCa patients. Inhibition of bone-induced c-kit expression in PCa cells transduced with lentiviral short hairpin RNA reduced intraosseous tumor incidence and growth. Overall, our results provide evidence of a novel pathway that links bone-induced c-kit expression in PCa cells to BRCA2 downregulation and supports bone metastasis. PMID:24798488

Factors for C-Kit Expression in Cardiac Outgrowth Cells and Human Heart Tissue.

PubMed

Matsushita, Satoshi; Minematsu, Kazuo; Yamamoto, Taira; Inaba, Hirotaka; Kuwaki, Kenji; Shimada, Akie; Yokoyama, Yasutaka; Amano, Atsushi

2017-12-12

We determined the factors associated with the expression of c-kit in the heart and the proliferation of c-kit-positive (c-kit pos ) cardiac stem cells among the outgrowth cells cultured from human cardiac explants.Samples of the right atrium (RA), left atrium (LA), and left ventricle obtained from patients during open-heart surgery were processed for cell culture of outgrowth cells and tissue analysis. The total number of growing cells and the population of c-kit pos cells were measured and compared with c-kit expression in native tissues and characteristics of the patients according to the region of the heart.We analyzed 452 samples from 334 patients. Atrial fibrillation (AF) in the patients reduced the number of outgrowth cells from the RA and LA, and aging was a co-factor for the LA. The c-kit pos population from the RA was associated with serum brain natriuretic peptide (BNP). C-kit expression in native tissue was also associated with BNP expression. However, we observed no relationship in expression between outgrowth cells and native tissue. In addition, the RA tissue provided the highest number of c-kit pos cells, and the left ventricle provided the lowest.C-kit was weakly expressed in response to damage. In addition, no correlation between outgrowth cells and native tissue was found for c-kit expression.

Identification of a polymorphism in the transmembrane domain of the protooncogene c-kit in healthy subjects.

PubMed

Nagata, H; Worobec, A S; Metcalfe, D D

1996-01-01

c-Kit is the receptor for stem cell factor (SCF) and is found on hematopoietic stem cells, mast cells, melanocytes, and germ cells. Aggregation of c-Kit by SCF regulates cell proliferation, differentiation, and survival. In the process of examining c-Kit, a polymorphism in the transmembrane domain of the protooncogene c-Kit was identified. This polymorphism consisted of an A-to-C transversion at nucleotide (nt) 1642, and was deduced to substitute leucine for methionine at codon 541. The frequency of the allele with 'C' at nt 1642 was 0.09 in 64 unrelated subjects. Analysis of a two-generation family with the polymorphism suggested that this polymorphism did not result in disease. This is the first report of a polymorphism in the transmembrane domain of c-Kit, and may be of value in understanding and following the function of c-Kit in normal subjects and in those with other abnormalities of c-Kit.

Comparison of the Effects of Carbon Ion and Photon Irradiation on the Angiogenic Response in Human Lung Adenocarcinoma Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamlah, Florentine, E-mail: Kamlah@staff.uni-marburg.de; Haenze, Joerg; Arenz, Andrea

2011-08-01

Purpose: Radiotherapy resistance is a commonly encountered problem in cancer treatment. In this regard, stabilization of endothelial cells and release of angiogenic factors by cancer cells contribute to this problem. In this study, we used human lung adenocarcinoma (A549) cells to compare the effects of carbon ion and X-ray irradiation on the cells' angiogenic response. Methods and Materials: A549 cells were irradiated with biologically equivalent doses for cell survival of either carbon ions (linear energy transfer, 170 keV/{mu}m; energy of 9.8 MeV/u on target) or X-rays and injected with basement membrane matrix into BALB/c nu/nu mice to generate a plug,more » allowing quantification of angiogenesis by blood vessel enumeration. The expression of angiogenic factors (VEGF, PlGF, SDF-1, and SCF) was assessed at the mRNA and secreted protein levels by using real-time reverse transcription-PCR and enzyme-linked immunosorbent assay. Signal transduction mediated by stem cell factor (SCF) was assessed by phosphorylation of its receptor c-Kit. For inhibition of SCF/c-Kit signaling, a specific SCF/c-Kit inhibitor (ISCK03) was used. Results: Irradiation of A549 cells with X-rays (6 Gy) but not carbon ions (2 Gy) resulted in a significant increase in blood vessel density (control, 20.71 {+-} 1.55; X-ray, 36.44 {+-} 3.44; carbon ion, 16.33 {+-} 1.03; number per microscopic field). Concordantly, irradiation with X-rays but not with carbon ions increased the expression of SCF and subsequently caused phosphorylation of c-Kit in endothelial cells. ISCK03 treatment of A549 cells irradiated with X-rays (6 Gy) resulted in a significant decrease in blood vessel density (X-ray, 36.44 {+-} 3.44; X-ray and ISCK03, 4.33 {+-} 0.71; number of microscopic field). These data indicate that irradiation of A549 cells with X-rays but not with carbon ions promotes angiogenesis. Conclusions: The present study provides evidence that SCF is an X-ray-induced mediator of angiogenesis in A549 cells, a phenomenon that could not be observed with carbon ion irradiation. Thus, in this model system evaluating angiogenesis, carbon ion irradiation may have a therapeutic advantage. This observation should be confirmed in orthotopic lung tumor models.« less

miR-137 downregulates c-kit expression in acute myeloid leukemia.

PubMed

Hu, Yanping; Dong, Xiaolong; Chu, Guoming; Lai, Guangrui; Zhang, Bijun; Wang, Leitong; Zhao, Yanyan

2017-06-01

The oncogene c-kit plays a vital role in the pathogenesis of acute myeloid leukemia (AML). However, the mechanism of microRNAs targeting c-kit in AML has not been determined in detail. Moreover, the role miR-137 in tumor cell proliferation remains controversial. The aim of this work was to verify whether miR-137 targets c-kit and to research the biological effects of restoring miR-137 expression in leukemia cells. We found that miR-137 binds specifically to the 3'-UTR of c-kit and suppresses the expression and activities of c-kit. There is a negative correlation between miR-137 and c-kit expression in both patients and cell lines determined by screening large clinical samples. We found that miR-137 can inhibit proliferation, promote apoptosis, and induce differentiation of c-kit+ AML cells. We determined that miR-137 can participate in the leukemogenesis by regulating c-kit, which could be used as a therapeutic target for acute myeloid leukemia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

PubMed

Monsanto, Megan M; White, Kevin S; Kim, Taeyong; Wang, Bingyan J; Fisher, Kristina; Ilves, Kelli; Khalafalla, Farid G; Casillas, Alexandria; Broughton, Kathleen; Mohsin, Sadia; Dembitsky, Walter P; Sussman, Mark A

2017-07-07

The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm 3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit + cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit - mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit + population is further enriched by selection for a CD133 + endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients. © 2017 American Heart Association, Inc.

Activated c-Kit receptor in the heart promotes cardiac repair and regeneration after injury

PubMed Central

Di Siena, S; Gimmelli, R; Nori, S L; Barbagallo, F; Campolo, F; Dolci, S; Rossi, P; Venneri, M A; Giannetta, E; Gianfrilli, D; Feigenbaum, L; Lenzi, A; Naro, F; Cianflone, E; Mancuso, T; Torella, D; Isidori, A M; Pellegrini, M

2016-01-01

The role of endogenous c-Kit receptor activation on cardiac cell homeostasis and repair remains largely unexplored. Transgenic mice carrying an activating point mutation (TgD814Y) in the kinase domain of the c-Kit gene were generated. c-KitTgD814Y receptor was expressed in the heart during embryonic development and postnatal life, in a similar timing and expression pattern to that of the endogenous gene, but not in the hematopoietic compartment allowing the study of a cardiac-specific phenotype. c-KitTgD814Y mutation produced a constitutive active c-Kit receptor in cardiac tissue and cells from transgenic mice as demonstrated by the increased phosphorylation of ERK1/2 and AKT, which are the main downstream molecular effectors of c-Kit receptor signaling. In adult transgenic hearts, cardiac morphology, size and total c-Kit+ cardiac cell number was not different compared with wt mice. However, when c-KitTgD814Y mice were subjected to transmural necrotic heart damage by cryoinjury (CI), all transgenic survived, compared with half of wt mice. In the sub-acute phase after CI, transgenic and wt mice showed similar heart damage. However, 9 days after CI, transgenic mice exhibited an increased number of c-Kit+CD31+ endothelial progenitor cells surrounding the necrotic area. At later follow-up, a consistent reduction of fibrotic area, increased capillary density and increased cardiomyocyte replenishment rate (as established by BrdU incorporation) were observed in transgenic compared with wt mice. Consistently, CD45−c-Kit+ cardiac stem cells isolated from transgenic c-KitTgD814Y mice showed an enhanced endothelial and cardiomyocyte differentiation potential compared with cells isolated from the wt. Constitutive activation of c-Kit receptor in mice is associated with an increased cardiac myogenic and vasculogenic reparative potential after injury, with a significant improvement of survival. PMID:27468693

Histone deacetylase inhibitor SAHA mediates mast cell death and epigenetic silencing of constitutively active D816V KIT in systemic mastocytosis.

PubMed

Lyberg, Katarina; Ali, Hani Abdulkadir; Grootens, Jennine; Kjellander, Matilda; Tirfing, Malin; Arock, Michel; Hägglund, Hans; Nilsson, Gunnar; Ungerstedt, Johanna

2017-02-07

Systemic mastocytosis (SM) is a clonal bone marrow disorder, where therapeutical options are limited. Over 90% of the patients carry the D816V point mutation in the KIT receptor that renders this receptor constitutively active. We assessed the sensitivity of primary mast cells (MC) and mast cell lines HMC1.2 (D816V mutated), ROSA (KIT WT) and ROSA (KIT D816V) cells to histone deacetylase inhibitor (HDACi) treatment. We found that of four HDACi, suberoyl anilide hydroxamic acid (SAHA) was the most effective in killing mutated MC. SAHA downregulated KIT, followed by major MC apoptosis. Primary SM patient MC cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas primary MC from healthy subjects were less affected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive SM, with almost 100% cell death among MC from the mast cell leukemia patient. Additionally, ROSA (KIT D816V) was more affected by HDACi than ROSA (KIT WT) cells. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/H3 decreased significantly in the KIT region. This epigenetic silencing was seen only in the KIT region and not in control genes upstream and downstream of KIT, indicating that the downregulation of KIT is exerted by specific epigenetic silencing. In conclusion, KIT D816V mutation sensitized MC to HDACi mediated killing, and SAHA may be of value as specific treatment for SM, although the specific mechanism of action requires further investigation.

Histone deacetylase inhibitor SAHA mediates mast cell death and epigenetic silencing of constitutively active D816V KIT in systemic mastocytosis

PubMed Central

Lyberg, Katarina; Ali, Hani Abdulkadir; Grootens, Jennine; Kjellander, Matilda; Tirfing, Malin; Arock, Michel; Hägglund, Hans

2017-01-01

Systemic mastocytosis (SM) is a clonal bone marrow disorder, where therapeutical options are limited. Over 90% of the patients carry the D816V point mutation in the KIT receptor that renders this receptor constitutively active. We assessed the sensitivity of primary mast cells (MC) and mast cell lines HMC1.2 (D816V mutated), ROSA (KIT WT) and ROSA (KIT D816V) cells to histone deacetylase inhibitor (HDACi) treatment. We found that of four HDACi, suberoyl anilide hydroxamic acid (SAHA) was the most effective in killing mutated MC. SAHA downregulated KIT, followed by major MC apoptosis. Primary SM patient MC cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas primary MC from healthy subjects were less affected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive SM, with almost 100% cell death among MC from the mast cell leukemia patient. Additionally, ROSA (KIT D816V) was more affected by HDACi than ROSA (KIT WT) cells. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/H3 decreased significantly in the KIT region. This epigenetic silencing was seen only in the KIT region and not in control genes upstream and downstream of KIT, indicating that the downregulation of KIT is exerted by specific epigenetic silencing. In conclusion, KIT D816V mutation sensitized MC to HDACi mediated killing, and SAHA may be of value as specific treatment for SM, although the specific mechanism of action requires further investigation. PMID:28038453

Transplantation of Epigenetically Modified Adult Cardiac c-Kit+ Cells Retards Remodeling and Improves Cardiac Function in Ischemic Heart Failure Model

PubMed Central

Zakharova, Liudmila; Nural-Guvener, Hikmet; Feehery, Lorraine; Popovic-Sljukic, Snjezana

2015-01-01

Cardiac c-Kit+ cells have a modest cardiogenic potential that could limit their efficacy in heart disease treatment. The present study was designed to augment the cardiogenic potential of cardiac c-Kit+ cells through class I histone deacetylase (HDAC) inhibition and evaluate their therapeutic potency in the chronic heart failure (CHF) animal model. Myocardial infarction (MI) was created by coronary artery occlusion in rats. c-Kit+ cells were treated with mocetinostat (MOCE), a specific class I HDAC inhibitor. At 3 weeks after MI, CHF animals were retrogradely infused with untreated (control) or MOCE-treated c-Kit+ cells (MOCE/c-Kit+ cells) and evaluated at 3 weeks after cell infusion. We found that class I HDAC inhibition in c-Kit+ cells elevated the level of acetylated histone H3 (AcH3) and increased AcH3 levels in the promoter regions of pluripotent and cardiac-specific genes. Epigenetic changes were accompanied by increased expression of cardiac-specific markers. Transplantation of CHF rats with either control or MOCE/c-Kit+ cells resulted in an improvement in cardiac function, retardation of CHF remodeling made evident by increased vascularization and scar size, and cardiomyocyte hypertrophy reduction. Compared with CHF infused with control cells, infusion of MOCE/c-Kit+ cells resulted in a further reduction in left ventricle end-diastolic pressure and total collagen and an increase in interleukin-6 expression. The low engraftment of infused cells suggests that paracrine effects might account for the beneficial effects of c-Kit+ cells in CHF. In conclusion, selective inhibition of class I HDACs induced expression of cardiac markers in c-Kit+ cells and partially augmented the efficacy of these cells for CHF repair. Significance The study has shown that selective class 1 histone deacetylase inhibition is sufficient to redirect c-Kit+ cells toward a cardiac fate. Epigenetically modified c-Kit+ cells improved contractile function and retarded remodeling of the congestive heart failure heart. This study provides new insights into the efficacy of cardiac c-Kit+ cells in the ischemic heart failure model. PMID:26240433

Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site.

PubMed

Herbst, R; Munemitsu, S; Ullrich, A

1995-01-19

The transforming gene of the Hardy-Zuckerman-4 strain of feline sarcoma virus, v-kit, arose by transduction of the cellular c-kit gene, which encodes the receptor tyrosine kinase (RTK) p145c-kit. To gain insight into the molecular basis of the v-kit transforming potential, we characterized the feline c-kit by cDNA cloning. Comparison of the feline v-kit and c-kit sequences revealed, in addition to deletions of the extracellular and transmembrane domains, three additional mutations in the v-kit oncogene product: deletion of tyrosine-569 and valine-570, the exchange of aspartate at position 761 to glycine, and replacement of the C-terminal 50 amino acids by five unrelated residues. Examinations of individual v-kit mutations in the context of chimeric receptors yielded inhibitory effects for some mutants on both autophosphorylation and substrate phosphorylation functions. In contrast, deletion of tyrosine-569 and valine-570 significantly enhanced transforming and mitogenic activities of p145c-kit, while the other mutations had no significant effects. Conservation in subclass III RTKs and the identification of the corresponding residue in beta PDGF-R, Y579, as a binding site for src family tyrosine kinases suggests an important role for Y568 in kit signal regulation and the definition of its oncogenic potential. Repositioning of Y571 by an inframe two codon deletion may be the crucial alteration resulting in enhancement of v-kit oncogenic activity.

The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions

PubMed Central

Pilloni, L.; Bianco, P.; Difelice, E.; Cabras, S.; Castellanos, M.E.; Atzori, L.; Ferreli, C.; Mulas, P.; Nemolato, S.; Faa, G.

2011-01-01

C-Kit (CD117), the receptor for the stem cell factor, a growth factor for melanocyte migration and proliferation, has shown differential immunostaining in various benign and malignant melanocytic lesions. The purpose of this study is to compare c-Kit immunostaining in benign nevi and in primary and metastatic malignant melanomas, to determine whether c-Kit can aid in the differential diagnosis of these lesions. c-Kit immunostaining was performed in 60 cases of pigmented lesions, including 39 benign nevi (5 blue nevi, 5 intra-dermal nevi, 3 junctional nevi, 15 cases of primary compound nevus, 11 cases of Spitz nevus), 18 cases of primary malignant melanoma and 3 cases of metastatic melanoma. The vast majority of nevi and melanomas examined in this study were positive for c-Kit, with minimal differences between benign and malignant lesions. C-Kit cytoplasmatic immunoreactivity in the intraepidermal proliferating nevus cells, was detected in benign pigmented lesions as well as in malignant melanoma, increasing with the age of patients (P=0.007) in both groups. The patient’s age at presentation appeared to be the variable able to cluster benign and malignant pigmented lesions. The percentage of c-Kit positive intraepidermal nevus cells was better associated with age despite other variables (P=0.014). The intensity and percentage of c-Kit positivity in the proliferating nevus cells in the dermis was significantly increased in malignant melanocytic lesions (P=0.015 and P=0.008) compared to benign lesions (compound melanocytic nevi, Spitz nevi, intradermal nevi, blue nevi). Immunostaning for c-Kit in metastatic melanomas was negative. Interestingly in two cases of melanoma occurring on a pre-existent nevus, the melanoma tumor cells showed strong cytoplasmatic and membranous positivity for c-kit, in contrast with the absence of any immunoreactivity in pre-existent intradermal nevus cells. C-Kit does not appear to be a strong immunohistochemical marker for distinguishing melanoma from melanocytic nevi, if we consider c-Kit expression in intraepidermal proliferating cells. The c-Kit expression in proliferating melanocytes in the dermis could help in the differential diagnosis between a superficial spreading melanoma (with dermis invasion) and a compound nevus or an intradermal nevus. Finally, c-Kit could be a good diagnostic tool for distinguishing benign compound nevi from malignant melanocytic lesions with dermis invasion and to differentiate metastatic melanoma from primary melanoma. PMID:22193299

FES kinase participates in KIT-ligand induced chemotaxis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voisset, Edwige, E-mail: Edwige.Voisset@inserm.fr; Institut Paoli-Calmettes, Marseille; Universite de la Mediterranee, Aix-Marseille II

2010-02-26

FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicatedmore » in cell migration.« less

Loss of c-Kit function impairs arteriogenesis in a mouse model of hindlimb ischemia.

PubMed

Hernandez, Diana R; Artiles, Adriana; Duque, Juan C; Martinez, Laisel; Pinto, Mariana T; Webster, Keith A; Velazquez, Omaida C; Vazquez-Padron, Roberto I; Lassance-Soares, Roberta M

2018-04-01

Arteriogenesis is a process whereby collateral vessels remodel usually in response to increased blood flow and/or wall stress. Remodeling of collaterals can function as a natural bypass to alleviate ischemia during arterial occlusion. Here we used a genetic approach to investigate possible roles of tyrosine receptor c-Kit in arteriogenesis. Mutant mice with loss of c-Kit function (Kit W/W-v ), and controls were subjected to hindlimb ischemia. Blood flow recovery was evaluated pre-, post-, and weekly after ischemia. Foot ischemic damage and function were assessed between days 1 to 14 post-ischemia while collaterals remodeling were measured 28 days post-ischemia. Both groups of mice also were subjected to wild type bone marrow cells transplantation 3 weeks before hindlimb ischemia to evaluate possible contributions of defective bone marrow c-Kit expression on vascular recovery. Kit W/W-v mice displayed impaired blood flow recovery, greater ischemic damage and foot dysfunction after ischemia compared to controls. Kit W/W-v mice also demonstrated impaired collateral remodeling consistent with flow recovery findings. Because arteriogenesis is a biological process that involves bone marrow-derived cells, we investigated which source of c-Kit signaling (bone marrow or vascular) plays a major role in arteriogenesis. Kit W/W-v mice transplanted with bone marrow wild type cells exhibited similar phenotype of impaired blood flow recovery, greater tissue ischemic damage and foot dysfunction as nontransplanted Kit W/W-v mice. This study provides evidence that c-Kit signaling is required during arteriogenesis. Also, it strongly suggests a vascular role for c-Kit signaling because rescue of systemic c-Kit activity by bone marrow transplantation did not augment the functional recovery of Kit W/W-v mouse hindlimbs. Copyright © 2017 Elsevier Inc. All rights reserved.

c-kit expression profile and regulatory factors during spermatogonial stem cell differentiation

PubMed Central

2013-01-01

Background It has been proven that c-kit is crucial for proliferation, migration, survival and maturation of spermatogenic cells. A periodic expression of c-kit is observed from primordial germ cells (PGCs) to spermatogenetic stem cells (SSCs), However, the expression profile of c-kit during the entire spermatogenesis process is still unclear. This study aims to reveal and compare c-kit expression profiles in the SSCs before and after the anticipated differentiation, as well as to examine its relationship with retinoic acid (RA) stimulation. Results We have found that there are more than 4 transcripts of c-kit expressed in the cell lines and in the testes. The transcripts can be divided into short and long categories. The long transcripts include the full-length canonical c-kit transcript and the 3′ end short transcript. Short transcripts include the 3.4 kb short transcript and several truncated transcripts (1.9-3.2 kb). In addition, the 3.4 kb transcript (starting from intron 9 and covering exons 10 ~ 21) is discovered to be specifically expressed in the spermatogonia. The extracellular domain of Kit is obtained in the spermatogonia stage, but the intracellular domain (50 kDa) is constantly expressed in both SSCs and spermatogonia. The c-kit expression profiles in the testis and the spermatogonial stem cell lines vary after RA stimulation. The wave-like changes of the quantitative expression pattern of c-kit (increase initially and decrease afterwards) during the induction process are similar to that of the in vivo male germ cell development process. Conclusions There are dynamic transcription and translation changes of c-kit before and after SSCs’ anticipated differentiation and most importantly, RA is a significant upstream regulatory factor for c-kit expression. PMID:24161026

The stem cell factor (SCF)/c-KIT signalling in testis and prostate cancer.

PubMed

Cardoso, Henrique J; Figueira, Marília I; Socorro, Sílvia

2017-12-01

The stem cell factor (SCF) is a cytokine that specifically binds the tyrosine kinase receptor c-KIT. The SCF/c-KIT interaction leads to receptor dimerization, activation of kinase activity and initiation of several signal transduction pathways that control cell proliferation, apoptosis, differentiation and migration in several tissues. The activity of SCF/c-KIT system is linked with the phosphatidylinositol 3-kinase (PI3-K), the Src, the Janus kinase/signal transducers and activators of transcription (JAK/STAT), the phospholipase-C (PLC-γ) and the mitogen-activated protein kinase (MAPK) pathways. Moreover, it has been reported that cancer cases display an overactivation of c-KIT due to the presence of gain-of-function mutations or receptor overexpression, which renders c-KIT a tempting target for cancer treatment. In the case of male cancers the most documented activated pathways are the PI3-K and Src, both enhancing abnormal cell proliferation. It is also known that the Src activity in prostate cancer cases depends on the presence of tr-KIT, the cytoplasmic truncated variant of c-KIT that is specifically expressed in tumour tissues and, thus, a very interesting target for drug development. The present review provides an overview of the signalling pathways activated by SCF/c-KIT and discusses the potential application of c-KIT inhibitors for treatment of testicular and prostatic cancers.

c-kit Positive Cardiac Outgrowth Cells Demonstrate Better Ability for Cardiac Recovery Against Ischemic Myopathy.

PubMed

Li, Chuan; Matsushita, Satoshi; Li, Zhengqing; Guan, Jianjun; Amano, Atsushi

2017-10-01

Resident cardiac stem cells are expected to be a therapeutic option for patients who suffer from severe heart failure. However, uncertainty remains over whether sorting cells for c-kit, a stem cell marker, improves therapeutic outcomes. Cardiac outgrowth cells cultured from explants of rat heart atrium were sorted according to their positivity (+) or negativity (-) for c-kit. These cells were exposed to hypoxia for 3 d, and subsequently harvested for mRNA expression measurement. The cell medium was also collected to assess cytokine secretion. To test for a functional benefit in animals, myocardial infarction (MI) was induced in rats, and c-kit+ or c-kit- cells were injected. The left ventricular ejection fraction (LVEF) was measured for up to 4 weeks, after which the heart was harvested for biological and histological analyses. Expression of the angiogenesis-related genes, VEGF and ANGPTL2, was significantly higher in c-kit+ cells after 3 d of hypoxic culture, although we found no such difference prior to hypoxia. Secretion of VEGF and ANGPTL2 was greater in the c-kit+ group than in the c-kit- group, while hypoxia tended to increase cytokine expression in both groups. In addition, IGF-1 was significantly increased in the c-kit+ group, consistent with the relatively low expression of cleaved-caspase 3 revealed by western blot assay, and the relatively low count of apoptotic cells revealed by histochemical analysis. Administration of c-kit+cells into the MI heart improved the LVEF and increased neovascularization. These results indicate that c-kit+cells may be useful in cardiac stem cell therapy.

Overexpression of c-kit(CD117), relevant with microvessel density, is an independent survival prognostic factor for patients with HBV-related hepatocellular carcinoma.

PubMed

Yan, Weiwei; Zhu, Zhenyu; Pan, Fei; Huang, Ang; Dai, Guang-Hai

2018-01-01

To explore new biomarkers for indicating the recurrence and prognosis in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients after tumor resection, we investigated the expression and prognostic value of c-kit(CD117) in HBV-related HCC. Immunohistochemistry was used to estimate the expression of c-kit(CD117) and CD34 in the liver cancer tissues. The correlations between the expression of these biomarkers and the clinicopathologic characteristics were analyzed. The positive rate of c-kit(CD117) expression in 206 HCC cases was 48.1%, and c-kit expression was significantly related with CD34-positive microvessel density. CD34-microvessel density numbers were much higher in c-kit(+) HCC tissues than in c-kit(-) HCC tissues (44.13±17.01 vs 26.87±13.16, P =0.003). The expression of c-kit was significantly higher in patients with Edmondson grade III-IV ( P <0.001) and TNM stage III ( P <0.001). Moreover, Kaplan-Meier survival analysis showed that c-kit ( P <0.001) expression was correlated with reduced disease-free survival (DFS). Multivariate analysis identified c-kit as an independent poor prognostic factor of DFS in HCC patients ( P <0.001). Increased c-kit expression could be considered as an independent unfavorable prognostic factor for predicting DFS in HBV-related HCC patients after surgery. These results could be used to identify patients at a higher risk of early tumor recurrence and poor prognosis.

Elevated expression of the proto-oncogene c-kit in patients with mastocytosis.

PubMed

Nagata, H; Worobec, A S; Semere, T; Metcalfe, D D

1998-02-01

The stem cell factor (SCF)c-kit receptor interaction plays a critical role in the development and survival of mast cells. Several studies have also associated c-kit receptor mutations with the human diseases, mastocytosis and piebaldism. Overexpression of c-kit has been reported to be associated with myeloproliferative disorders and myelodysplastic syndromes. Using peripheral blood mononuclear cells (PBMCs) from 11 patients with indolent mastocytosis (category I), mastocytosis with an associated hematologic disorder (category II), or aggressive mastocytosis (category III); a patient with CMML unassociated with mastocytosis, and PBMCs from 13 normal subjects, we examined the level of expression of c-kit mRNA along with other c-kit isoforms to determine if overexpression of the c-kit receptor was associated with mastocytosis. Using quantitative competitive PCR, c-kit mRNA levels on average were found to be statistically elevated in the five patients with mastocytosis with an associated hematologic disorder and in the patient with aggressive mastocytosis as compared with controls, but not elevated in patients with indolent mastocytosis. The relative mRNA expression for the two c-kit isoforms was not significantly different in the mastocytosis patients compared with controls. This demonstration of the overexpression of c-kit mRNA in mastocytosis, and particularly those patients with clinical evidence of myelodysplastic syndrome, adds evidence to support the conclusion that mastocytosis, at least in some patients, is a feature of myelodysplasia; and suggests that determination of c-kit mRNA expression in PBMCs may provide an additional approach to assessing prognosis.

Evaluation of tyrosine-kinase receptor c-KIT (c-KIT) mutations, mRNA and protein expression in canine leukemia: might c-KIT represent a therapeutic target?

PubMed

Giantin, M; Aresu, L; Aricò, A; Gelain, M E; Riondato, F; Martini, V; Comazzi, S; Dacasto, M

2013-04-15

The tyrosine-kinase receptor c-KIT (c-KIT) plays an important role in proliferation, survival and differentiation of progenitor cells in normal hematopoietic cells. In human hematological malignancies, c-KIT is mostly expressed by progenitor cell neoplasia and seldom by those involving mature cells. Tyrosine kinase inhibitors (TKIs) are actually licensed for the first- and second-line treatment of human hematologic disorders. Aim of the present study was to evaluate c-KIT mRNA and protein expression and complementary DNA (cDNA) mutations in canine leukemia. Eleven acute lymphoblastic leukemia (ALL) and acute undifferentiated leukemia (AUL) and 12 chronic lymphocytic leukemia (CLL) were enrolled in this study. The amounts of c-KIT mRNA and protein were determined, in peripheral blood samples, by using quantitative real time RT-PCR, flow cytometry and immunocytochemistry, respectively. The presence of mutations on c-KIT exons 8-11 and 17 were investigated by cDNA sequencing. Higher amounts of c-KIT mRNA were found in ALL/AUL compared to CLL, and this latter showed a lower pattern of gene expression. Transcriptional data were confirmed at the protein level. No significant gain-of-function mutations were ever observed in both ALL/AUL and CLL. Among canine hematological malignancies, ALL/AUL typically show a very aggressive biological behavior, partly being attributable to the lack of efficacious therapeutic options. The high level of c-KIT expression found in canine ALL/AUL might represent the rationale for using TKIs in future clinical trials. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparison of lesional skin c-KIT mutations with clinical phenotype in patients with mastocytosis.

PubMed

Chan, I J; Tharp, M D

2018-06-01

Activating c-KIT mutations cause abnormal mast cell growth and appear to play a role in mastocytosis. However, the correlation of c-KIT mutations with disease phenotypes is poorly characterized. To evaluate the correlation of c-KIT mutations with clinical presentations and laboratory findings. Total cellular RNA was isolated from the skin lesions of 43 adults and 7 children with mastocytosis, and PCR amplicons of cDNA were sequenced for c-KIT mutations. The most common activating mutation, KIT-D816V, was identified in 72% of adults and 57% of children. Additional activating mutations, namely, V560G and the internal tandem duplications (ITDs) 502-503dupAY, were detected in 12% of adults and 8% of children. V560G occurred more commonly in our patients than previously reported, and it appeared to be associated with more advanced disease. Otherwise, the presence or absence of activating mutations did not correlate with skin lesion morphology, disease extent or total serum tryptase levels. Four adults had expression only of wild-type KIT, while two others had expression of a truncated KIT lacking tyrosine kinase activity; yet these patients were clinically indistinguishable from those patients with activating c-KIT mutations. Activating c-KIT mutations exist in a significant portion of patients with mastocytosis, but not all patients showed expression of these mutations. Except for V560G, the presence or absence of activating c-KIT mutations did not predict the extent of disease. These observations suggest that although activating c-KIT mutations are associated with mast cell growth, other genes probably play a role in the cause of mastocytosis. © 2018 British Association of Dermatologists.

Tyrosine Kinase Inhibitors Induce Down-Regulation of c-Kit by Targeting the ATP Pocket

PubMed Central

Descarpentries, Clotilde; Frisan, Emilie; Adam, Kevin; Verdier, Frederique; Floquet, Célia; Dubreuil, Patrice; Lacombe, Catherine; Fontenay, Michaela; Mayeux, Patrick; Kosmider, Olivier

2013-01-01

The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket. PMID:23637779

Loss of c-KIT expression in thyroid cancer cells.

PubMed

Franceschi, Sara; Lessi, Francesca; Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria

2017-01-01

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.

Loss of c-KIT expression in thyroid cancer cells

PubMed Central

Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria

2017-01-01

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression. PMID:28301608

Spatial Distribution and Receptor Specificity of Zebrafish Kit System - Evidence for a Kit-Mediated Bi-Directional Communication System in the Preovulatory Ovarian Follicle

PubMed Central

Yao, Kai; Ge, Wei

2013-01-01

Consisting of Kit ligand and receptor Kit, the Kit system is involved in regulating many ovarian functions such as follicle activation, granulosa cell proliferation, and oocyte growth and maturation. In mammals, Kit ligand is derived from the granulosa cells and Kit receptor is expressed in the oocyte and theca cells. In the zebrafish, the Kit system contains two ligands (Kitlga and Kitlgb) and two receptors (Kita and Kitb). Interestingly, Kitlga and Kitb are localized in the somatic follicle cells, but Kitlgb and Kita are expressed in the oocyte. Using recombinant zebrafish Kitlga and Kitlgb, we demonstrated that Kitlga preferentially activated Kita whereas Kitlgb specifically activated Kitb by Western analysis for receptor phosphorylation. In support of this, Kitlgb triggered a stronger and longer MAPK phosphorylation in follicle cells than Kitlga, whereas Kitlga but not Kitlgb activated MAPK in the denuded oocytes, in agreement with the distribution of Kita and Kitb in the follicle and their specificity for Kitlga and Kitlgb. Further analysis of the interaction between Kit ligands and receptors by homology modeling showed that Kitlga-Kita and Kitlgb-Kitb both have more stable electrostatic interaction than Kitlgb-Kita or Kitlga-Kitb. A functional study of Kit involvement in final oocyte maturation showed that Kitlga and Kitlgb both suppressed the spontaneous maturation significantly; in contrast, Kitlgb but not Kitlga significantly promoted 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) -induced oocyte maturation. Our results provided strong evidence for a Kit-mediated bi-directional communication system in the zebrafish ovarian follicle, which could be part of the complex interplay between the oocyte and the follicle cells in the development of follicles. PMID:23409152

[Expression of c-kit in North African nasopharyngeal carcinomas: correlation with age and LMP1].

PubMed

Charfi, S; Khabir, A; Ayadi, L; Mseddi, M; Makni, H; Gorbel, A; Daoud, J; Frikha, M; Jlidi, R; Busson, P; Boudawara, T S

2007-09-01

To determine the level and prognostic significance of c-kit expression in the two age groups of North African nasopharyngeal carcinomas. A retrospective study of 99 NPC specimens from Tunisian patients was investigated by immunohistochemistry. Immunohistochemical data were correlated with Epstein-Barr virus LMP1 expression and pathological, clinical and survival parameters. c-kit was detected in 79% of the cases for patients under 30 years of age (juvenile form) but in only 56% of specimens in patients over 30 years (P=0.039) and was significantly over-expressed for patients with lymph node involvement (P=0.015). LMP1 score was 5.78 (+/-1.84) for c-kit negative tumors compared to 8,23 (+/-2.39) for c-kit positive tumors (P=0.002). Multivariate analysis including age, lymph nodes involvement and LMP1 expression as co-variables, showed that only age (P=0.027) and LMP1 expression (P=0.005) were significantly correlated to the c-kit expression. c-kit is highly expressed in the juvenile form of North African nasopharyngeal carcinomas. There is a significant association between LMP1 and c-kit expression. The contrasted levels of C-kit expression in the two age groups strengthen the hypothesis that these clinical forms result from distinct oncogenic mechanisms.

SDF1 gradient associates with the distribution of c-Kit+ cardiac cells in the heart.

PubMed

Renko, Outi; Tolonen, Anna-Maria; Rysä, Jaana; Magga, Johanna; Mustonen, Erja; Ruskoaho, Heikki; Serpi, Raisa

2018-01-18

Identification of the adult cardiac stem cells (CSCs) has offered new therapeutic possibilities for treating ischemic myocardium. CSCs positive for the cell surface antigen c-Kit are known as the primary source for cardiac regeneration. Accumulating evidence shows that chemokines play important roles in stem cell homing. Here we investigated molecular targets to be utilized in modulating the mobility of endogenous CSCs. In a four week follow-up after experimental acute myocardial infarction (AMI) with ligation of the left anterior descending (LAD) coronary artery of Sprague-Dawley rats c-Kit+ CSCs redistributed in the heart. The number of c-Kit+ CSCs in the atrial c-Kit niche was diminished, whereas increased amount was observed in the left ventricle and apex. This was associated with increased expression of stromal cell-derived factor 1 alpha (SDF1α), and a significant positive correlation was found between c-Kit+ CSCs and SDF1α expression in the heart. Moreover, the migratory capacity of isolated c-Kit+ CSCs was induced by SDF1 treatment in vitro. We conclude that upregulation of SDF1α after AMI associates with increased expression of endogenous c-Kit+ CSCs in the injury area, and show induced migration of c-Kit+ cells by SDF1.

The stem cell factor (SCF)/c-KIT system in carcinogenesis of reproductive tissues: What does the hormonal regulation tell us?

PubMed

Figueira, Marília I; Cardoso, Henrique J; Correia, Sara; Maia, Cláudio J; Socorro, Sílvia

2017-10-01

The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF) are expressed in several tissues of male and female reproductive tract, playing an important role in the regulation of basic biological processes. The activation of c-KIT by SCF controls, cell survival and death, cell differentiation and migration. Also, the SCF/c-KIT system has been implicated in carcinogenesis of reproductive tissues due to its altered expression pattern or overactivation in consequence of gain-of-functions mutations. Over the years, it has also been shown that hormones, the primary regulators of reproductive function and causative agents in the case of hormone-dependent cancers, are also able to control the SCF/c-KIT tissue levels. Therefore, it is liable to suppose that disturbed SCF/c-KIT expression driven by (de)regulated hormone actions can be a relevant step towards carcinogenesis. The present review describes the SCF and c-KIT expression in cancers of reproductive tissues, discussing the implications of the hormonal regulation of the SCF/c-KIT system in cancer development. Understanding the relationship between hormonal imbalance and the SCF/c-KIT expression and activity would be relevant in the context of novel therapeutic approaches in reproductive cancers. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells With D816V-KIT

PubMed Central

Bandara, Geethani; Muñoz-Cano, Rosa; Tobío, Araceli; Yin, Yuzhi; Komarow, Hirsh D.; Desai, Avanti; Metcalfe, Dean D.; Olivera, Ana

2018-01-01

Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 depletion, and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK isoforms reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DDR in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation. PMID:29643855

Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells With D816V-KIT.

PubMed

Bandara, Geethani; Muñoz-Cano, Rosa; Tobío, Araceli; Yin, Yuzhi; Komarow, Hirsh D; Desai, Avanti; Metcalfe, Dean D; Olivera, Ana

2018-01-01

Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 depletion, and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK isoforms reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DDR in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.

Increased c-kit and stem cell factor expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PubMed

Takahashi, Toshiaki; Friedmacher, Florian; Zimmer, Julia; Puri, Prem

2016-05-01

Persistent pulmonary hypertension(PPH) in congenital diaphragmatic hernia (CDH) is caused by increased vascular cell proliferation and endothelial cell (EC) dysfunction, thus leading to obstructive changes in the pulmonary vasculature. C-Kit and its ligand, stem cell factor(SCF), are expressed by ECs in the developing lung mesenchyme, suggesting an important role during lung vascular formation. Conversely, absence of c-Kit expression has been demonstrated in ECs of dysplastic alveolar capillaries. We hypothesized that c-Kit and SCF expression is increased in the pulmonary vasculature of nitrofen-induced CDH. Timed-pregnant rats received nitrofen or vehicle on gestational day 9(D9). Fetuses were sacrificed on D15, D18, and D21, and divided into control and CDH group. Pulmonary gene expression levels of c-Kit and SCF were analyzed by qRT-PCR. Immunofluorescence double staining for c-Kit and SCF was combined with CD34 to evaluate protein expression in ECs of the pulmonary vasculature. Relative mRNA levels of c-Kit and SCF were significantly increased in lungs of CDH fetuses on D15, D18, and D21 compared to controls. Confocal laser scanning microscopy confirmed markedly increased vascular c-Kit and SCF expression in mesenchymal ECs of CDH lungs on D15, D18, and D21 compared to controls. Increased expression of c-Kit and SCF in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that increased c-Kit signaling during lung vascular formation may contribute to vascular remodeling and thus to PPH. Copyright © 2016 Elsevier Inc. All rights reserved.

Immunohistochemical study of C-kit expression in subtypes of renal cell carcinoma.

PubMed

Norouzinia, Farahnaz; Abbasi, Fariba; Dindarian, Sina; Mohammadi, Sedra; Meisami, Farid; Bagheri, Mahdi; Mohammadi, Hozan

2018-01-01

Renal cell carcinomas (RCCs) include about 2% of adult neoplasms and 90-95% of all renal tumors. Mostly, it is possible to distinguish RCC subtypes using hematoxylin-eosin staining. However, overlapping morphologic features cause some difficulties in making a precise diagnosis. In order to render an accurate diagnosis, additional methods such as immunohistochemical staining for c-kit have been recommended. In this study, we aimed to investigate c-kit gene expression in various subtypes of RCC. We reviewed 65 diagnosed RCC cases. Formalin- fixed, paraffin- embedded specimens were available for the cases. The expression of c-kit was evaluated using immunohistochemistry. The correlation between c-kit expression and clinicopathological parameters including patients' age and gender in addition to grade, stage, and size of the tumor were investigated. Six cases of 39 clear cell types (15.4%), 8 of 13 papillary types (61.5%), 11 of 12 chromophobe types (91.7%), and no sarcomatoid type were positive for c-kit expression. Based on chi-square test results, there was a significant relationship between RCC subtypes and c-kit expression (p=0.001). There was no significant correlation between age, sex, grade, stage, and size of the tumor and c-kit expression. The expression of c-kit in RCC may have diagnostic significance in subtypes of RCC especially papillary and chromophobe subtypes of RCC.

c-kit+ Cells Minimally Contribute Cardiomyocytes to the Heart

PubMed Central

van Berlo, Jop H.; Kanisicak, Onur; Maillet, Marjorie; Vagnozzi, Ronald J.; Karch, Jason; Lin, Suh-Chin J.; Middleton, Ryan C.; Marbán, Eduardo; Molkentin, Jeffery D.

2014-01-01

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine if endogenous c-kit+ cells contribute differentiated cardiomyocytes to the heart during development, with aging or after injury in adulthood. A cDNA encoding either Cre recombinase or a tamoxifen inducible MerCreMer chimeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit+ cells did produce new cardiomyocytes within the heart, although at a percentage of ≈0.03% or less, and if a preponderance towards cellular fusion is considered, the percentage falls below ≈0.008%. In contrast, c-kit+ cells amply generated cardiac endothelial cells. Thus, endogenous c-kit+ cells can generate cardiomyocytes within the heart, although likely at a functionally insignificant level. PMID:24805242

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pedersen, Malin; Loefstedt, Tobias; Sun Jianmin

Signaling by the receptor for stem cell factor (SCF), c-Kit, is of major importance for hematopoiesis, melanogenesis and reproduction, and the biological responses are commonly proliferation and cell survival. Thus, constitutive activation due to c-Kit mutations is involved in the pathogenesis of several forms of cancer, e.g. leukemias, gastrointestinal stromal tumors and testicular tumors. Tumor survival requires oxygen supply through induced neovascularization, a process largely mediated by the vascular endothelial growth factor (VEGF), a prominent target of the transcription factors hypoxia-inducible factor-1 (HIF-1) and HIF-2. Using Affymetrix microarrays we have identified genes that are upregulated following SCF stimulation. Interestingly, manymore » of the genes induced were found to be related to a hypoxic response. These findings were corroborated by our observation that SCF stimulation of the hematopoietic cell lines M-07e induces HIF-1{alpha} and HIF-2{alpha} protein accumulation at normoxia. In addition, SCF-induced HIF-1{alpha} was transcriptionally active, and transcribed HIF-1 target genes such as VEGF, BNIP3, GLUT1 and DEC1, an effect that could be reversed by siRNA against HIF-1{alpha}. We also show that SCF-induced accumulation of HIF-1{alpha} is dependent on both the PI-3-kinase and Ras/MEK/Erk pathways. Our data suggest a novel mechanism of SCF/c-Kit signaling in angiogenesis and tumor progression.« less

[4-t-butylphenyl]-N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03) inhibits SCF/c-kit signaling in 501mel human melanoma cells and abolishes melanin production in mice and brownish guinea pigs.

PubMed

Na, Yong Joo; Baek, Heung Su; Ahn, Soo Mi; Shin, Hyun Jung; Chang, Ih-Seop; Hwang, Jae Sung

2007-09-01

It is well known that c-kit is related to pigmentation as well as to the oncology target protein. The objective of this study was to discover a skin-whitening agent that regulates c-kit activity. We have developed a high-throughput screening system using recombinant human c-kit protein. Approximately 10,000 synthetic compounds were screened for their effect on c-kit activity. Phenyl-imidazole sulfonamide derivatives showed inhibitory activity on c-kit phosphorylation in vitro. The effects of one derivative, [4-t-butylphenyl]-N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03), on stem-cell factor (SCF)/c-kit cellular signaling in 501mel human melanoma cells were examined further. Pretreatment of 501mel cells with ISCK03 inhibited SCF-induced c-kit phosphorylation dose dependently. ISCK03 also inhibited p44/42 ERK mitogen-activated protein kinase (MAPK) phosphorylation, which is known to be involved in SCF/c-kit downstream signaling. However ISCK03 did not inhibit hepatocyte growth factor (HGF)-induced phosphorylation of p44/42 ERK proteins. To determine the in vivo potency of ISCK03, it was orally administered to depilated C57BL/6 mice. Interestingly, oral administration of ISCK03 induced the dose-dependent depigmentation of newly regrown hair, and this was reversed with cessation of ISCK03 treatment. Finally, to investigate whether the inhibitory effect of ISCK03 on SCF/c-kit signaling abolished UV-induced pigmentation, ISCK03 was applied to UV-induced pigmented spots on brownish guinea pig skin. The topical application of ISCK03 promoted the depigmentation of UV-induced hyperpigmented spots. Fontana-Masson staining analysis showed epidermal melanin was diminished in spots treated with ISCK03. These results indicate that phenyl-imidazole sulfonamide derivatives are potent c-kit inhibitors and might be used as skin-whitening agents.

Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.

PubMed Central

Nagata, H; Worobec, A S; Oh, C K; Chowdhury, B A; Tannenbaum, S; Suzuki, Y; Metcalfe, D D

1995-01-01

Both stem cells and mast cells express c-kit and proliferate after exposure to c-kit ligand. Mutations in c-kit may enhance or interfere with the ability of c-kit receptor to initiate the intracellular pathways resulting in cell proliferation. These observations suggested to us that mastocytosis might in some patients result from mutations in c-kit. cDNA synthesized from peripheral blood mononuclear cells of patients with indolent mastocytosis, mastocytosis with an associated hematologic disorder, aggressive mastocytosis, solitary mastocytoma, and chronic myelomonocytic leukemia unassociated with mastocytosis was thus screened for a mutation of c-kit. This analysis revealed that four of four mastocytosis patients with an associated hematologic disorder with predominantly myelodysplastic features had an A-->T substitution at nt 2468 of c-kit mRNA that causes an Asp-816-->Val substitution. One of one patient examined who had mastocytosis with an associated hematologic disorder had the corresponding mutation in genomic DNA. Identical or similar amino acid substitutions in mast cell lines result in ligand-independent autophosphorylation of the c-kit receptor. This mutation was not identified in the patients within the other disease categories or in 67 of 67 controls. The identification of the point mutation Asp816Val in c-kit in patients with mastocytosis with an associated hematologic disorder provides insight not only into the pathogenesis of this form of mastocytosis but also into how hematopoiesis may become dysregulated and may serve to provide a means of confirming the diagnosis, assessing prognosis, and developing intervention strategies. Images Fig. 1 Fig. 2 Fig. 3 PMID:7479840

Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.

PubMed

Nagata, H; Worobec, A S; Oh, C K; Chowdhury, B A; Tannenbaum, S; Suzuki, Y; Metcalfe, D D

1995-11-07

Both stem cells and mast cells express c-kit and proliferate after exposure to c-kit ligand. Mutations in c-kit may enhance or interfere with the ability of c-kit receptor to initiate the intracellular pathways resulting in cell proliferation. These observations suggested to us that mastocytosis might in some patients result from mutations in c-kit. cDNA synthesized from peripheral blood mononuclear cells of patients with indolent mastocytosis, mastocytosis with an associated hematologic disorder, aggressive mastocytosis, solitary mastocytoma, and chronic myelomonocytic leukemia unassociated with mastocytosis was thus screened for a mutation of c-kit. This analysis revealed that four of four mastocytosis patients with an associated hematologic disorder with predominantly myelodysplastic features had an A-->T substitution at nt 2468 of c-kit mRNA that causes an Asp-816-->Val substitution. One of one patient examined who had mastocytosis with an associated hematologic disorder had the corresponding mutation in genomic DNA. Identical or similar amino acid substitutions in mast cell lines result in ligand-independent autophosphorylation of the c-kit receptor. This mutation was not identified in the patients within the other disease categories or in 67 of 67 controls. The identification of the point mutation Asp816Val in c-kit in patients with mastocytosis with an associated hematologic disorder provides insight not only into the pathogenesis of this form of mastocytosis but also into how hematopoiesis may become dysregulated and may serve to provide a means of confirming the diagnosis, assessing prognosis, and developing intervention strategies.

Opposing roles of KIT and ABL1 in the therapeutic response of gastrointestinal stromal tumor (GIST) cells to imatinib mesylate.

PubMed

Rausch, Jessica L; Boichuk, Sergei; Ali, Areej A; Patil, Sneha S; Liu, Lijun; Lee, Donna M; Brown, Matthew F; Makielski, Kathleen R; Liu, Ying; Taguchi, Takahiro; Kuan, Shih-Fan; Duensing, Anette

2017-01-17

Most gastrointestinal stromal tumors (GISTs) are caused by activating mutations of the KIT receptor tyrosine kinase. The small molecule inhibitor imatinib mesylate was initially developed to target the ABL1 kinase, which is constitutively activated through chromosomal translocation in BCR-ABL1-positive chronic myeloid leukemia. Because of cross-reactivity of imatinib against the KIT kinase, the drug is also successfully used for the treatment of GIST. Although inhibition of KIT clearly has a major role in the therapeutic response of GIST to imatinib, the contribution of concomitant inhibition of ABL in this context has never been explored. We show here that ABL1 is expressed in the majority of GISTs, including human GIST cell lines. Using siRNA-mediated knockdown, we demonstrate that depletion of KIT in conjunction with ABL1 - hence mimicking imatinib treatment - leads to reduced apoptosis induction and attenuated inhibition of cellular proliferation when compared to depletion of KIT alone. These results are explained by an increased activity of the AKT survival kinase, which is mediated by the cyclin-dependent kinase CDK2, likely through direct phosphorylation. Our results highlight that distinct inhibitory properties of targeted agents can impede antitumor effects and hence provide insights for rational drug development. Novel KIT-targeted agents to treat GIST should therefore comprise an increased specificity for KIT while at the same time displaying a reduced ability to inhibit ABL1.

"String theory" of c-kit(pos) cardiac cells: a new paradigm regarding the nature of these cells that may reconcile apparently discrepant results.

PubMed

Keith, Matthew C L; Bolli, Roberto

2015-03-27

Although numerous preclinical investigations have consistently demonstrated salubrious effects of c-kit(pos) cardiac cells administered after myocardial infarction, the mechanism of action remains highly controversial. We and others have found little or no evidence that these cells differentiate into mature functional cardiomyocytes, suggesting paracrine effects. In this review, we propose a new paradigm predicated on a comprehensive analysis of the literature, including studies of cardiac development; we have (facetiously) dubbed this conceptual construct "string theory" of c-kit(pos) cardiac cells because it reconciles multifarious and sometimes apparently discrepant results. There is strong evidence that, during development, the c-kit receptor is expressed in different pools of cardiac progenitors (some capable of robust cardiomyogenesis and others with little or no contribution to myocytes). Accordingly, c-kit positivity, in itself, does not define the embryonic origins, lineage capabilities, or differentiation capacities of specific cardiac progenitors. C-kit(pos) cells derived from the first heart field exhibit cardiomyogenic potential during development, but these cells are likely depleted shortly before or after birth. The residual c-kit(pos) cells found in the adult heart are probably of proepicardial origin, possess a mesenchymal phenotype (resembling bone marrow mesenchymal stem/stromal cells), and are capable of contributing significantly only to nonmyocytic lineages (fibroblasts, smooth muscle cells, and endothelial cells). If these 2 populations (first heart field and proepicardium) express different levels of c-kit, the cardiomyogenic potential of first heart field progenitors might be reconciled with recent results of c-kit(pos) cell lineage tracing studies. The concept that c-kit expression in the adult heart identifies epicardium-derived, noncardiomyogenic precursors with a mesenchymal phenotype helps to explain the beneficial effects of c-kit(pos) cell administration to ischemically damaged hearts despite the observed paucity of cardiomyogenic differentiation of these cells. © 2015 American Heart Association, Inc.

Synthesis and biological evaluation of di-aryl urea derivatives as c-Kit inhibitors.

PubMed

Ravez, Séverine; Arsenlis, Stéphane; Barczyk, Amélie; Dupont, Anthony; Frédérick, Raphaël; Hesse, Stéphanie; Kirsch, Gilbert; Depreux, Patrick; Goossens, Laurence

2015-11-15

Inhibition of receptor tyrosine kinases (RTKs) continued to be a successful approach for the treatment of many types of human cancers and many potent small molecules kinase inhibitors have been discovered the last decade. In the present study, we describe the synthesis of thienopyrimidine derivatives and their pharmacological evaluation against nine kinases (EGFR, PDGFR-ß, c-Kit, c-Met, Src, Raf, VEGFR-1, -2 and -3). Most of the synthesized compounds showed from moderate to potent activities against c-Kit with IC50 values in the nanomolar range. Among them, 4-anilino(urea)thienopyrimidine analogs showed selectivity and potent c-Kit inhibition with IC50 values less than 6 nM. Docking simulation was performed for the most promising compound 9 into the c-Kit active site to determine the potential binding mode. This study reveal that the 4-anilino(urea)thienopyrimidine is an interesting scaffold to design novel potent and selective c-Kit inhibitors which may make promising candidates for cancers where c-Kit receptors are overexpressed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Pharmacological effects of a C-phycocyanin-based multicomponent nutraceutical in an in-vitro canine chondrocyte model of osteoarthritis

PubMed Central

Martinez, Stephanie E.; Chen, Yufei; Ho, Emmanuel A.; Martinez, Steven A.; Davies, Neal M.

2015-01-01

Multicomponent nutraceuticals are becoming increasingly popular treatments or adjunctive therapies for osteoarthritis in veterinary medicine despite lack of evidence of efficacy for many products. The objective of this study was to evaluate the anti-inflammatory and antioxidant activities of a commercially available C-phycocyanin-based nutraceutical and select constituent ingredients in an in-vitro model of canine osteoarthritis. Normal canine articular chondrocytes were used in an in-vitro model of osteoarthritis. Inflammatory conditions were induced using interleukin-1β. The nutraceutical preparation as a whole, its individual constituents, as well as carprofen were evaluated at concentrations of 0 to 250 μg/mL for reduction of the following inflammatory mediators and indicators of catabolism of the extracellular matrix: prostaglandin E2 (PGE2), tumor necrosis factor-α (TFN-α), interleukin-6 (IL-6), metalloproteinase-3 (MMP-3), nitric oxide, and sulfated glycosaminoglycans (sGAGs). Validated, commercially available assay kits were used for quantitation of inflammatory mediators. The antioxidant capacities, as well as cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and lipoxygenase (LOX) inhibitory activities of the whole nutraceutical preparation and select constituents, were also assessed using validated commercially available assay kits. The antioxidant capacity of the nutraceutical and constituents was concentration-dependent. The nutraceutical and constituents appear to display anti-inflammatory activity primarily through the inhibition of COX-2. The nutraceutical displayed similar strength to carprofen in reducing TNF-α, IL-6, MMP-3, nitric oxide, and sGAGs at select concentration ranges. The C-phycocyanin (CPC)-based nutraceutical and constituents may be able to mediate 3 primary pathogenic mechanisms of osteoarthritis: inflammation, chondral degeneration, and oxidative stress in vitro. The nutraceutical may be clinically useful in veterinary medicine and its efficacy should be further investigated in vivo. PMID:26130858

Improved amplification results following episodes of failure to amplify at the Amelogenin Locus using PowerPlex® ESI 16 Fast System.

PubMed

Berlyne, Sigal; Oz, Carla; Einot, Naftaly; Avraham, Shlomit; Ram, Tanya; Goldberg, Miri D; Gafny, Ron

2017-07-01

In 2012 the Israel Police DNA Casework laboratory adopted the 16 STR PowerPlex ® ESI kit for routine use. The Promega Company updated this kit and developed the PowerPlex ® ESI 16 Fast System in which all autosomal primer pairs remained identical to the original set, except at the amelogenin site. The master mix was improved and optimized which allowed for direct, faster and more robust amplification. Prior to implementing the PowerPlex ® ESI 16 Fast System in our lab, we conducted a preliminary assay where 213 casework samples were amplified using the new kit. These samples had previously been extracted by one of two extraction kits employed by our lab. (the PrepFiler ExpressTM and PrepFiler BTATM Forensic DNA Extraction Kits). The amplification results from these samples were compared to samples amplified using the original PowerPlex ® ESI 16 kit. Multiple incidents of failure to amplify at the amelogenin locus were noted using the new system with the recommended protocol at a rate of 13% (28 of 213 samples). Experiments were performed to understand whether these amplification failures could be a result of primer binding site mutations, extraction method reagents and/or inhibitors. The conclusions reached following these experiments, in conjunction with consultation with the manufacturer, led to the trial of a modified amplification protocol where the suggested annealing temperature was reduced by 2 degrees. To evaluate the efficiency of this altered protocol, a comparison study was undertaken where 88 additional casework samples were chosen and amplified using both the modified 58°C and the recommended 60°C annealing temperatures. We concluded that the most effective method in our laboratory for achieving a consistent and balanced amplification at the amelogenin locus was to reduce the annealing temperature from the manufacturer's recommended 60°C to 58°C. This modification resulted in a reduction of the failure to amplify at the amelogenin locus from 13% (28/213) to 1.1% (1/88) without any observed changes to the autosomal STR amplification results. Copyright © 2017 Elsevier B.V. All rights reserved.

Nociceptive tuning by stem cell factor/c-Kit signaling.

PubMed

Milenkovic, Nevena; Frahm, Christina; Gassmann, Max; Griffel, Carola; Erdmann, Bettina; Birchmeier, Carmen; Lewin, Gary R; Garratt, Alistair N

2007-12-06

The molecular mechanisms regulating the sensitivity of sensory circuits to environmental stimuli are poorly understood. We demonstrate here a central role for stem cell factor (SCF) and its receptor, c-Kit, in tuning the responsiveness of sensory neurons to natural stimuli. Mice lacking SCF/c-Kit signaling displayed profound thermal hypoalgesia, attributable to a marked elevation in the thermal threshold and reduction in spiking rate of heat-sensitive nociceptors. Acute activation of c-Kit by its ligand, SCF, resulted in a reduced thermal threshold and potentiation of heat-activated currents in isolated small-diameter neurons and thermal hyperalgesia in mice. SCF-induced thermal hyperalgesia required the TRP family cation channel TRPV1. Lack of c-Kit signaling during development resulted in hypersensitivity of discrete mechanoreceptive neuronal subtypes. Thus, c-Kit can now be grouped with a small family of receptor tyrosine kinases, including c-Ret and TrkA, that control the transduction properties of sensory neurons.

XMOS XC-2 Development Board for Mechanical Control and Data Collection

NASA Technical Reports Server (NTRS)

Jarnot, Robert F.; Bowden, William J.

2011-01-01

The scanning microwave limb sounder (SMLS) will use technological improvements in low-noise mixers to provide precise data on the Earth s atmospheric composition with high spatial resolution. This project focuses on the design and implementation of a realtime control system needed for airborne engineering tests of the SMLS. The system must coordinate the actuation of optical components using four motors with encoder readback, while collecting synchronized telemetric data from a GPS receiver and 3-axis gyrometric system. A graphical user interface for testing the control system was also designed using Python. Although the system could have been implemented with an FPGA(fieldprogrammable gate array)-based setup, a processor development kit manufactured by XMOS was chosen. The XMOS architecture allows parallel execution of multiple tasks on separate threads, making it ideal for this application. It is easily programmed using XC (a subset of C). The necessary communication interfaces were implemented in software, including Ethernet, with significant cost and time reduction compared to an FPGA-based approach. A simple approach to control the chopper, calibration mirror, and gimbal for the airborne SMLS was needed. The XMOS board allows for multiple threads and real-time data acquisition. The XC-2 development kit is an attractive choice for synchronized, real-time, event-driven applications. The XMOS is based on the transputer microprocessor architecture developed for parallel computing, which is being revamped in this new platform. The XMOS device has multiple cores capable of running parallel applications on separate threads. The threads communicate with each other via user-defined channels capable of transmitting data within the device. XMOS provides a C-based development environment using XC, which eliminates the need for custom tool kits associated with FPGA programming. The XC-2 has four cores and necessary hardware for Ethernet I/O.

Kit W-sh Mutation Prevents Cancellous Bone Loss during Calcium Deprivation.

PubMed

Lotinun, Sutada; Suwanwela, Jaijam; Poolthong, Suchit; Baron, Roland

2018-01-01

Calcium is essential for normal bone growth and development. Inadequate calcium intake increases the risk of osteoporosis and fractures. Kit ligand/c-Kit signaling plays an important role in regulating bone homeostasis. Mice with c-Kit mutations are osteopenic. The present study aimed to investigate whether impairment of or reduction in c-Kit signaling affects bone turnover during calcium deprivation. Three-week-old male WBB6F1/J-Kit W /Kit W-v /J (W/W v ) mice with c-Kit point mutation, Kit W-sh /HNihrJaeBsmJ (W sh /W sh ) mice with an inversion mutation in the regulatory elements upstream of the c-Kit promoter region, and their wild-type controls (WT) were fed either a normal (0.6% calcium) or a low calcium diet (0.02% calcium) for 3 weeks. μCT analysis indicated that both mutants fed normal calcium diet had significantly decreased cortical thickness and cancellous bone volume compared to WT. The low calcium diet resulted in a comparable reduction in cortical bone volume and cortical thickness in the W/W v and W sh /W sh mice, and their corresponding controls. As expected, the low calcium diet induced cancellous bone loss in the W/W v mice. In contrast, W sh /W sh cancellous bone did not respond to this diet. This c-Kit mutation prevented cancellous bone loss by antagonizing the low calcium diet-induced increase in osteoblast and osteoclast numbers in the W sh /W sh mice. Gene expression profiling showed that calcium deficiency increased Osx, Ocn, Alp, type I collagen, c-Fms, M-CSF, and RANKL/OPG mRNA expression in controls; however, the W sh mutation suppressed these effects. Our findings indicate that although calcium restriction increased bone turnover, leading to osteopenia, the decreased c-Kit expression levels in the W sh /W sh mice prevented the low calcium diet-induced increase in cancellous bone turnover and bone loss but not the cortical bone loss.

NI-1: a novel canine mastocytoma model for studying drug resistance and IgER-dependent mast cell activation

PubMed Central

Hadzijusufovic, E; Peter, B; Herrmann, H; Rülicke, T; Cerny-Reiterer, S; Schuch, K; Kenner, L; Thaiwong, T; Yuzbasiyan-Gurkan, V; Pickl, W F; Willmann, M; Valent, P

2012-01-01

Background Advanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organs, mediator-related symptoms, and a poor prognosis. Kit mutations supposedly contribute to abnormal growth and drug resistance in these patients. Methods We established a novel canine mastocytoma cell line, NI-1, from a patient suffering from MC leukemia. Results NI-1 cells were found to form mastocytoma lesions in NOD/SCID IL-2Rgammanull mice and to harbor several homozygous Kit mutations, including missense mutations at nucleotides 107(C→T) and 1187(A→G), a 12-bp duplication (nucleotide 1263), and a 12-bp deletion (nucleotide 1550). NI-1 cells expressed several MC differentiation antigens, including tryptase, Kit, and a functional IgE receptor. Compared to the C2 mastocytoma cell line harboring a Kit exon 11 mutation, NI-1 cells were found to be less responsive against the Kit tyrosine kinase inhibitors (TKI) masitinib and imatinib, but were even more sensitive against proliferation-inhibitory effects of the mammalian target of rapamycin (mTOR) blocker RAD001 and PI3-kinase/mTOR blocker NVP-BEZ235. The Kit-targeting multikinase inhibitors PKC412 and dasatinib were also found to override TKI resistance in NI-1 cells, and produced growth inhibition with reasonable IC50 values (<0.1 μM). Conclusion NI-1 may serve as a useful tool to investigate IgE-dependent reactions and mechanisms of abnormal growth and drug resistance in neoplastic MC in advanced mastocytosis. PMID:22583069

Oncogenic Kit signals on endolysosomes and endoplasmic reticulum are essential for neoplastic mast cell proliferation

PubMed Central

Obata, Yuuki; Toyoshima, Shota; Wakamatsu, Ei; Suzuki, Shunichi; Ogawa, Shuhei; Esumi, Hiroyasu; Abe, Ryo

2014-01-01

Kit is a receptor-type tyrosine kinase found on the plasma membrane. It can transform mast cells through activating mutations. Here, we show that a mutant Kit from neoplastic mast cells from mice, Kit(D814Y), is permanently active and allows cells to proliferate autonomously. It does so by activating two signalling pathways from different intracellular compartments. Mutant Kit from the cell surface accumulates on endolysosomes through clathrin-mediated endocytosis, which requires Kit’s kinase activity. Kit(D814Y) is constitutively associated with phosphatidylinositol 3-kinase, but the complex activates Akt only on the cytoplasmic surface of endolysosomes. It resists destruction because it is under-ubiquitinated. Kit(D814Y) also appears in the endoplasmic reticulum soon after biosynthesis, and there, can activate STAT5 aberrantly. These mechanisms of oncogenic signalling are also seen in rat and human mast cell leukemia cells. Thus, oncogenic Kit signalling occurs from different intracellular compartments, and the mutation acts by altering Kit trafficking as well as activation. PMID:25493654

Comparison of single cell sequencing data between two whole genome amplification methods on two sequencing platforms.

PubMed

Chen, DaYang; Zhen, HeFu; Qiu, Yong; Liu, Ping; Zeng, Peng; Xia, Jun; Shi, QianYu; Xie, Lin; Zhu, Zhu; Gao, Ya; Huang, GuoDong; Wang, Jian; Yang, HuanMing; Chen, Fang

2018-03-21

Research based on a strategy of single-cell low-coverage whole genome sequencing (SLWGS) has enabled better reproducibility and accuracy for detection of copy number variations (CNVs). The whole genome amplification (WGA) method and sequencing platform are critical factors for successful SLWGS (<0.1 × coverage). In this study, we compared single cell and multiple cells sequencing data produced by the HiSeq2000 and Ion Proton platforms using two WGA kits and then comprehensively evaluated the GC-bias, reproducibility, uniformity and CNV detection among different experimental combinations. Our analysis demonstrated that the PicoPLEX WGA Kit resulted in higher reproducibility, lower sequencing error frequency but more GC-bias than the GenomePlex Single Cell WGA Kit (WGA4 kit) independent of the cell number on the HiSeq2000 platform. While on the Ion Proton platform, the WGA4 kit (both single cell and multiple cells) had higher uniformity and less GC-bias but lower reproducibility than those of the PicoPLEX WGA Kit. Moreover, on these two sequencing platforms, depending on cell number, the performance of the two WGA kits was different for both sensitivity and specificity on CNV detection. The results can help researchers who plan to use SLWGS on single or multiple cells to select appropriate experimental conditions for their applications.

Characterization of a mast cell line that lacks the extracellular domain of membrane c-kit.

PubMed

Mekori, Y A; Oh, C K; Dastych, J; Goff, J P; Adachi, S; Bianchine, P J; Worobec, A; Semere, T; Pierce, J H; Metcalfe, D D

1997-04-01

Expression of the c-kit proto-oncogene receptor on mast cells is essential for their normal proliferation and maturation as well as for several biological responses such as chemotaxis and attachment. In the present study we report that the interleukin-3 (IL-3)-dependent mast cell line CFTL-15 lacks the extracellular domain of the c-kit receptor. This observation was made after noting that the c-kit ligand stem cell factor (SCF) could not prevent IL-3 deprivation-induced mast cell apoptosis and that CFTL-15 cells did not proliferate in response to SCF. Flow cytometric analysis employing monoclonal anti-c-kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequently showed a diminished expression of c-kit on CFTL-15 cells. There was no identifiable message for the extracellular domain of c-kit in these cells, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). These previously unrecognized properties of the CFTL-15 mast cell line allowed the examination of other biological consequences of the lack of c-kit on mast cells. Analysing the ability of these cells to adhere to surface-bound fibronectin, it was found that addition of SCF did not increase their adhesion to this substrate, in opposition to what is reported with other mast cells. Similarly, CFTL-15 mast cells did not adhere to fibroblasts, which is known to require c-kit expression. Also, there was no protein tyrosine phosphorylation in these cells in response to SCF. CFTL-15 cells underwent apoptosis on removal of IL-3 coincident with a decrease in endogenous Bcl-2 mRNA. Overexpression of Bcl-2 cDNA prolonged survival of Bcl-2-transfected CFTL-15 cells upon withdrawal of IL-3. Thus, the CFTL-15 cell line that lacks surface c-kit is not able to proliferate in response to SCF, undergoes apoptosis in the presence of SCF, and does not adhere to fibroblasts. These results confirm earlier studies on the functional consequences of c-kit and provide a novel experimental model for further investigation.

Characterization of a mast cell line that lacks the extracellular domain of membrane c-kit.

PubMed Central

Mekori, Y A; Oh, C K; Dastych, J; Goff, J P; Adachi, S; Bianchine, P J; Worobec, A; Semere, T; Pierce, J H; Metcalfe, D D

1997-01-01

Expression of the c-kit proto-oncogene receptor on mast cells is essential for their normal proliferation and maturation as well as for several biological responses such as chemotaxis and attachment. In the present study we report that the interleukin-3 (IL-3)-dependent mast cell line CFTL-15 lacks the extracellular domain of the c-kit receptor. This observation was made after noting that the c-kit ligand stem cell factor (SCF) could not prevent IL-3 deprivation-induced mast cell apoptosis and that CFTL-15 cells did not proliferate in response to SCF. Flow cytometric analysis employing monoclonal anti-c-kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequently showed a diminished expression of c-kit on CFTL-15 cells. There was no identifiable message for the extracellular domain of c-kit in these cells, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). These previously unrecognized properties of the CFTL-15 mast cell line allowed the examination of other biological consequences of the lack of c-kit on mast cells. Analysing the ability of these cells to adhere to surface-bound fibronectin, it was found that addition of SCF did not increase their adhesion to this substrate, in opposition to what is reported with other mast cells. Similarly, CFTL-15 mast cells did not adhere to fibroblasts, which is known to require c-kit expression. Also, there was no protein tyrosine phosphorylation in these cells in response to SCF. CFTL-15 cells underwent apoptosis on removal of IL-3 coincident with a decrease in endogenous Bcl-2 mRNA. Overexpression of Bcl-2 cDNA prolonged survival of Bcl-2-transfected CFTL-15 cells upon withdrawal of IL-3. Thus, the CFTL-15 cell line that lacks surface c-kit is not able to proliferate in response to SCF, undergoes apoptosis in the presence of SCF, and does not adhere to fibroblasts. These results confirm earlier studies on the functional consequences of c-kit and provide a novel experimental model for further investigation. Images Figure 4 Figure 6 Figure 7 Figure 8 PMID:9176104

Cell type-specific deficiency of c-kit gene expression in mutant mice of mi/mi genotype.

PubMed Central

Isozaki, K.; Tsujimura, T.; Nomura, S.; Morii, E.; Koshimizu, U.; Nishimune, Y.; Kitamura, Y.

1994-01-01

The mi locus of mice encodes a novel member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called mi factor). In addition to microphthalmus, osteopetrosis, and lack of melanocytes, mice of mi/mi genotype are deficient in mast cells. Since the c-kit receptor tyrosine kinase plays an important role in the development of mast cells, and since the c-kit expression by cultured mast cells from mi/mi mice is deficient in both mRNA and protein levels, the mast cell deficiency of mi/mi mice has been attributed at least in part to the deficient expression of c-kit. However, it remained to be examined whether the c-kit expression was also deficient in tissues of mi/mi mice. In the present study, we examined the c-kit expression by mi/mi skin mast cells using in situ hybridization and immunohistochemistry. Moreover, we examined the c-kit expression by various cells other than mast cells in tissues of mi/mi mice. We found that the c-kit expression was deficient in mast cells but not in erythroid precursors, testicular germ cells, and neurons of mi/mi mice. This suggested that the regulation of the c-kit transcription by the mi factor was dependent on cell types. Mice of mi/mi genotype appeared to be a useful model to analyze the function of transcription factors in the whole-animal level. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7524330

Survivin is a novel transcription regulator of KIT and is downregulated by miRNA‐494 in gastrointestinal stromal tumors

PubMed Central

Yun, SeongJu; Kim, Won Kyu; Kwon, Yujin; Jang, Mi; Bauer, Sebastian

2018-01-01

Gain‐of‐function mutations of KIT are pathognomonic in sporadic gastrointestinal stromal tumors (GISTs). Several microRNAs have been shown to be dysregulated in GISTs and impact KIT expression. Little is known though on KIT‐independent targets of KIT‐regulating mRNAs. We sought to investigate how miR‐494 inhibits GIST proliferation and to identify novel target gene. We used microarray‐based gene expression analyses to identify pathways and target genes affected by miR‐494. The expressional relationship between survivin and miR‐494 was determined in 35 GIST tissues. Cell proliferation assay, FACS analysis, colony formation assay, promoter assays and chromatin immunoprecipitation (ChiP) were performed to clarify the roles of survivin in GIST progression. Gene expression microarray analysis revealed that miR‐494 inhibited GISTs by affecting multiple genes in the cell cycle pathway. Survivin (BIRC5) was a key target of miR‐494, and its expression showed an inverse correlation with miR‐494 expression in 35 GIST tissues (Pearson's correlation coefficient, r = −0.418, p = 0.012). Downregulation of survivin inhibited proliferation and colony formation, and resulted in cell cycle alteration. Induced survivin overexpression relieved miR‐494‐mediated inhibition of GIST progression. Targeting PI3K effectively suppressed proliferation of GISTs with downregulation of survivin. Survivin also regulated KIT expression at the transcription level. Immunohistochemical analysis using 113 GISTs revealed that survivin expression was significantly correlated with overall survival of GIST patients (p = 0.004). Our findings indicated that miR‐494 synergistically suppressed GISTs by concomitantly targeting survivin and KIT. PMID:29277888

Synthesis and evaluation of 7-substituted-5,6-dihydrobenzo[c]acridine derivatives as new c-KIT promoter G-quadruplex binding ligands.

PubMed

Guo, Qian-Liang; Su, Hua-Fei; Wang, Ning; Liao, Sheng-Rong; Lu, Yu-Ting; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Huang, Zhi-Shu

2017-04-21

It has been shown that treatment of cancer cells with c-KIT G-quadruplex binding ligands can reduce their c-KIT expression levels thus inhibiting cell proliferation and inducing cell apoptosis. Herein, a series of new 7-substituted-5,6-dihydrobenzo[c]acridine derivatives were designed and synthesized. Subsequent biophysical evaluation demonstrated that the derivatives could effectively bind to and stabilize c-KIT G-quadruplex with good selectivity against duplex DNA. It was found that 12-N-methylated derivatives with a positive charge introduced at 12-position of 5,6-dihydrobenzo[c]acridine ring had similar binding affinity but lower stabilizing ability to c-KIT G-quadruplex DNA, compared with those of nonmethylated derivatives. Further molecular modeling studies showed possible binding modes of G-quadruplex with the ligands. RT-PCR assay and Western blot showed that compound 2b suppressed transcription and translation of c-KIT gene in K562 cells, which was consistent with the property of an effective G-quadruplex binding ligand targeting c-KIT oncogene promoter. Further biological evaluation showed that compound 2b could induce apoptosis through activation of the caspase-3 cascade pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, M.; Takeishi, Takashi; Geissler, E.N.

1991-07-15

The authors investigated the effects of a newly recognized multifunctional growth factor, the c-kit ligand stem cell factor (SCF), on mouse mast cell proliferation and phenotype. Recombinant rat SCF{sup 164} (rrSCF{sup 164}) induced the development of large numbers of dermal mast cells in normal mice in vivo. Many of these mast cells had features of connective tissue-type mast cells (CTMC), in that they were reactive both with the heparin-binding fluorescent dye berberine sulfate and with safranin. In vitro, rrSCF{sup 164} induced the proliferation of cloned interleukin 3 (IL-3)-dependent mouse mast cells and primary populations of IL-3-dependent, bone marrow-derived cultured mastmore » cells (BMCMC), which represent immature mast cells, and purified peritoneal mast cells, which represent a type of mature CTMC> BMCMC maintained in rrSCF{sup 164} not only proliferated but also matured. These findings identify SCF as a single cytokine that can induce immature, IL-3-dependent mast cells to mature and to acquire multiple characteristics of CTMC. These findings also directly demonstrate that SCF can regulate the development of a cellular lineage expressing c-kit through effects on both proliferation and maturation.« less

SCF/C-Kit/JNK/AP-1 Signaling Pathway Promotes Claudin-3 Expression in Colonic Epithelium and Colorectal Carcinoma.

PubMed

Wang, Yaxi; Sun, Tingyi; Sun, Haimei; Yang, Shu; Li, Dandan; Zhou, Deshan

2017-04-06

Claudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and c-kit loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with CLDN-3 promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the c-Kit loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development.

Mast cells regulate myofilament calcium sensitization and heart function after myocardial infarction.

PubMed

Ngkelo, Anta; Richart, Adèle; Kirk, Jonathan A; Bonnin, Philippe; Vilar, Jose; Lemitre, Mathilde; Marck, Pauline; Branchereau, Maxime; Le Gall, Sylvain; Renault, Nisa; Guerin, Coralie; Ranek, Mark J; Kervadec, Anaïs; Danelli, Luca; Gautier, Gregory; Blank, Ulrich; Launay, Pierre; Camerer, Eric; Bruneval, Patrick; Menasche, Philippe; Heymes, Christophe; Luche, Elodie; Casteilla, Louis; Cousin, Béatrice; Rodewald, Hans-Reimer; Kass, David A; Silvestre, Jean-Sébastien

2016-06-27

Acute myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Inflammatory cells orchestrate postischemic cardiac remodeling after MI. Studies using mice with defective mast/stem cell growth factor receptor c-Kit have suggested key roles for mast cells (MCs) in postischemic cardiac remodeling. Because c-Kit mutations affect multiple cell types of both immune and nonimmune origin, we addressed the impact of MCs on cardiac function after MI, using the c-Kit-independent MC-deficient (Cpa3(Cre/+)) mice. In response to MI, MC progenitors originated primarily from white adipose tissue, infiltrated the heart, and differentiated into mature MCs. MC deficiency led to reduced postischemic cardiac function and depressed cardiomyocyte contractility caused by myofilament Ca(2+) desensitization. This effect correlated with increased protein kinase A (PKA) activity and hyperphosphorylation of its targets, troponin I and myosin-binding protein C. MC-specific tryptase was identified to regulate PKA activity in cardiomyocytes via protease-activated receptor 2 proteolysis. This work reveals a novel function for cardiac MCs modulating cardiomyocyte contractility via alteration of PKA-regulated force-Ca(2+) interactions in response to MI. Identification of this MC-cardiomyocyte cross-talk provides new insights on the cellular and molecular mechanisms regulating the cardiac contractile machinery and a novel platform for therapeutically addressable regulators. ©2016 Ngkelo et al.

Prognostic significance of c-KIT in vulvar cancer: bringing this molecular marker from bench to bedside

PubMed Central

2012-01-01

Background Vulvar carcinomas are rare tumors, and there is limited data regarding molecular alterations. To our knowledge there are no published studies on c-KIT and squamous cell carcinomas of the vulva (VSCC). Although there are a significant number of other tumor types which express c-KIT, there remains controversy as to its relationship to patient outcome. Thus, we wished to investigate such controversial findings to determine the prognostic importance of c-KIT by evaluating its protein and mRNA expression in VSCCs, correlating these findings with clinicopathological features and Human Papillomavirus (HPV) infection. Methods c-KIT expression was scored by immunohistochemistry (IHC) as positive or negative in 139 formalin-fixed paraffin-embedded (FFPE) cases of vulvar carcinomas arrayed in a tissue microarray (TMA) using the DAKO® A4502 rabbit polyclonal c-KIT antibody (diluted 1:100). c-KIT mRNA was evaluated by qRT-PCR in 34 frozen samples from AC Camargo Hospital Biobank (17 tumoral and 17 non-tumoral samples) using TaqMan probes–Applied Biosystems [Hs00174029_m1]. HPV genotyping was assessed in 103 samples using Linear Array® HPV Genotyping Test kit (Roche Molecular Diagnostics, Basel, Switzerland). All results obtained were correlated with clinical and pathological data of the patients. Results c-KIT protein was positive by immunohistochemistry in 70.5% of the cases and this was associated with a higher global survival (p = 0.007), a higher recurrence-free survival (p < 0.0001), an absence of associated lesions (p = 0.001), lymph node metastasis (p = 0.0053), and HPV infection (p = 0.034). Furthermore, c-KIT mRNA quantitation revealed higher levels of transcripts in normal samples compared to tumor samples (p = 0,0009). Conclusions Our findings indicate that those vulvar tumors staining positively for c-KIT present better prognosis. Thus, positivity of c-KIT as evaluated by IHC may be a good predictor for use of more conservative surgery techniques and lymph node dissection in vulvar cancer. So part of the essence of our study is to see the possibility of translating our current results from the bench to the bedside. This will help provide patients a more appropriate, less mutilating treatment, in order to keep the maximum physical and psychic quality as possible to these women. PMID:22839358

Detection of novel polymorphisms in the ckit gene of canine patients with lymphoma, melanoma, haemangiosarcoma, and osteosarcoma.

PubMed

Gramer, Irina; Kessler, Martin; Geyer, Joachim

2016-06-01

Tyrosine kinase inhibitors (TKIs) that specifically target cKIT represent a therapeutic approach for non-resectable canine mast cell tumours (MCTs) grade II/III. The therapeutic benefit of TKIs has been investigated in other tumours based on clinical response rates and identification of gain-of-function mutations. In the present study, cKIT expression in 14 dogs with osteosarcoma, melanoma, haemangiosarcoma, lymphoma, and fibrosarcoma was analysed. Tissue samples were used for cKIT sequencing to (I) detect the cKIT transcript and to (II) identify gain-of-function mutations. The cKIT transcript was detected in ten patients. Four novel amino acid substitutions and five silent polymorphisms were identified. Furthermore, an insertion mutation (GNSK) was discovered in the tissue, but not in the blood sample of one dog. CKIT expression was identified in a variety of canine tumours and, therefore, TKIs might have a broader therapeutic indication apart from treatment of MCTs. Further investigations will be necessary to localize the cKIT protein in the respective tumours and to evaluate the functional consequence of the cKIT variants identified in the present study.

Association of paediatric mastocytosis with a polymorphism resulting in an amino acid substitution (M541L) in the transmembrane domain of c-KIT.

PubMed

Foster, R; Byrnes, E; Meldrum, C; Griffith, R; Ross, G; Upjohn, E; Braue, A; Scott, R; Varigos, G; Ferrao, P; Ashman, L K

2008-11-01

The receptor tyrosine kinase c-KIT plays a key role in normal mast cell development. Point mutations in c-KIT have been associated with sporadic or familial mastocytosis. Two unrelated pairs of apparently identical twins affected by cutaneous mastocytosis attending the Mastocytosis Clinic at the Royal Children's Hospital, Melbourne, provided an opportunity to assess the possible contribution of c-KIT germline mutations or polymorphisms in this disease. Tissue biopsy, blood and/or buccal swab specimens were collected from 10 children with mastocytosis. To detect germline mutations/polymorphisms in c-KIT, we studied all coding exons by denaturing high pressure liquid chromatography. Exons showing mismatches were examined by direct sequencing. The influence of the substitution identified was further examined by expressing the variant form of c-KIT in factor-dependent FDC-P1 cells. In both pairs of twins, a heterozygous ATG to CTG transition in codon 541 was observed, resulting in the substitution of a methionine residue in the transmembrane domain by leucine (M541L). In each case, one parent was also heterozygous for this allele. Expression of M541L KIT in FDC-P1 cells enabled them to grow in human KIT ligand (stem cell factor, SCF) but did not confer factor independence. Compared with cells expressing wild-type KIT at a similar level, M541L KIT-expressing cells displayed enhanced growth at low levels of SCF, and heightened sensitivity to the KIT inhibitor, imatinib mesylate. The data suggest that the single nucleotide polymorphism resulting in the substitution M541L may predispose to paediatric mastocytosis.

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

PubMed

Roskoski, Robert

2005-11-11

Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of interaction with several enzymes and adaptor proteins.

Cell proliferation and inhibition of apoptosis are related to c-Kit activation in leukaemic lymphoblasts.

PubMed

Reyes-Sebastian, Josefina; Montiel-Cervantes, Laura Arcelia; Reyes-Maldonado, Elba; Dominguez-Lopez, Maria Lilia; Ortiz-Butron, Rocio; Castillo-Alvarez, Aida; Lezama, Ruth Angélica

2018-03-01

Receptor tyrosine kinase (RTK) activity may contribute to carcinogenesis. The c-Kit receptor, a member of the RTK family, is expressed in immature haematopoietic system cells. Acute lymphoblastic leukaemia (ALL) presents incompletely differentiated lymphoblasts, and consequently, c-Kit expression can be detected in these cells. The BCR-ABL kinase, which is usually present in both ALL and chronic myeloid leukaemia, can trigger signalling pathways with neoplastic effects. However, a certain number of ALL patients and chronic myeloid leukaemia patients do not express this kinase, raising the question of which other proteins that intervene in signalling pathways may be involved in the development of these diseases. To test whether c-Kit has proliferative effects and affects the inhibition of apoptosis of leukaemic lymphoblasts that do not express BCR-ABL. We cultured RS4:11 lymphoblasts and analysed the expression and activation of c-Kit by immunofluorescence, and flow cytometry, evaluation of cell proliferation, apoptosis, cyclin D1 and Bak expression were carried out by flow cytometry; activation of AKT and survivin expression were tested by immunoblot. The c-Kit receptor was found to induce proliferation and to increase the expression of cyclin D1 via the PI3K/AKT/NF-kB signalling pathway. Additionally, the c-Kit/PI3K/AKT pathway increased the inhibition of apoptosis and survivin expression. Similarly, c-Kit was observed to reduce the expression of the pro-apoptotic Bak protein. These results suggest that, in leukaemic lymphoblasts, c-Kit triggers a signalling pathway with proliferative and anti-apoptotic effects; information to this effect has not yet been reported in the literature.

Identification of a secondary mutation in the KIT kinase domain correlated with imatinib-resistance in a canine mast cell tumor.

PubMed

Nakano, Yuko; Kobayashi, Masato; Bonkobara, Makoto; Takanosu, Masamine

2017-06-01

Imatinib-resistance is a major therapeutic problem in human chronic myeloid leukemia, human gastrointestinal stromal tumors, and canine mast cell tumors. In the present study, we identified the secondary mutation c.2006C>T in c-KIT exon 14 in a mast cell tumor obtained from a dog carrying c.1663-1671del in exon 11 and showing resistance to imatinib. The mutation in exon 14 resulted in substitution of threonine with isoleucine at position 669, which was located at the center of the ATP binding site as a gatekeeper and played an important role in binding to imatinib. Transfectants were constructed to survey the functions of these mutations in exons 11 and 14. The transfectant with mutant KIT encoded by c-KIT carrying c.1663-1671del showed constitutive ligand-independent phosphorylation that was suppressed by imatinib, indicating a gain-of-function mutation. Furthermore, the transfectant with mutant KIT encoded by c-KIT carrying both c.1663-1671del and c.2006C>T caused ligand-independent phosphorylation, which was not suppressed by imatinib. From these results, we concluded that the mutation c.2006C>T in c-KIT exon 14 was an imatinib-resistance mutation in a canine mast cell tumor. These findings revealed, for the first time, a mechanism of imatinib resistance in a clinical case of canine mast cell tumor. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of the surface expression of c-kit and occurrence of the c-kit Asp816Val activating mutation in T cells, B cells, and myelomonocytic cells in patients with mastocytosis.

PubMed

Akin, C; Kirshenbaum, A S; Semere, T; Worobec, A S; Scott, L M; Metcalfe, D D

2000-02-01

The Asp816Val c-kit activating mutation is detectable in the peripheral blood cells of some patients with mastocytosis and in lesional skin biopsies obtained from adult patients with urticaria pigmentosa. These observations led to the conclusion that this mutation is present in mast cells and mast cell precursors that express c-kit. However, the distribution of the Asp816Val mutation among hematopoietic lineages is unknown. To determine the distribution of the Asp816Val mutation among hematopoietic lineages and to explore its relationship to clinical disease, we examined cells bearing differentiation markers for myelomonocytic cells as well as T and B lymphocytes, in both peripheral blood and bone marrow obtained from patients with mastocytosis. The presence of Asp816Val c-kit mutation in cells magnetically sorted from peripheral blood or bone marrow according to surface differentiation markers was studied by reverse transcriptase polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) analysis. The surface expression of c-kit was determined by flow cytometry. The mutation was detectable by RT-PCR in at least one cell lineage in the bone marrow in 7 of 7 patients examined and in the peripheral blood of 11 of 11 adult patients with urticaria pigmentosa and indolent disease. The mutation was identified most frequently in B cells and myeloid cells. Flow cytometric analysis demonstrated that the differentiated cells expressing mutated c-kit were negative for surface KIT. These results are consistent with the conclusion that the c-kit Asp816Val mutation occurs in an early progenitor cell and is carried by myelomonocytic cells, T cells, and B cells in addition to mast cells. However, unlike mast cells, these myelomonocytic cells, T cells, and B cells do not concomitantly express surface c-kit and thus may be less susceptible to the effects of this mutation.

C-MORE Science Kits as a Classroom Learning Tool

ERIC Educational Resources Information Center

Foley, J. M.; Bruno, B. C.; Tolman, R. T.; Kagami, R. S.; Hsia, M. H.; Mayer, B.; Inazu, J. K.

2013-01-01

To support teachers in enhancing ocean literacy, the Center for Microbial Oceanography: Research and Education (C-MORE) has developed a series of portable, hands-on science kits on selected topics in oceanography. This paper provides an overview of kit content, describes how the kits were developed, and evaluates their efficacy as a curriculum…

Imatinib mesylate inhibits Leydig cell tumor growth: evidence for in vitro and in vivo activity.

PubMed

Basciani, Sabrina; Brama, Marina; Mariani, Stefania; De Luca, Gabriele; Arizzi, Mario; Vesci, Loredana; Pisano, Claudio; Dolci, Susanna; Spera, Giovanni; Gnessi, Lucio

2005-03-01

Leydig cell tumors are usually benign tumors of the male gonad. However, if the tumor is malignant, no effective treatments are currently available. Leydig cell tumors express platelet-derived growth factor (PDGF), kit ligand and their respective receptors, PDGFR and c-kit. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the c-kit and PDGFR tyrosine kinases, on the growth of rodent Leydig tumor cell lines in vivo and in vitro, and examined, in human Leydig cell tumor samples, the expression of activated PDGFR and c-kit and the mutations in exons of the c-kit gene commonly associated with solid tumors. Imatinib caused concentration-dependent decreases in the viability of Leydig tumor cell lines, which coincided with apoptosis and inhibition of proliferation and ligand-stimulated phosphorylation of c-kit and PDGFRs. Mice bearing s.c. allografts of a Leydig tumor cell line treated with imatinib p.o., had an almost complete inhibition of tumor growth, less tumor cell proliferation, increased apoptosis, and a lesser amount of tumor-associated mean vessel density compared with controls. No drug-resistant tumors appeared during imatinib treatment but tumors regrew after drug withdrawal. Human Leydig cell tumors showed an intense expression of the phosphorylated form of c-kit and a less intense expression of phosphorylated PDGFRs. No activating mutations in common regions of mutation of the c-kit gene were found. Our studies suggest that Leydig cell tumors might be a potential target for imatinib therapy.

Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment.

PubMed

Arai, Yasuyuki; Choi, Uimook; Corsino, Cristina I; Koontz, Sherry M; Tajima, Masaki; Sweeney, Colin L; Black, Mary A; Feldman, Steven A; Dinauer, Mary C; Malech, Harry L

2018-05-02

We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (<1%-13.1%). This resulted in significant depletion of the BM c-kit + population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue. Published by Elsevier Inc.

Gata4-Dependent Differentiation of c-Kit+ Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the Heart.

PubMed

Maliken, Bryan D; Kanisicak, Onur; Karch, Jason; Khalil, Hadi; Fu, Xing; Boyer, Justin G; Prasad, Vikram; Zheng, Yi; Molkentin, Jeffery D

2018-04-17

Background -While c-Kit + adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells (CPCs). Methods - Kit allele-dependent lineage tracing and fusion analysis was performed in mice following simultaneous Gata4 and Gata6 cell-type specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit + cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2 CreERT2 transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. Results -Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2 CreERT2 transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artefact of greater leukocyte-cardiomyocyte cellular fusion due to defective endothelial cell differentiation in the absence of Gata4 Conclusions -Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit + cells using Kit allele lineage tracing appears to be an artefact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit + endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and capillary network integrity.

A framework for measurement and harmonization of pediatric multiple sclerosis etiologic research studies: The Pediatric MS Tool-Kit.

PubMed

Magalhaes, Sandra; Banwell, Brenda; Bar-Or, Amit; Fortier, Isabel; Hanwell, Heather E; Lim, Ming; Matt, Georg E; Neuteboom, Rinze F; O'Riordan, David L; Schneider, Paul K; Pugliatti, Maura; Shatenstein, Bryna; Tansey, Catherine M; Wassmer, Evangeline; Wolfson, Christina

2018-06-01

While studying the etiology of multiple sclerosis (MS) in children has several methodological advantages over studying etiology in adults, studies are limited by small sample sizes. Using a rigorous methodological process, we developed the Pediatric MS Tool-Kit, a measurement framework that includes a minimal set of core variables to assess etiological risk factors. We solicited input from the International Pediatric MS Study Group to select three risk factors: environmental tobacco smoke (ETS) exposure, sun exposure, and vitamin D intake. To develop the Tool-Kit, we used a Delphi study involving a working group of epidemiologists, neurologists, and content experts from North America and Europe. The Tool-Kit includes six core variables to measure ETS, six to measure sun exposure, and six to measure vitamin D intake. The Tool-Kit can be accessed online ( www.maelstrom-research.org/mica/network/tool-kit ). The goals of the Tool-Kit are to enhance exposure measurement in newly designed pediatric MS studies and comparability of results across studies, and in the longer term to facilitate harmonization of studies, a methodological approach that can be used to circumvent issues of small sample sizes. We believe the Tool-Kit will prove to be a valuable resource to guide pediatric MS researchers in developing study-specific questionnaire.

MNK1/2 inhibition limits oncogenicity and metastasis of KIT-mutant melanoma

PubMed Central

Zhan, Yao; Guo, Jun; Yang, William; Goncalves, Christophe; Rzymski, Tomasz; Dreas, Agnieszka; Żyłkiewicz, Eliza; Mikulski, Maciej; Brzózka, Krzysztof; Golas, Aniela; Kong, Yan; Ma, Meng; Huang, Fan; Huor, Bonnie; Guo, Qianyu; da Silva, Sabrina Daniela; Torres, Jose; Cai, Yutian; Topisirovic, Ivan; Su, Jie; Bijian, Krikor; Alaoui-Jamali, Moulay A.; Huang, Sidong; Journe, Fabrice; Ghanem, Ghanem E.; Miller, Wilson H.

2017-01-01

Melanoma can be stratified into unique subtypes based on distinct pathologies. The acral/mucosal melanoma subtype is characterized by aberrant and constitutive activation of the proto-oncogene receptor tyrosine kinase C-KIT, which drives tumorigenesis. Treatment of these melanoma patients with C-KIT inhibitors has proven challenging, prompting us to investigate the downstream effectors of the C-KIT receptor. We determined that C-KIT stimulates MAP kinase–interacting serine/threonine kinases 1 and 2 (MNK1/2), which phosphorylate eukaryotic translation initiation factor 4E (eIF4E) and render it oncogenic. Depletion of MNK1/2 in melanoma cells with oncogenic C-KIT inhibited cell migration and mRNA translation of the transcriptional repressor SNAI1 and the cell cycle gene CCNE1. This suggested that blocking MNK1/2 activity may inhibit tumor progression, at least in part, by blocking translation initiation of mRNAs encoding cell migration proteins. Moreover, we developed an MNK1/2 inhibitor (SEL201), and found that SEL201-treated KIT-mutant melanoma cells had lower oncogenicity and reduced metastatic ability. Clinically, tumors from melanoma patients harboring KIT mutations displayed a marked increase in MNK1 and phospho-eIF4E. Thus, our studies indicate that blocking MNK1/2 exerts potent antimelanoma effects and support blocking MNK1/2 as a potential strategy to treat patients positive for KIT mutations. PMID:29035277

MNK1/2 inhibition limits oncogenicity and metastasis of KIT-mutant melanoma.

PubMed

Zhan, Yao; Guo, Jun; Yang, William; Goncalves, Christophe; Rzymski, Tomasz; Dreas, Agnieszka; Żyłkiewicz, Eliza; Mikulski, Maciej; Brzózka, Krzysztof; Golas, Aniela; Kong, Yan; Ma, Meng; Huang, Fan; Huor, Bonnie; Guo, Qianyu; da Silva, Sabrina Daniela; Torres, Jose; Cai, Yutian; Topisirovic, Ivan; Su, Jie; Bijian, Krikor; Alaoui-Jamali, Moulay A; Huang, Sidong; Journe, Fabrice; Ghanem, Ghanem E; Miller, Wilson H; Del Rincón, Sonia V

2017-11-01

Melanoma can be stratified into unique subtypes based on distinct pathologies. The acral/mucosal melanoma subtype is characterized by aberrant and constitutive activation of the proto-oncogene receptor tyrosine kinase C-KIT, which drives tumorigenesis. Treatment of these melanoma patients with C-KIT inhibitors has proven challenging, prompting us to investigate the downstream effectors of the C-KIT receptor. We determined that C-KIT stimulates MAP kinase-interacting serine/threonine kinases 1 and 2 (MNK1/2), which phosphorylate eukaryotic translation initiation factor 4E (eIF4E) and render it oncogenic. Depletion of MNK1/2 in melanoma cells with oncogenic C-KIT inhibited cell migration and mRNA translation of the transcriptional repressor SNAI1 and the cell cycle gene CCNE1. This suggested that blocking MNK1/2 activity may inhibit tumor progression, at least in part, by blocking translation initiation of mRNAs encoding cell migration proteins. Moreover, we developed an MNK1/2 inhibitor (SEL201), and found that SEL201-treated KIT-mutant melanoma cells had lower oncogenicity and reduced metastatic ability. Clinically, tumors from melanoma patients harboring KIT mutations displayed a marked increase in MNK1 and phospho-eIF4E. Thus, our studies indicate that blocking MNK1/2 exerts potent antimelanoma effects and support blocking MNK1/2 as a potential strategy to treat patients positive for KIT mutations.

Multiple malignant extragastrointestinal stromal tumors of the greater omentum and results of immunohistochemistry and mutation analysis: A case report

PubMed Central

Kim, Jong-Han; Boo, Yoon-Jung; Jung, Cheol-Woong; Park, Sung-Soo; Kim, Seung-Joo; Mok, Young-Jae; Kim, Sang-Dae; Chae, Yang-Suk; Kim, Chong-Suk

2007-01-01

To report an extragastrointestinal stromal tumor (EGIST) that occurs outside the gastrointestinal tract and shows unique clinicopathologic and immunohistochemical features. In our case, we experienced multiple soft tissue tumors that originate primarily in the greater omentum, and in immunohistochemical analysis, the tumors showed features that correspond to malignant EGIST. Two large omental masses measured 15 cm x 10 cm and 5 cm × 4 cm sized and several small ovoid fragments were attached to small intestine, mesentery and peritoneum. On histologic findings, the masses were separated from small bowel serosa and had high mitotic count (115/50 HPFs). In the results of immunohistochemical stains, the tumor showed CD117 (c-kit) positive reactivity and high Ki-67 labeling index. On mutation analysis, the c-kit gene mutation was found in the juxtamembrane domain (exon 11) and it was heterozygote. Platelet-derived growth factor receptor (PDGFR) gene mutation was also found in the juxtamemembrane (exon 12) and it was polymorphism. From above findings, we proposed that there may be several mutational pathways to malignant EGIST, so further investigations could be needed to approach this unfavorable disease entity. PMID:17659683

Isolation of pancreatic progenitor cells with the surface marker of hematopoietic stem cells.

PubMed

Ma, Fengxia; Chen, Fang; Chi, Ying; Yang, Shaoguang; Lu, Shihong; Han, Zhongchao

2012-01-01

To isolate pancreatic progenitor cells with the surface markers of hematopoietic stem cells, the expression of stem cell antigen (Sca-1) and c-Kit and the coexpression of them with pancreatic duodenal homeobox-1 (PDX-1), neurogenin 3 (Ngn3), and insulin were examined in murine embryonic pancreas. Then different pancreatic cell subpopulations were isolated by magnet-activated cell sorting. Isolated cells were cultured overnight in hanging drops. When cells formed spheres, they were laid on floating filters at the air/medium interface. With this new culture system, pancreatic progenitor cells were induced to differentiate to endocrine and exocrine cells. It was shown that c-Kit and Sca-1 were expressed differently in embryonic pancreas at 12.5, 15.5, and 17.5 days of gestation. The expression of c-Kit and Sca-1 was the highest at 15.5 days of gestation. c-Kit rather than Sca-1 coexpressed with PDX-1, Ngn3, and insulin. Cells differentiated from c-Kit-positive cells contained more insulin-producing cells and secreted more insulin in response to glucose stimulation than that from c-Kit-negative cells. These results suggested that c-Kit could be used to isolate pancreatic progenitor cells and our new culture system permitted pancreatic progenitor cells to differentiate to mature endocrine cells.

Expression of c-kit receptor in human cholangiocarcinoma and in vivo treatment with imatinib mesilate in chimeric mice

PubMed Central

Kamenz, Thomas; Caca, Karel; Blüthner, Thilo; Tannapfel, Andrea; Mössner, Joachim; Wiedmann, Marcus

2006-01-01

AIM: To investigate the c-kit expression in biliary tract cancer cell lines and histological sections from patients with extrahepatic cholangiocarcinoma (CC) and to evaluate the efficacy of in vitro and in vitro treatment with imatinib mesilate. METHODS: The protein expression of c-kit in the human biliary tract cancer cell lines Mz-ChA-2 and EGI-1 and histological sections from 19 patients with extrahepatic CC was assessed by immunoblotting, immunocytochemistry, and immunohistochemistry. The anti-proliferative effect of imatinib mesilate on biliary tract cancer cell lines Mz-ChA-2 and EGI-1 was studied in vitro by automated cell counting. In addition, immunodeficient NMRI mice (TaconicTM) were subcutaneously injected with 5 x 106 cells of cell lines MzChA-2 and EGI-1. After having reached a tumour volume of 200 mm3, daily treatment was started intraperitoneally with imatinib mesilate at a dose of 50 mg/kg or normal saline (NS). Tumor volume was calculated with a Vernier caliper. After 14 d, mice were sacrificed with tumors excised and tumor mass determined. RESULTS: Immunoblotting revealed presence of c-kit in Mz-ChA-2 and absence in EGI-1 cells. Immunocytochemistry with c-kit antibodies displayed a cytoplasmatic and membraneous localization of receptor protein in Mz-ChA-2 cells and absence of c-kit in EGI-1 cells. c-kit was expressed in 7 of 19 (37%) extrahepatic human CC tissue samples, 2 showed a moderate and 5 a rather weak immunostaining. Imatinib mesilate at a low concentration of 5 µmol/L caused a significant growth inhibition in the c-kit positive cell line Mz-ChA-2 (31%), but not in the c-kit negative cell line EGI-1 (0%) (P < 0.05). Imatinib mesilate at an intermediate concentration of 10 µmol/L inhibited cellular growth of both cell lines (51% vs 57%). Imatinib mesilate at a higher concentration of 20 µmol/L seemed to have a general toxic effect on both cell lines. The IC50 values were 9.7 µmol/L and 11 µmol/L, respectively. After 14 d of in vitro treatment with imatinib mesilate, using the chimeric mouse model, c-kit positive Mz-ChA-2 tumors had a significantly reduced volume and mass as compared to NS treatment (P < 0.05). In contrast to that, treatment of mice bearing c-kit negative EGI-1 tumors did not result in any change of tumor volume and mass as compared to NS treatment. CONCLUSION: c-kit expression is detectable at a moderate to low protein level in biliary tract cancer. Imatinib mesilate exerts marked effects on tumor growth in vitro and in vitro dependent on the level of c-kit expression. PMID:16570351

Direct association of Csk homologous kinase (CHK) with the diphosphorylated site Tyr568/570 of the activated c-KIT in megakaryocytes.

PubMed

Price, D J; Rivnay, B; Fu, Y; Jiang, S; Avraham, S; Avraham, H

1997-02-28

The Csk homologous kinase (CHK), formerly MATK, has previously been shown to bind to activated c-KIT. In this report, we characterize the binding of SH2(CHK) to specific phosphotyrosine sites on the c-KIT protein sequence. Phosphopeptide inhibition of the in vitro interaction of SH2(CHK)-glutathione S-transferase fusion protein/c-KIT from SCF/KL-treated Mo7e megakaryocytic cells indicated that two sites on c-KIT were able to bind SH2(CHK). These sites were the Tyr568/570 diphosphorylated sequence and the monophosphorylated Tyr721 sequence. To confirm this, we precipitated native CHK from cellular extracts using phosphorylated peptides linked to Affi-Gel 15. In addition, purified SH2(CHK)-glutathione S-transferase fusion protein was precipitated with the same peptide beads. All of the peptide bead-binding studies were consistent with the direct binding of SH2(CHK) to phosphorylated Tyr568/570 and Tyr721 sites. Binding of FYN and SHC to the diphosphorylated Tyr568/570 site was observed, while binding of Csk to this site was not observed. The SH2(CHK) binding to the two sites is direct and not through phosphorylated intermediates such as FYN or SHC. Site-directed mutagenesis of the full-length c-KIT cDNA followed by transient transfection indicated that only the Tyr568/570, and not the Tyr721, is able to bind SH2(CHK). This indicates that CHK binds to the same site on c-KIT to which FYN binds, possibly bringing the two into proximity on associated c-KIT subunits and leading to the down-regulation of FYN by CHK.

c-Kit Mutation and Localization Status as Response Predictors in Mast Cell Tumors in Dogs Treated with Prednisone and Toceranib or Vinblastine.

PubMed

Weishaar, K M; Ehrhart, E J; Avery, A C; Charles, J B; Elmslie, R E; Vail, D M; London, C A; Clifford, C A; Eickhoff, J C; Thamm, D H

2018-01-01

KIT inhibitors, such as toceranib (TOC), and vinblastine (VBL) have not been prospectively compared in the treatment of macroscopic mast cell tumors (MCTs). Also, it is unknown whether VBL or TOC is superior for treating MCT without c-kit mutations. To determine the value of KIT genotyping and localization in treatment decisions for dogs with macroscopic MCT. We hypothesized that c-kit mutated MCT would have a better response to TOC than VBL. Eighty-eight client-owned dogs with macroscopic MCT. Prospective, randomized trial. Dogs were randomized to TOC (2.75 mg/kg EOD) or VBL (2.5 mg/m 2 weekly × 4 then EOW) by KIT localization and c-kit mutation status using an adaptive randomization scheme. Sixty dogs were allocated to TOC and 28 to VBL. Of the dogs receiving TOC, 20% had c-kit mutations, compared to 30% receiving VBL (P = 0.74). Overall response rates were 46% (TOC) and 30% (VBL) (odds ratio = 1.56 [0.62-3.92]; P = 0.28). Median progression-free survival (PFS) for dogs receiving VBL was 78 days (7-1,521) and for TOC 95.5 (14-990); hazard ratio (HR) = 1.34 [0.72-2.50]; P = 0.36. Median overall survival (OS) was 241.5 days (10-1,521) for the VBL group and 159 (20-990) for the TOC group; HR = 0.80 ([0.45-1.41]; P = 0.44). Neither PFS nor OS was significantly different between treatment groups. As the proportion of dogs with c-kit mutations was not different between treatment groups in this population of dogs, c-kit mutation status did not predict treatment response. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

C-kit-positive gastric metastasis of lobular carcinoma of the breast masquerading as gastrointestinal stromal tumor.

PubMed

Vennapusa, Bharathi; Oman, Sarah A; Parasher, Gulshan; Cerilli, Lisa A

2010-10-01

A 61-year-old woman with no significant past history underwent gastric biopsies demonstrating a strongly c-kit-positive epithelioid malignancy, initially thought to represent gastrointestinal stromal tumor (GIST). Subsequent clinical and immunohistochemical evaluation proved the neoplasm to represent metastatic lobular carcinoma. This case illustrates that although c-kit is highly specific and sensitive for GIST, its expression may occur in a variety of other neoplasms, some of which morphologically resemble GIST and may present in the gastrointestinal tract as metastases. Therefore, a review of other c-kit-positive lesions is also highlighted.

Protein crystallography prescreen kit

DOEpatents

Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

2007-10-02

A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

Protein crystallography prescreen kit

DOEpatents

Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

2005-07-12

A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

Evaluation and structure-activity relationship analysis of a new series of arylnaphthalene lignans as potential anti-tumor agents.

PubMed

Luo, Jiaoyang; Hu, Yichen; Kong, Weijun; Yang, Meihua

2014-01-01

Arylnaphthalene lignan lactones have attracted considerable interest because of their anti-tumor and anti-hyperlipidimic activities. However, to our knowledge, few studies have explored the effects of these compounds on human leukemia cell lines. In this study, five arylnaphthalene lignans including 6'-hydroxy justicidin A (HJA), 6'-hydroxy justicidin B (HJB), justicidin B (JB), chinensinaphthol methyl ether (CME) and Taiwanin E methyl ether (TEME) were isolated from Justicia procumbens and their effects on the proliferation and apoptosis of the human leukemia K562 cell line were investigated then used to assess structure-activity relationships. To achieve these aims, cytotoxicity was assayed using the MTT assay, while intracellular SOD activity was detected using the SOD Activity Assay kit. Apoptosis was measured by both the using a cycle TEST PLUS DNA reagent kit as well as the FITC Annexin V apoptosis detection kit in combination with flow cytometry. Activation of caspase-mediated apoptosis was evaluated using a FITC active Caspase-3 apoptosis kit and flow cytometry. The results indicated that HJB, HJA and JB significantly inhibited the growth of K562 cells by decreasing both proliferation and SOD activity and inducing apoptosis. The sequence of anti-proliferative activity induced by the five tested arylnaphthalenes by decreasing strength was HJB > HJA > JB > CME > TEME. HJB, HJA and JB also decreased SOD activity and induced apoptosis in a dose-dependent manner. Activation of caspase-3 further indicated that HJB, HJA and JB induced caspase-dependent intrinsic and/or extrinsic apoptosis pathways. Together, these assays suggest that arylnaphthalene lignans derived from Justicia procumbens induce apoptosis to varying degrees, through a caspase-dependent pathway in human leukemia K562 cells. Furthermore, analysis of structure-activity relationships suggest that hydroxyl substitution at C-1 and C-6' significantly increased the antiproliferative activity of arylnaphthalene lignans while a methoxyl at C-1 significantly decreased the effect.

Complex interactions in EML cell stimulation by stem cell factor and IL-3.

PubMed

Ye, Zhi-jia; Gulcicek, Erol; Stone, Kathryn; Lam, Tukiet; Schulz, Vincent; Weissman, Sherman M

2011-03-22

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34- cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone.

Complex interactions in EML cell stimulation by stem cell factor and IL-3

PubMed Central

Ye, Zhi-jia; Gulcicek, Erol; Stone, Kathryn; Lam, Tukiet; Schulz, Vincent; Weissman, Sherman M.

2011-01-01

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34− cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone. PMID:21383156

Anal mucosal melanoma with KIT-activating mutation and response to imatinib therapy--case report and review of the literature.

PubMed

Satzger, Imke; Küttler, Uta; Völker, Bernward; Schenck, Florian; Kapp, Alexander; Gutzmer, Ralf

2010-01-01

Previously an increased frequency of KIT aberrations in mucosal melanomas was reported, whereas c-KIT in most types of cutaneous melanomas does not appear to be of pathogenetic importance. Imatinib has become the standard of care in other cancers with KIT mutations such as gastrointestinal stromal tumors. Recently 12 cases of metastatic melanoma and KIT-activating mutations have been published to be successfully treated with c-KIT blockers such as imatinib, sunitinib, dasatinib or sorafenib. We report here on one of our patients with KIT-activating mutation in metastatic anal mucosal melanoma, who showed a response to imatinib therapy and summarize the available literature regarding this new therapeutic option. Copyright 2009 S. Karger AG, Basel.

Expression profiling of c-kit and its impact after esiRNA silencing during gonadal development in catfish.

PubMed

Laldinsangi, C; Senthilkumaran, B

2018-04-03

C-kit receptor is a member of a family of growth factor receptors that have tyrosine kinase activity, and are involved in the transduction of growth regulatory signals across plasma membrane by activation of its ligand, kitl/scf. The present study analysed mRNA and protein expression profiles of c-kit in the gonads of catfish, Clarias gariepinus, using real time PCR, in situ hybridization and immunohistochemistry. Tissue distribution analysis revealed higher expression mainly in the catfish gonads. Ontogeny studies showed minimal expression during early developmental stages and highest during 50-75 days post hatch, and the dimorphic expression in gonads decreased gradually till adulthood, which might suggest an important role for this gene around later stages of sex differentiation and gonadal development. Expression of C-kit was analysed at various phases of gonadal cycle in both male and female, which showed minimal expression during the resting phase, and higher expression in male compared to females during the pre-spawning phase. In vitro and in vivo induction using human chorionic gonadotropin elevated the expression of c-kit indicating the regulatory influence of hypothalamo-hypophyseal axis. In vivo transient gene silencing using c-kit-esiRNA in adult catfish during gonadal recrudescence showed a decrease in c-kit expression, which affected the expression level of germ cell meiotic marker sycp3, as well as several factors and steroidogenic enzyme genes involved in germ cell development. Decrease in the levels of serum 11-KT and T were also observed after esiRNA silencing. The findings of this study suggest that c-kit has an important role in the process of germ cell proliferation, development and maturation during gonadal development and recrudescence in catfish. Copyright © 2018. Published by Elsevier Inc.

c-Kit mutation reduce intestinal epithelial cell proliferation and migration, but not influence intestinal permeability stimulated by lipopolysaccharide.

PubMed

Xue, Hong; Wang, Feng Yun; Kang, Qian; Tang, Xu Dong

2018-06-20

The proto-oncogene c-kit, as a marker of interstitial cells of Cajal (ICCs) in the gastrointestinal tract, plays an important role in the ICCs. Although limited evidences showed c-kit is present in the colonic epithelium but its roles remain unclear. In the present study, we aimed to investigate the expression, location and function of c-kit in the intestinal epithelium. Immunofluorescence, western blotting, and RT-PCR were performed to detect the expression and location of c-kit in the intestinal mucosa of WT mice. We investigated intestinal epithelial proliferation and migration in vivo by performing 5-Bromodeoxyuridine (BrdU) incorporation and Ki-67 staining in WT and Wads m/m mice. An Ussing chamber with fluorescein-isothiocyanate dextran 4000 was used to detect the transepithelial electric resistance (TER), short circuit current (ISC) and permeability across ex vivo colon segments under control and endotoxaemia conditions. We demonstrated that c-kit was located and expressed in the gut crypt compartment in WT mice, which was demonstrated in the c-kit mutant mice (Wads m/m ). In addition, both the number of proliferating cells and the percentage of the distance migrated were lower in the Wads m/m mice than those in the WT mice. Moreover, the intestinal permeability, TER and tight junction were unaltered in the Wads m/m mice under endotoxic conditions compared with those in both the control condition and the WT mice. Altogether, these observations imply that the expression of c-kit in the colonic epithelium is involved in the proliferation and permeability of the colonic epithelium. Copyright © 2018. Published by Elsevier GmbH.

Conditional Deletion of Kit in Melanocytes: White Spotting Phenotype Is Cell Autonomous.

PubMed

Aoki, Hitomi; Tomita, Hiroyuki; Hara, Akira; Kunisada, Takahiro

2015-07-01

It is well established that cell-intrinsic signaling through the receptor tyrosine kinase KIT is critical for the development of neural crest-derived melanocytes. Nevertheless, it is not entirely clear whether Kit acts exclusively in a melanocyte-autonomous manner or in addition indirectly through other cell types. To address this question in vivo, we generated a targeted allele of Kit that allowed for CRE recombinase-mediated deletion of the transmembrane domain of KIT. Mice carrying one copy of the targeted allele and expressing CRE under the melanoblast/melanocyte-specific tyrosinase promoter exhibited a white spotting phenotype that was even more extensive compared with that found in mice heterozygous for a Kit-null allele. This phenotype is unlikely the result of sequestration of KIT ligand by neighboring cells or by potentially secreted forms of KIT because the spotting phenotype could not be rescued by overexpression of KITL. Likewise, overexpression of endothelin-3 or hepatocyte growth factor was unable to rescue melanocytes in these mice. Although the severity of the observed phenotype remains to be explained, the findings indicate that melanocyte-selective impairment of Kit is sufficient to interfere with normal melanocyte development.

Imatinib in systemic mastocytosis: a phase IV clinical trial in patients lacking exon 17 KIT mutations and review of the literature

PubMed Central

Alvarez-Twose, Iván; Matito, Almudena; Morgado, José Mário; Sánchez-Muñoz, Laura; Jara-Acevedo, María; García-Montero, Andrés; Mayado, Andrea; Caldas, Carolina; Teodósio, Cristina; Muñoz-González, Javier Ignacio; Mollejo, Manuela; Escribano, Luis; Orfao, Alberto

2017-01-01

Resistance to imatinib has been recurrently reported in systemic mastocytosis (SM) carrying exon 17 KIT mutations. We evaluated the efficacy and safety of imatinib therapy in 10 adult SM patients lacking exon 17 KIT mutations, 9 of which fulfilled criteria for well-differentiated SM (WDSM). The World Health Organization 2008 disease categories among WDSM patients were mast cell (MC) leukemia (n = 3), indolent SM (n = 3) and cutaneous mastocytosis (n = 3); the remainder case had SM associated with a clonal haematological non-MC disease. Patients were given imatinib for 12 months −400 or 300 mg daily depending on the presence vs. absence of > 30% bone marrow (BM) MCs and/or signs of advanced disease–. Absence of exon 17 KIT mutations was confirmed in highly-purified BM MCs by peptide nucleic acid-mediated PCR, while mutations involving other exons were investigated by direct sequencing of purified BM MC DNA. Complete response (CR) was defined as resolution of BM MC infiltration, skin lesions, organomegalies and MC-mediator release-associated symptoms, plus normalization of serum tryptase. Criteria for partial response (PR) included ≥ 50% reduction in BM MC infiltration and improvement of skin lesions and/or organomegalies. Treatment was well-tolerated with an overall response rate of 50%, including early and sustained CR in four patients, three of whom had extracellular mutations of KIT, and PR in one case. This later patient and all non-responders (n = 5) showed wild-type KIT. These results together with previous data from the literature support the relevance of the KIT mutational status in selecting SM patients who are candidates for imatinib therapy. PMID:28978170

Imatinib in systemic mastocytosis: a phase IV clinical trial in patients lacking exon 17 KIT mutations and review of the literature.

PubMed

Alvarez-Twose, Iván; Matito, Almudena; Morgado, José Mário; Sánchez-Muñoz, Laura; Jara-Acevedo, María; García-Montero, Andrés; Mayado, Andrea; Caldas, Carolina; Teodósio, Cristina; Muñoz-González, Javier Ignacio; Mollejo, Manuela; Escribano, Luis; Orfao, Alberto

2017-09-15

Resistance to imatinib has been recurrently reported in systemic mastocytosis (SM) carrying exon 17 KIT mutations. We evaluated the efficacy and safety of imatinib therapy in 10 adult SM patients lacking exon 17 KIT mutations, 9 of which fulfilled criteria for well-differentiated SM (WDSM). The World Health Organization 2008 disease categories among WDSM patients were mast cell (MC) leukemia ( n = 3), indolent SM ( n = 3) and cutaneous mastocytosis ( n = 3); the remainder case had SM associated with a clonal haematological non-MC disease. Patients were given imatinib for 12 months -400 or 300 mg daily depending on the presence vs. absence of > 30% bone marrow (BM) MCs and/or signs of advanced disease-. Absence of exon 17 KIT mutations was confirmed in highly-purified BM MCs by peptide nucleic acid-mediated PCR, while mutations involving other exons were investigated by direct sequencing of purified BM MC DNA. Complete response (CR) was defined as resolution of BM MC infiltration, skin lesions, organomegalies and MC-mediator release-associated symptoms, plus normalization of serum tryptase. Criteria for partial response (PR) included ≥ 50% reduction in BM MC infiltration and improvement of skin lesions and/or organomegalies. Treatment was well-tolerated with an overall response rate of 50%, including early and sustained CR in four patients, three of whom had extracellular mutations of KIT , and PR in one case. This later patient and all non-responders ( n = 5) showed wild-type KIT . These results together with previous data from the literature support the relevance of the KIT mutational status in selecting SM patients who are candidates for imatinib therapy.

Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications.

PubMed

Jara-Acevedo, Maria; Teodosio, Cristina; Sanchez-Muñoz, Laura; Álvarez-Twose, Ivan; Mayado, Andrea; Caldas, Carolina; Matito, Almudena; Morgado, José M; Muñoz-González, Javier I; Escribano, Luis; Garcia-Montero, Andrés C; Orfao, Alberto

2015-08-01

Recent studies have found the KIT D816V mutation in peripheral blood of virtually all adult systemic mastocytosis patients once highly sensitive PCR techniques were used; thus, detection of the KIT D816V mutation in peripheral blood has been proposed to be included in the diagnostic work-up of systemic mastocytosis algorithms. However, the precise frequency of the mutation, the biological significance of peripheral blood-mutated cells and their potential association with involvement of bone marrow hematopoietic cells other than mast cells still remain to be investigated. Here, we determined the frequency of peripheral blood involvement by the KIT D816V mutation, as assessed by two highly sensitive PCR methods, and investigated its relationship with multilineage involvement of bone marrow hematopoiesis. Overall, our results confirmed the presence of the KIT D816V mutation in peripheral blood of most systemic mastocytosis cases (161/190; 85%)--with an increasing frequency from indolent systemic mastocytosis without skin lesions (29/44; 66%) to indolent systemic mastocytosis with skin involvement (124/135; 92%), and more aggressive disease subtypes (11/11; 100%)--as assessed by the allele-specific oligonucleotide-qPCR method, which was more sensitive (P<.0001) than the peptide nucleic acid-mediated PCR approach (84/190; 44%). Although the presence of the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide-qPCR technique, did not accurately predict for multilineage bone marrow involvement of hematopoiesis, the allele-specific oligonucleotide-qPCR allele burden and the peptide nucleic acid-mediated-PCR approach did. These results suggest that both methods provide clinically useful and complementary information through the identification and/or quantification of the KIT D816V mutation in peripheral blood of patients suspected of systemic mastocytosis.

Molecular biological characteristics of the recruitment of hematopoietic stem cells from bone marrow niche in chronic myeloid leukemia

PubMed Central

Zhu, Biao; Zhang, Jianbo; Chen, Jiao; Li, Chenglong; Wang, Xiaodong

2015-01-01

Chronic myeloid leukemia (CML) can be contextualized as a disease of unregulated self-renewal of stem cells which exist in a quiescent state and are instructed to differentiate and mobilize to circulation under pathologic circumstances leading to tumor invasion and metastasis. Here we found that matrix metalloproteinase-9 (MMP-9), induced by TGF-β1, upregulated s-KitL and s-ICAM-1, permitting the transfer of c-kit+ hematopoietic stem cells (HSCs) from the quiescent to proliferative niche in CML. Further study showed that this MMP-9 production was raised by CML specific BCR/ABL+ oncogene mediated TGF-β1. Besides, phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was evidenced to govern this stem cell recruitment in CML pathogenesis. Overall, our observations defined a novel critical role for TGF-β1 induced PI3K/Akt/NF-κB signaling pathway in the recruitment of the malignant cells in CML by releasing s-KitL and s-ICAM-1 and this was through a distinct PI3K/Akt/NF-κB signaling pathway. PMID:26722450

Kit receptor tyrosine kinase dysregulations in feline splenic mast cell tumours.

PubMed

Sabattini, S; Barzon, G; Giantin, M; Lopparelli, R M; Dacasto, M; Prata, D; Bettini, G

2017-09-01

This study investigated Kit receptor dysregulations (cytoplasmic immunohistochemical expression and/or c-KIT mutations) in cats affected with splenic mast cell tumours. Twenty-two cats were included. Median survival time was 780 days (range: 1-1219). An exclusive splenic involvement was significantly (P = 0.042) associated with longer survival (807 versus 120 days). Eighteen tumours (85.7%) showed Kit cytoplasmic expression (Kit pattern 2, 3). Mutation analysis was successful in 20 cases. Fourteen missense mutations were detected in 13 out of 20 tumours (65%). Eleven (78.6%) were located in exon 8, and three (21.6%) in exon 9. No mutations were detected in exons 11 and 17. Seven mutations corresponded to the same internal tandem duplication in exon 8 (c.1245_1256dup). Although the association between Kit cytoplasmic expression and mutations was significant, immunohistochemistry cannot be considered a surrogate marker for mutation analysis. No correlation was observed between c-Kit mutations and tumour differentiation, mitotic activity or survival. © 2016 John Wiley & Sons Ltd.

15 CFR 742.2 - Proliferation of chemical and biological weapons.

Code of Federal Regulations, 2014 CFR

2014-01-01

..., analytical, diagnostic, and food testing kits that consist of pre-packaged materials of defined composition... purposes: (1) Test kits containing no more than 300 grams of any chemical controlled by ECCN 1C350.b or .c... the EAR). Such test kits are controlled by ECCN 1C395 for CB and CW reasons, to States not Party to...

Unraveling the spectrum of KIT mutations in gastrointestinal stromal tumors: An Indian Tertiary Cancer Center Experience.

PubMed

Pai, Trupti; Bal, Munita; Shetty, Omshree; Gurav, Mamta; Ostwal, Vikas; Ramaswamy, Anant; Ramadwar, Mukta; Desai, Sangeeta

2017-01-01

Primary mutations in the KIT gene are the driving force for gastrointestinal stromal tumors (GIST) tumorigenesis. Predictive role of KIT mutation status aids oncologists in patient management. There is a paucity of comprehensive data on the frequency of mutations in the KIT gene in GIST affecting Indian patients. The aims of this study were to determine the frequency and spectrum of molecular alterations affecting the KIT gene and assess their association with clinicopathologic features in a cohort of patients of GIST. Morphological and immunohistochemically confirmed GIST cases ( n = 114) accessioned from August 2014-June 2015 were analyzed for mutations in KIT exons 9, 11, 13, and 17 and subjected to Sanger sequencing onto the ABI 3500 Genetic Analyzer. The sequences were analyzed using sequence analysis software: SeqScape ® and Chromas Lite. KIT mutations were seen in 70% of cases and the majority of KIT mutations involved exon 11 (57%), followed by exon 9 (10%), exon 13 (3%), and exon 17 (1%). Most common exon 11 mutations were in-frame deletions (61.4%) followed by substitution mutations (19.3%). Exon 9 mutations showed identical duplication of Ala-Tyr at codons 502-503. Simultaneous mutations affecting exon 11 and 13 were discovered. Novel variations, namely, p.Q556E (c.1666C>G), p.Q556dup (c.1666_1668dupCAG), p.K558_V559delinsS (c.1672_1677delAAGGTTinsAGT), p.Y503_F504insTY (c.1509_1510insACCTAT), and p.K642R (c.1925A>G) involving exons 11, 9, and 13, respectively, were observed. First study with complete analysis of all 4 exons of KIT (exons 9, 11, 13, and 17) in Indian GIST patients. Along with well-described KIT mutations, several rare double mutations as well as novel alterations were reported in this series.

Cardiac hypertrophy limits infarct expansion after myocardial infarction in mice.

PubMed

Iismaa, Siiri E; Li, Ming; Kesteven, Scott; Wu, Jianxin; Chan, Andrea Y; Holman, Sara R; Calvert, John W; Haq, Ahtesham Ul; Nicks, Amy M; Naqvi, Nawazish; Husain, Ahsan; Feneley, Michael P; Graham, Robert M

2018-04-17

We have previously demonstrated that adult transgenic C57BL/6J mice with CM-restricted overexpression of the dominant negative W v mutant protein (dn-c-kit-Tg) respond to pressure overload with robust cardiomyocyte (CM) cell cycle entry. Here, we tested if outcomes after myocardial infarction (MI) due to coronary artery ligation are improved in this transgenic model. Compared to non-transgenic littermates (NTLs), adult male dn-c-kit-Tg mice displayed CM hypertrophy and concentric left ventricular (LV) hypertrophy in the absence of an increase in workload. Stroke volume and cardiac output were preserved and LV wall stress was markedly lower than that in NTLs, leading to a more energy-efficient heart. In response to MI, infarct size in adult (16-week old) dn-c-kit-Tg hearts was similar to that of NTL after 24 h but was half that in NTL hearts 12 weeks post-MI. Cumulative CM cell cycle entry was only modestly increased in dn-c-kit-Tg hearts. However, dn-c-kit-Tg mice were more resistant to infarct expansion, adverse LV remodelling and contractile dysfunction, and suffered no early death from LV rupture, relative to NTL mice. Thus, pre-existing cardiac hypertrophy lowers wall stress in dn-c-kit-Tg hearts, limits infarct expansion and prevents death from myocardial rupture.

Genetic and Biochemical Evidence That Haploinsufficiency of the Nf1 Tumor Suppressor Gene Modulates Melanocyte and Mast Cell Fates in Vivo

PubMed Central

Ingram, David A.; Yang, Feng-Chun; Travers, Jeffrey B.; Wenning, Mary Jo; Hiatt, Kelly; New, Sheryl; Hood, Antoinette; Shannon, Kevin; Williams, David A.; Clapp, D. Wade

2000-01-01

Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's “two hit” model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1−/− murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W41 mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras–mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W41) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types. PMID:10620616

Genetic and biochemical evidence that haploinsufficiency of the Nf1 tumor suppressor gene modulates melanocyte and mast cell fates in vivo.

PubMed

Ingram, D A; Yang, F C; Travers, J B; Wenning, M J; Hiatt, K; New, S; Hood, A; Shannon, K; Williams, D A; Clapp, D W

2000-01-03

Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's "two hit" model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1-/- murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W(41) mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras-mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W(41)) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types.

Masitinib Antagonizes ATP-Binding Cassette Subfamily C Member 10-Mediated Paclitaxel Resistance: A Preclinical Study

PubMed Central

Kathawala, Rishil J.; Sodani, Kamlesh; Chen, Kang; Patel, Atish; Abuznait, Alaa H.; Anreddy, Nagaraju; Sun, Yue-Li; Kaddoumi, Amal; Ashby, Charles R.; Chen, Zhe-Sheng

2014-01-01

Paclitaxel displays clinical activity against a wide variety of solid tumors. However, resistance to paclitaxel significantly attenuates the response to chemotherapy. The ABC transporter subfamily C member 10 (ABCC10), also known as multi-drug resistance protein 7 (MRP7) efflux transporter, is a major mediator of paclitaxel resistance. In this study, we show that masitinib, a small molecule stem-cell growth factor receptor (c-Kit) tyrosine kinase inhibitor, at non-toxic concentrations, significantly attenuates paclitaxel resistance in HEK293 cells transfected with ABCC10. Our in vitro studies indicated that masitinib (2.5 μM) enhanced the intracellular accumulation and decreased the efflux of paclitaxel by inhibiting the ABCC10 transport activity without altering the expression level of ABCC10 protein. Furthermore, masitinib, in combination with paclitaxel, significantly inhibited the growth of ABCC10-expressing tumors in nude athymic mice in vivo. Masitinib administration also resulted in a significant increase in the levels of paclitaxel in the plasma, tumors and lungs compared to paclitaxel alone. In conclusion, the combination of paclitaxel and masitinib could serve as a novel and useful therapeutic strategy to reverse paclitaxel resistance mediated by ABCC10. PMID:24431074

Transfected Cell Microarrays for the Expression of Membrane-Displayed Single-Chain Antibodies

DTIC Science & Technology

2011-01-01

v) yeast extract, 0.005% (w/v) NaCl, and 50 μg/ml kanamycin. The broth was stored at 4◦C for up to 3 months. 4. QIAGEN Plasmid Midi kit (Qiagen) or...ampi- cillin. The broth was stored at 4◦C for up to 3 months. 16. QIAprep spin miniprep kit and QIAGEN Plasmid Midi kit (Qiagen) or PureYield Plasmid...was stored at 4◦C for up to 3 months. 8. QIAGEN Plasmid Midi kit (Qiagen) or PureYield Plasmid Midiprep System (Promega Corp.) was stored at room tem

Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuzawa,S.; Opatowsky, Y.; Zhang, Z.

2007-01-01

Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4more » interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.« less

The AskA Starter Kit: How To Build and Maintain Digital Reference Services.

ERIC Educational Resources Information Center

Lankes, R. David; Kasowitz, Abby S.

This Starter Kit is designed to help organizations and individuals who wish to offer human-mediated information services via the Internet to users in the K-12 community. A six-step process is proposed for organizations to follow in creating an "AskA" service. This process addresses all aspects involved in building and maintaining an AskA…

Performance Tested Method multiple laboratory validation study of ELISA-based assays for the detection of peanuts in food.

PubMed

Park, Douglas L; Coates, Scott; Brewer, Vickery A; Garber, Eric A E; Abouzied, Mohamed; Johnson, Kurt; Ritter, Bruce; McKenzie, Deborah

2005-01-01

Performance Tested Method multiple laboratory validations for the detection of peanut protein in 4 different food matrixes were conducted under the auspices of the AOAC Research Institute. In this blind study, 3 commercially available ELISA test kits were validated: Neogen Veratox for Peanut, R-Biopharm RIDASCREEN FAST Peanut, and Tepnel BioKits for Peanut Assay. The food matrixes used were breakfast cereal, cookies, ice cream, and milk chocolate spiked at 0 and 5 ppm peanut. Analyses of the samples were conducted by laboratories representing industry and international and U.S governmental agencies. All 3 commercial test kits successfully identified spiked and peanut-free samples. The validation study required 60 analyses on test samples at the target level 5 microg peanut/g food and 60 analyses at a peanut-free level, which was designed to ensure that the lower 95% confidence limit for the sensitivity and specificity would not be <90%. The probability that a test sample contains an allergen given a prevalence rate of 5% and a positive test result using a single test kit analysis with 95% sensitivity and 95% specificity, which was demonstrated for these test kits, would be 50%. When 2 test kits are run simultaneously on all samples, the probability becomes 95%. It is therefore recommended that all field samples be analyzed with at least 2 of the validated kits.

Resveratrol Improves Cell Cycle Arrest in Chronic Prostatitis Rats, by C-kit/SCF Suppression.

PubMed

He, Yi; Zeng, Huizhi; Yu, Yang; Zhang, Jiashu; Zeng, Xiaona; Gong, Fengtao; Liu, Qi; Yang, Bo

2017-08-01

Chronic prostatitis (CP) with complex pathogenesis is difficult for treatment. c-kit has been associated with the control of cell proliferation of prostate cells. This study aims to evaluate the role of resveratrol, an activator of Sirt1, in regulating the expression of c-kit in CP and investigate the consequent effects on cell cycle. Rat model of CP was established through subcutaneous injections of diphtheria-pertussis-tetanus vaccine and subsequently treated with resveratrol. Hematoxylin and eosin staining was performed to identify the histopathological changes in prostates. Western blotting and immunohistochemical staining examined the expression level of c-kit, stem cell factor (SCF), Sirt1, and cell cycle-associated proteins. The model group exhibited severe diffuse chronic inflammation, characterized by leukocyte infiltration and papillary frond protrusion into the gland cavities, and a notable increase in prostatic epithelial height. Gland lumen diameter was also significantly smaller; the activity of c-kit/SCF in the CP rats was increased significantly compared to the control group. Meanwhile, the cell cycle proteins are dysregulated significantly in CP rats. Resveratrol treatment significantly improved these factors by Sirt1 activation. Dysregulation of cell cycle was involved in the pathological processes of CP, which was improved after resveratrol treatment by the downregulation of c-kit/SCF by activating Sirt1.

c-KIT positive schistosomal urinary bladder carcinoma are frequent but lack KIT gene mutations.

PubMed

Shams, Tahany M; Metawea, Mokhtar; Salim, Elsayed I

2013-01-01

Urinary bladder squamous cell carcinoma (SCC), one of the most common neoplasms in Egypt, is attributed to chronic urinary infection with Schistosoma haematobium (Schistosomiasis). The proto-oncogene c-KIT, encoding a tyrosine kinase receptor and implicated in the development of a number of human malignancies, has not been studied so far in schistosomal urinary bladder SCCs. We therefore determined immunohistochemical (IHC) expression of c-KIT in paraffin sections from 120 radical cystectomies of SCCs originally obtained from the Pathology Department of Suez Canal University (Ismailia, Egypt). Each slide was evaluated for staining intensity where the staining extent of >10% of cells was considered positive. c-KIT overexpression was detected in 78.3% (94/120) of the patients, the staining extents in the tumor cells were 11-50% and >50% in 40 (42.6%) and 54 (57.4%) respectively. The positive cases had 14.9%, 63.8%, 21.3% as weak, moderate and strong intensity respectively. Patients with positive bilharzial ova had significantly higher c-KIT expression than patients without (95.2% vs. 38.9%, P=0.000). Mutation analysis of exons 9-13 was negative in thirty KIT positive cases. The high rate of positivity in SBSCC was one of the striking findings; However, CD117 may be a potential target for site specific immunotherapy to improve the outcome of this tumor.

Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody.

PubMed

He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Yang, Xiqin; Bai, Guanzhong; Ling, Shigan; Zhang, Heqiu

2011-08-01

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

Radiosensitization Effect of STI-571 on Pancreatic Cancer Cells In Vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chung, Hye Won; Wen, Jing; Lim, Jong-Baeck

2009-11-01

Purpose: To examine STI-571-induced radiosensitivity in human pancreatic cancer cells in vitro. Methods and Materials: Three human pancreatic cancer cell lines (Bxpc-3, Capan-1, and MiaPaCa-2) exhibiting different expression levels of c-Kit and platelet-derived growth factor receptor beta (PDGFRbeta) and showing different K-ras mutation types were used. For evaluation of the antitumor activity of STI-571 in combination with radiation, clonogenic survival assays, Western blot analysis, and the annexin V/propidium iodide assay with microscopic evaluation by 4',6-diamidino-2-phenylindole were conducted. Results: Dramatic phosphorylated (p)-c-Kit and p-PDGFRbeta attenuation, a modest dose- and time-dependent growth inhibition, and significant radiosensitization were observed after STI-571 treatment inmore » view of apoptosis, although the levels of growth inhibition and increased radiosensitization were different according to cell lines. The grades of radiosensitivity corresponded to the attenuation levels of p-c-Kit and p-PDGFRbeta by STI-571, particularly to those of p-c-Kit, and the radiosensitivity was partially affected by K-ras mutation in pancreatic cancer cells. Among downstream pathways associated with c-Kit or PDGFRbeta, p-PLCgamma was more closely related to radiosensitivity compared with p-Akt1 or p-extracellular signal-regulated kinase 1. Conclusion: STI-571 enhances radiation response in pancreatic cancer cells. This effect is affected by the attenuation levels of p-c-Kit or p-PDGFRbeta, and K-ras mutation status. Among them, p-c-Kit plays more important roles in the radiosensitivity in pancreatic cancer compared with p-PDGFRbeta or K-ras mutation status.« less

Prevalence of c-KIT Mutations in Gonadoblastoma and Dysgerminomas of Patients with Disorders of Sex Development (DSD) and Ovarian Dysgerminomas

PubMed Central

Hersmus, Remko; Stoop, Hans; van de Geijn, Gert Jan; Eini, Ronak; Biermann, Katharina; Oosterhuis, J. Wolter; DHooge, Catharina; Schneider, Dominik T.; Meijssen, Isabelle C.; Dinjens, Winand N. M.; Dubbink, Hendrikus Jan; Drop, Stenvert L. S.; Looijenga, Leendert H. J.

2012-01-01

Activating c-KIT mutations (exons 11 and 17) are found in 10–40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent ∼3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY. PMID:22937135

In phyllodes tumour of the breast expression of c-kit but not of ALDH1A1 is associated with adverse clinico-pathological features.

PubMed

Chougule, Abhijit; Bal, Amanjit; Das, Ashim; Kohli, Pavneet Singh; Singh, Gurpreet

2016-12-01

Attempts at identification of an ideal prognostic/predictive biomarker in phyllodes tumour (PT) have not been fruitful so far. Studies evaluating c-kit expression in PT have shown contradictory results. Recently aldehyde dehydrogenase 1A1 (ALDH1A1) was proposed as a stem cell marker for malignant PT but its expression has not been studied in benign and borderline tumours. We aimed to evaluate expression and prognostic significance of c-kit and ALDH1A1 in different grades of PT. Epithelial and stromal c-kit and ALDH1A1 expression were studied in 104 PT cases (86 primary and 18 recurrent tumours) and compared with different clinico-pathological features and recurrence rates. Stromal c-kit expression at 1 % cutoff correlated with increasing tumour grade, larger tumour size, hypercellularity, nuclear atypia, stromal overgrowth, infiltrative margins and mitotic count. These associations, however, were lost with higher (5 or 10 %) cutoffs. Conversely, decreased c-kit expression in the epithelial component correlated with increasing tumour grade, regardless of the cutoffs used. Stromal ALDH1A1 expression did not have significant associations with tumour grade or other adverse clinico-pathological features, regardless of different cutoffs. None of the cases showed significant epithelial ALDH1A1 expression. Expression of c-kit was associated with poorer overall survival (p = 0.011), while ALDH1A1 expression was associated with shorter recurrence-free survival (p = 0.036). In conclusion, c-kit expression was associated with higher tumour grade and adverse clinico-pathological features. However, these associations are cutoff dependent, partly explaining the variability in previously reported studies. ALDH1A1 expression did not have significant correlations with tumour grade and adverse clinico-pathological variables.

mRNA Expression of Platelet-Derived Growth Factor Receptor-{beta} and C-KIT: Correlation With Pathologic Response to Cetuximab-Based Chemoradiotherapy in Patients With Rectal Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erben, Philipp; Horisberger, Karoline; Muessle, Benjamin

2008-12-01

Purpose: Deviant expression of platelet-derived growth factor receptor-{beta} (PDGFR{beta}) and c-kit was shown in patients with colorectal cancer. In the present study, mRNA expression of PDGFR{beta} and c-kit in 33 patients with locally advanced rectal cancer undergoing preoperative chemoradiotherapy with cetuximab/capecitabine/irinotecan in correlation with the tumor regression rate was investigated. Methods and Materials: Pretherapeutic biopsy cores and tumor material from the resected specimens were collected in parallel with normal rectal mucosa. The expression levels of PDGFR{beta} and c-kit were measured by quantitative polymerase chain reaction. Tumors were classified as good responders (tumor regression grade [TRG], 2-3) or poor responders (TRG,more » 0-1). Results: The TRG evaluation of the resected specimen was TRG 0-1 in 11 and TRG 2-3 in 22. The median normalized ratios in the pretreatment mucosa vs. tumor biopsy cores was as follows: PDGFR{beta} ratio of 15.2 vs. 49.5 (p <0.0001) and c-kit ratio of 0.94 vs. 0.67 (p = 0.014). The same tendency was observed for the median PDGFR{beta} ratios after chemoradiotherapy completion: 34.2 vs. 170.0 (p <0.0001). The PDGFR{beta} and c-kit mRNA expression values in the pretreatment tumor biopsy cores were lower than the values in the resected specimens: PDGFR{beta} ratio 49.5 vs. 170.0 (p = 0.0002) and c-kit ratio 0.67 vs. 1.1 (p = 0.0003). Nevertheless, no correlation was seen between the pretherapeutic PDGFR{beta} and c-kit mRNA expression and the pathologic regression rate. Conclusion: Cetuximab-based chemoradiotherapy increased PDGFR{beta} levels even further compared with the pretreatment samples and deserves further investigation.« less

First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential.

PubMed

Balbi, Carolina; Piccoli, Martina; Barile, Lucio; Papait, Andrea; Armirotti, Andrea; Principi, Elisa; Reverberi, Daniele; Pascucci, Luisa; Becherini, Pamela; Varesio, Luigi; Mogni, Massimo; Coviello, Domenico; Bandiera, Tiziano; Pozzobon, Michela; Cancedda, Ranieri; Bollini, Sveva

2017-05-01

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT + hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O 2 versus 1% O 2 preconditioning). The capacity of the c-KIT + hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, Smn F7/F7 mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT + hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340-1355. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

C/EBPβ Mediates Growth Hormone-Regulated Expression of Multiple Target Genes

PubMed Central

Cui, Tracy X.; Lin, Grace; LaPensee, Christopher R.; Calinescu, Anda-Alexandra; Rathore, Maanjot; Streeter, Cale; Piwien-Pilipuk, Graciela; Lanning, Nathan; Jin, Hui; Carter-Su, Christin; Qin, Zhaohui S.

2011-01-01

Regulation of c-Fos transcription by GH is mediated by CCAAT/enhancer binding protein β (C/EBPβ). This study examines the role of C/EBPβ in mediating GH activation of other early response genes, including Cyr61, Btg2, Socs3, Zfp36, and Socs1. C/EBPβ depletion using short hairpin RNA impaired responsiveness of these genes to GH, as seen for c-Fos. Rescue with wild-type C/EBPβ led to GH-dependent recruitment of the coactivator p300 to the c-Fos promoter. In contrast, rescue with C/EBPβ mutated at the ERK phosphorylation site at T188 failed to induce GH-dependent recruitment of p300, indicating that ERK-mediated phosphorylation of C/EBPβ at T188 is required for GH-induced recruitment of p300 to c-Fos. GH also induced the occupancy of phosphorylated C/EBPβ and p300 on Cyr61, Btg2, and Socs3 at predicted C/EBP-cAMP response element-binding protein motifs in their promoters. Consistent with a role for ERKs in GH-induced expression of these genes, treatment with U0126 to block ERK phosphorylation inhibited their GH-induced expression. In contrast, GH-dependent expression of Zfp36 and Socs1 was not inhibited by U0126. Thus, induction of multiple early response genes by GH in 3T3-F442A cells is mediated by C/EBPβ. A subset of these genes is regulated similarly to c-Fos, through a mechanism involving GH-stimulated ERK 1/2 activation, phosphorylation of C/EBPβ, and recruitment of p300. Overall, these studies suggest that C/EBPβ, like the signal transducer and activator of transcription proteins, regulates multiple genes in response to GH. PMID:21292824

Ethanolic extract of Moringa oleifera Lam. leaves protect the pre-pubertal spermatogonial cells from cyclophosphamide-induced damage.

PubMed

Nayak, Guruprasad; Honguntikar, Sachin D; Kalthur, Sneha Guruprasad; D'Souza, Antony Sylvan; Mutalik, Srinivas; Setty, Manjunath M; Kalyankumar, Raksha; Krishnamurthy, Hanumanthappa; Kalthur, Guruprasad; Adiga, Satish Kumar

2016-04-22

Moringa oleifera Lam. is widely cultivated in Asian and African countries for its medicinal and dietary significance. The leaves are highly nutritious and are known to possess various biological activities. Pre-pubertal Swiss albino male mice were injected with single dose of cyclophosphamide (CP, 200mg/kg body weight) or ethanolic extract of Moringa oleifera leaves (MOE, 100mg/kg body weight) intraperitoneally. In combination group, MOE was administered 24h prior to CP injection. CP induced a significant decrease in testicular weight (p<0.01) and depletion of germ cells (p<0.001) and higher level of DNA damage (p<0.001) compared to control. The expression of P53, Bax, Cytochrome C (Cyt C) was increased while there was a decrease in the expression of Bcl2, c-Kit and Oct4. Administration of MOE 24h prior to CP treatment ameliorated the depletion (p<0.001), DNA damage (p<0.001) and apoptosis (p<0.01) of germ cells induced by CP. The mitigating effect of MOE appears to be mediated by up-regulating the expression of c-Kit and Oct4 transcripts in P53-independent manner. MOE protects the spermatogonial cells from CP-induced damage by modulating the apoptotic response elicited by CP and therefore can be considered as an efficient method of male fertility preservation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A variant c-KIT mutation, D816H, fundamental to the sequential development of an ovarian mixed germ cell tumor and systemic mastocytosis with chronic myelomonocytic leukemia.

PubMed

Mitchell, Sarah G; Bunting, Silvia T; Saxe, Debra; Olson, Thomas; Keller, Frank G

2017-04-01

An activating point mutation of the c-KIT tyrosine kinase receptor gene, D816H, has been described in germ cell tumors (GCTs). We report an adolescent diagnosed with an ovarian mixed GCT and systemic mastocytosis with chronic myelomonocytic leukemia (SM-CMML). The teratoma and dysgerminoma differed by copy number aberrations via single nucleotide polymorphism (SNP) microarray, but were inclusive of the same c-KIT D816H point mutation (c.2446G>C) also identified in blood and bone marrow mast cells. These findings indicate not only a clonal origin of the GCT and hematologic malignancy, but also suggest a rare KIT mutation may be playing a fundamental role in malignancy development. © 2016 Wiley Periodicals, Inc.

Phosphorylation of the Activation Loop Tyrosine 823 in c-Kit Is Crucial for Cell Survival and Proliferation*

PubMed Central

Agarwal, Shruti; Kazi, Julhash U.; Rönnstrand, Lars

2013-01-01

The receptor tyrosine kinase c-Kit, also known as the stem cell factor receptor, plays a key role in several developmental processes. Activating mutations in c-Kit lead to alteration of these cellular processes and have been implicated in many human cancers such as gastrointestinal stromal tumors, acute myeloid leukemia, testicular seminomas and mastocytosis. Regulation of the catalytic activity of several kinases is known to be governed by phosphorylation of tyrosine residues in the activation loop of the kinase domain. However, in the case of c-Kit phosphorylation of Tyr-823 has been demonstrated to be a late event that is not required for kinase activation. However, because phosphorylation of Tyr-823 is a ligand-activated event, we sought to investigate the functional consequences of Tyr-823 phosphorylation. By using a tyrosine-to-phenylalanine mutant of tyrosine 823, we investigated the impact of Tyr-823 on c-Kit signaling. We demonstrate here that Tyr-823 is crucial for cell survival and proliferation and that mutation of Tyr-823 to phenylalanine leads to decreased sustained phosphorylation and ubiquitination of c-Kit as compared with the wild-type receptor. Furthermore, the mutated receptor was, upon ligand-stimulation, quickly internalized and degraded. Phosphorylation of the E3 ubiquitin ligase Cbl was transient, followed by a substantial reduction in phosphorylation of downstream signaling molecules such as Akt, Erk, p38, Shc, and Gab2. Thus, we propose that activation loop tyrosine 823 is crucial for activation of both the MAPK and PI3K pathways and that its disruption leads to a destabilization of the c-Kit receptor and decreased survival of cells. PMID:23803604

Oct4-GFP expression during transformation of gonocytes into spermatogonial stem cells in the perinatal mouse testis.

PubMed

Li, Ruili; Vannitamby, Amanda; Zhang, Jian-Guo; Fehmel, Emma L; Southwell, Bridget R; Hutson, John M

2015-12-01

In cryptorchidism perinatal failure to switch off Oct4, a germ cell (GC) marker, may lead to carcinoma in situ. We aimed to analyze Oct4 expression during mouse gonocyte transformation into spermatogonial stem cells (SSC). Testes from OG2 (Oct4-promoter driven eGFP) mice at embryonic day (E) 17 and postnatal day P0-10 underwent immunohistochemistry and immunoblotting. Antibodies against MVH, AMH, Ki67, and c-Kit were visualized by confocal microscopy. Numbers of Oct4-GFP(+) GC and Oct4-GFP(-) GC/tubule were counted using ImageJ. Data were analyzed using nonparametric one-way ANOVA. GC from E17-P4 were Oct4-GFP(+). Numbers of Oct4-GFP(-) GC/tubule increased from P6-10, whereas Oct4-GFP(+) GC/tubule numbers remained similar between P6 and P10. Sertoli cells proliferated from E17-P10, whereas GC only proliferated from P2. Gonocytes (Oct4-GFP(+)/c-Kit(-)) central in tubules migrated to the basement membrane to become prospermatogonia (Oct4-GFP(+)/c-Kit(-)) and then SSC (Oct4-GFP(+)/c-Kit(+)) from day 4 and further developed into Oct4-GFP(-)/c-Kit(+) at P6. In Oct4-GFP mice both centrally located gonocytes and prospermatogonia located at the tubular basement membrane were Oct4-GFP(+)/c-Kit(-) before further developing into SSC (Oct4-GFP(+)/c-Kit(+)). This indicates that Oct4 is important in gonocyte transformation into SSC. Understanding this process will aid GC tumor diagnostics and fertility potential in boys with UDT undergoing orchidopexy. Copyright © 2015 Elsevier Inc. All rights reserved.

Gastroschisis in the rat model is associated with a delayed maturation of intestinal pacemaker cells and smooth muscle cells.

PubMed

Midrio, P; Faussone-Pellegrini, M S; Vannucchi, M G; Flake, A W

2004-10-01

A pacemaker system is required for peristalsis generation. The interstitial cells of Cajal (ICC) are considered the intestinal pacemaker, and are identified by expression of the c-kit gene--encoded protein. Gastroschisis is characterized by a severe gastrointestinal dysmotility in newborns. In spite of this clinical picture, few studies have focused on smooth muscle cells (SMC) morphology and none on ICC. Therefore, their morphology has been studied in fetuses at term in the rat model of gastroschisis. At 18.5 day's gestation (E18.5), 10 rat fetuses were killed, 10 underwent surgical creation of gastroschisis, and 10 underwent manipulation only. The small intestine of the latter 2 groups was harvested at E21.5. Specimens were processed for H&E, c-kit and actin (alpha smooth muscle antibody [alpha-SMA]) immunohistochemistry, and transmission electron microscopy (TEM). In the controls, SMC were c-kit+ and alpha-SMA+, with labeling intensity increasing by age. At E21.5, some cells around the Auerbach's plexus were more intensely c-kit+, and differentiating ICC were seen under TEM at this level. Gastroschisis fetuses had no c-kit+ cells referable to ICC. In the more damaged loops, SMC were very faintly c-kit+ and alpha-SMA+. Under TEM, there were few differentiated SMC and no presumptive ICC. In the less-damaged loops, SMC were faintly c-kit+ and alpha-SMA+ and had ultrastructural features intermediate between those of E18.5 and E21.5 controls; ICC were very immature. ICC and SMC differentiation is delayed in gastroschisis with the most damaged loops showing the most incomplete picture. These findings might help in understanding the delayed onset of peristalsis and the variable time-course of the recover seen in babies affected by gastroschisis.

Discovery of Aryl Aminoquinazoline Pyridones as Potent, Selective, and Orally Efficacious Inhibitors of Receptor Tyrosine Kinase c-Kit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Essa; Tasker, Andrew; White, Ryan D.

2008-12-09

Inhibition of c-Kit has the potential to treat mast cell associated fibrotic diseases. We report the discovery of several aminoquinazoline pyridones that are potent inhibitors of c-Kit with greater than 200-fold selectivity against KDR, p38, Lck, and Src. In vivo efficacy of pyridone 16 by dose-dependent inhibition of histamine release was demonstrated in a rodent pharmacodynamic model of mast cell activation.

Generation of Functional Blood Vessels from a Single c-kit+ Adult Vascular Endothelial Stem Cell

PubMed Central

Fang, Shentong; Wei, Jing; Pentinmikko, Nalle; Leinonen, Hannele; Salven, Petri

2012-01-01

In adults, the growth of blood vessels, a process known as angiogenesis, is essential for organ growth and repair. In many disorders including cancer, angiogenesis becomes excessive. The cellular origin of new vascular endothelial cells (ECs) during blood vessel growth in angiogenic situations has remained unknown. Here, we provide evidence for adult vascular endothelial stem cells (VESCs) that reside in the blood vessel wall endothelium. VESCs constitute a small subpopulation within CD117+ (c-kit+) ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity. Isolated VESCs can produce tens of millions of endothelial daughter cells in vitro. A single transplanted c-kit-expressing VESC by the phenotype lin−CD31+CD105+Sca1+CD117+ can generate in vivo functional blood vessels that connect to host circulation. VESCs also have long-term self-renewal capacity, a defining functional property of adult stem cells. To provide functional verification on the role of c-kit in VESCs, we show that a genetic deficit in endothelial c-kit expression markedly decreases total colony-forming VESCs. In vivo, c-kit expression deficit resulted in impaired EC proliferation and angiogenesis and retardation of tumor growth. Isolated VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization after ischemic events. VESCs also provide a novel cellular target to block pathological angiogenesis and cancer growth. PMID:23091420

Tyrosine Kinase Inhibition in HPV-related Squamous Cell Carcinoma Reveals Beneficial Expression of cKIT and Src.

PubMed

Kramer, Benedikt; Kneissle, Marcel; Birk, Richard; Rotter, Nicole; Aderhold, Christoph

2018-05-01

Therapeutic options of locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC) are limited. Src and cKIT are key protein regulators for local tumor progression. The aim of the study was to investigate the therapeutic potential of targeted therapies in human squamous cell carcinoma (HNSCC) in vitro. Therefore, the influence of the selective tyrosine kinase inhibitors niotinib, dasatinib, erlotinib, gefitinib and afatinib on Src and cKIT expression in Human papilloma virus (HPV)-positive and HPV-negative squamous cancer cells (SCC) was analyzed in vitro. ELISA was performed to evaluate the expression of Src and cKIT under the influence of nilotinib, dasatinib, erlotinib, gefitinib and afatinib (10 μmol/l) in HPV-negative and HPV-positive SCC (24-96 h of incubation). Gefitinib significantly increased cKIT expression in HPV-positive and HPV-negative cells whereas nilotinib and afatinib decreased cKIT expression in HPV-positive SCC. The influence of tyrosine kinase inhibitors in HPV-negative SCC was marginal. Surprisingly, Src expression was significantly increased by all tested tyrosine kinase inhibitors in HPV-positive SCC. The results revealed beneficial and unexpected information concerning the interaction of selective tyrosine kinase inhibitors and the tumor biology of HNSCC. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Abdominal Manual Therapy Repairs Interstitial Cells of Cajal and Increases Colonic c-Kit Expression When Treating Bowel Dysfunction after Spinal Cord Injury

PubMed Central

Zhang, Wenyi; Zhu, Zhaojin; Xie, Bin; Yu, Jun

2017-01-01

Background This study aimed to evaluate the therapeutic effects of abdominal manual therapy (AMT) on bowel dysfunction after spinal cord injury (SCI), investigating interstitial cells of Cajal (ICCs) and related c-kit expression. Methods Model rats were divided as SCI and SCI with drug treatment (intragastric mosapride), low-intensity (SCI + LMT; 50 g, 50 times/min), and high-intensity AMT (SCI + HMT; 100 g, 150 times/min). After 14 days of treatment, weight, improved Basso-Beattie-Bresnahan (BBB) locomotor score, and intestinal movement were evaluated. Morphological structure of spinal cord and colon tissues were examined. Immunostaining, RT-PCR, and western blot were used to assess c-kit expression. Results In SCI rats, AMT could not restore BBB, but it significantly increased weight, shortened time to defecation, increased feces amounts, and improved fecal pellet traits and colon histology. AMT improved the number, distribution, and ultrastructure of colonic ICCs, increasing colonic c-kit mRNA and protein levels. Compared with the SCI + Drug and SCI + LMT groups, the SCI + HMT group showed better therapeutic effect in improving intestinal transmission function and promoting c-kit expression. Conclusions AMT is an effective therapy for recovery of intestinal transmission function. It could repair ICCs and increase c-kit expression in colon tissues after SCI, in a frequency-dependent and pressure-dependent manner. PMID:29349063

Evaluation of rapid post-mortem test kits for bovine spongiform encephalopathy (BSE) screening in Japan: Their analytical sensitivity to atypical BSE prions.

PubMed

Hagiwara, Ken'ichi; Iwamaru, Yoshifumi; Tabeta, Naoko; Yokoyama, Takashi; Tobiume, Minoru

2017-03-04

A classical type of bovine spongiform encephalopathy (C-BSE), recognized in 1987, had a large impact on public health due to its zoonotic link to variant Creutzfeldt-Jakob disease by the human consumption of dietary products contaminated with the C-BSE prion. Thus, a number of countries implemented BSE surveillance using rapid post-mortem test kits that were approved for detection of the C-BSE prion in the cattle brain. However, as atypical BSE (L- and H-BSE) cases emerged in subsequent years, the efficacy of the kits for the detection of atypical BSE prions became a matter of concern. In response to this, laboratories in the European Union and Canada evaluated the kits used in their countries. Here, we carried out an evaluation study of NippiBL®, a kit currently used for BSE screening in Japan. By applying the kit to cattle brains of field cases of C-BSE and L-BSE, and an experimental case of H-BSE, we showed its comparable sensitivities to C, L-, and H-BSE prions, and satisfactory performance required by the European Food Safety Authority. In addition to NippiBL®, two kits (TeSeE® and FRELISA®) formerly used in Japan were effective for detection of the L-BSE prion, although the two kits were unable to be tested for the H-BSE prion due to the discontinuation of domestic sales during this study. These results indicate that BSE screening in Japan is as effective as those in other countries, and it is unlikely that cases of atypical BSE have been overlooked.

Hydrogen-Rich Water Ameliorates Total Body Irradiation-Induced Hematopoietic Stem Cell Injury by Reducing Hydroxyl Radical

PubMed Central

Xue, Xiaolei; Han, Xiaodan; Li, Yuan; Lu, Lu; Li, Deguan

2017-01-01

We examined whether consumption of hydrogen-rich water (HW) could ameliorate hematopoietic stem cell (HSC) injury in mice with total body irradiation (TBI). The results indicated that HW alleviated TBI-induced HSC injury with respect to cell number alteration and to the self-renewal and differentiation of HSCs. HW specifically decreased hydroxyl radical (∙OH) levels in the c-kit+ cells of 4 Gy irradiated mice. Proliferative bone marrow cells (BMCs) increased and apoptotic c-kit+ cells decreased in irradiated mice uptaken with HW. In addition, the mean fluorescence intensity (MFI) of γ-H2AX and percentage of 8-oxoguanine positive cells significantly decreased in HW-treated c-kit+ cells, indicating that HW can alleviate TBI-induced DNA damage and oxidative DNA damage in c-kit+ cells. Finally, the cell cycle (P21), cell apoptosis (BCL-XL and BAK), and oxidative stress (NRF2, HO-1, NQO1, SOD, and GPX1) proteins were significantly altered by HW in irradiated mouse c-kit+ cells. Collectively, the present results suggest that HW protects against TBI-induced HSC injury. PMID:28243358

Hydrogen-Rich Water Ameliorates Total Body Irradiation-Induced Hematopoietic Stem Cell Injury by Reducing Hydroxyl Radical.

PubMed

Zhang, Junling; Xue, Xiaolei; Han, Xiaodan; Li, Yuan; Lu, Lu; Li, Deguan; Fan, Saijun

2017-01-01

We examined whether consumption of hydrogen-rich water (HW) could ameliorate hematopoietic stem cell (HSC) injury in mice with total body irradiation (TBI). The results indicated that HW alleviated TBI-induced HSC injury with respect to cell number alteration and to the self-renewal and differentiation of HSCs. HW specifically decreased hydroxyl radical ( ∙ OH) levels in the c-kit + cells of 4 Gy irradiated mice. Proliferative bone marrow cells (BMCs) increased and apoptotic c-kit + cells decreased in irradiated mice uptaken with HW. In addition, the mean fluorescence intensity (MFI) of γ -H2AX and percentage of 8-oxoguanine positive cells significantly decreased in HW-treated c-kit + cells, indicating that HW can alleviate TBI-induced DNA damage and oxidative DNA damage in c-kit + cells. Finally, the cell cycle (P21), cell apoptosis (BCL-XL and BAK), and oxidative stress (NRF2, HO-1, NQO1, SOD, and GPX1) proteins were significantly altered by HW in irradiated mouse c-kit + cells. Collectively, the present results suggest that HW protects against TBI-induced HSC injury.

C-kit+ cells isolated from developing kidneys are a novel population of stem cells with regenerative potential

PubMed Central

Rangel, Erika B; Gomes, Samirah A; Dulce, Raul A; Premer, Courtney; Rodrigues, Claudia O; Kanashiro-Takeuchi, Rosemeire M; Oskouei, Behzad; Carvalho, Decio A; Ruiz, Phillip; Reiser, Jochen; Hare, Joshua M

2013-01-01

The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit+ cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit+ cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex-vivo expanded c-kit+ cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together these findings document a novel neonatal rat kidney c-kit+ stem cell population that can be isolated, expanded, cloned, differentiated, and employed for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications. PMID:23733311

15 CFR 742.2 - Proliferation of chemical and biological weapons.

Code of Federal Regulations, 2011 CFR

2011-01-01

... medical, analytical, diagnostic, and food testing kits that consist of pre-packaged materials of defined... health purposes: (1) Test kits containing no more than 300 grams of any chemical controlled by ECCN 1C350... part 745 of the EAR). Such test kits are controlled by ECCN 1C395 for CB and CW reasons, to States not...

15 CFR 742.2 - Proliferation of chemical and biological weapons.

Code of Federal Regulations, 2013 CFR

2013-01-01

... medical, analytical, diagnostic, and food testing kits that consist of pre-packaged materials of defined... health purposes: (1) Test kits containing no more than 300 grams of any chemical controlled by ECCN 1C350... part 745 of the EAR). Such test kits are controlled by ECCN 1C395 for CB and CW reasons, to States not...

Simultaneous detection of human CYP2C19 polymorphisms and antibiotic resistance of Helicobacter pylori using a personalised diagnosis kit.

PubMed

Zhang, Jun; Zhong, Jing; Ding, Jian; Shi, Jiemin; Tang, Tao; Liu, Qiqi; Huang, Huilian; Dai, Licheng; Yang, Ningmin

2018-06-01

A personalised diagnosis kit for Helicobacter pylori that employs visual gene chip technology for the simultaneous detection of CYP2C19 polymorphisms and clarithromycin/levofloxacin antibiotic resistance was evaluated. Gastric antrum mucosa biopsy specimens of 394 patients were tested using the kit. DNA sequencing and antibiotic susceptibility testing of the H. pylori were also performed. In total, 267 (67.8%) of the 394 specimens were positive for H. pylori using the kit and DNA sequencing, and 136 (34.5%) were positive by culturing. For human CYP2C19 and the bacterial 23S rRNA and gyrA genes, the concordance rates were 92.4% (364/394), 96.6% (258/267) and 97.0% (259/267) between the kit and DNA sequencing results, respectively. For clarithromycin and levofloxacin resistance, the concordance rates were 90.4% (123/136) and 81.6% (111/136) between the kit and antibiotic susceptibility testing results. The personalised diagnosis kit for H. pylori provides useful information for the choice of proton pump inhibitor and antibiotic in combination therapy. Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Evaluation of commercial kit based on loop-mediated isothermal amplification for rapid detection of low levels of uninjured and injured Salmonella on duck meat, bean sprouts, and fishballs in Singapore.

PubMed

Lim, Hazel Sin Yue; Zheng, Qianwang; Miks-Krajnik, Marta; Turner, Matthew; Yuk, Hyun-Gyun

2015-06-01

The objective of this study was to evaluate performance of the commercial kit based on loop-mediated isothermal amplification (LAMP) in comparison with the International Organization for Standardization method for detecting uninjured and sublethally injured Salmonella cells artificially inoculated at levels of 10(0) and 10(1) CFU/25 g on raw duck wing, raw mung bean sprouts, and processed fishballs. Injured cells were prepared by a heat treatment for duck wings and fishball samples and a chlorine treatment for bean sprout samples. Additionally, a validation study was performed on naturally contaminated food samples sold in Singapore. A total of 110 samples of each commodity were analyzed in this study. Regardless of inoculum levels, the detection by the commercial LAMP kit showed 100% sensitivity and specificity for both inoculated and uninoculated samples compared with the International Organization for Standardization method, with the exception of bean sprout samples. Only 20% of bean sprout samples inoculated with 10(0) CFU/25 g injured Salmonella cells were positive by using the commercial LAMP-based kit. However, all negative samples became positive following a secondary enrichment in Rappaport-Vassiliadis medium with soy broth or after concentration by centrifugation. These results suggest that secondary enrichment or centrifugation should be considered as an additional step to increase the sensitivity of the commercial LAMP-based kit with low numbers of injured target cells in samples with high background microflora (such as mung bean sprouts). The validation study also showed that the commercial LAMP-based kit provided 91% sensitivity and 95% specificity for naturally contaminated samples. Thus, this study demonstrates that the commercial LAMP-based kit might be a cost-effective method, as this system could provide rapid, accurate detection of both uninjured and injured Salmonella cells on raw duck wings, raw mung bean sprouts, and processed fishballs in less than 26 h.

Validation of spot-testing kits to determine iodine content in salt.

PubMed Central

Pandav, C. S.; Arora, N. K.; Krishnan, A.; Sankar, R.; Pandav, S.; Karmarkar, M. G.

2000-01-01

Iodine deficiency disorders are a major public health problem, and salt iodization is the most widely practised intervention for their elimination. For the intervention to be successful and sustainable, it is vital to monitor the iodine content of salt regularly. Iodometric titration, the traditional method for measuring iodine content, has problems related to accessibility and cost. The newer spot-testing kits are inexpensive, require minimal training, and provide immediate results. Using data from surveys to assess the availability of iodized salt in two states in India, Madhya Pradesh and the National Capital Territory of Delhi, we tested the suitability of such a kit in field situations. Salt samples from Delhi were collected from 30 schools, chosen using the Expanded Programme on Immunization (EPI) cluster sampling technique. A single observer made the measurement for iodine content using the kit. Salt samples from Madhya Pradesh were from 30 rural and 30 urban clusters, identified by using census data and the EPI cluster sampling technique. In each cluster, salt samples were collected from 10 randomly selected households and all retailers. The 15 investigators performing the survey estimated the iodine content of salt samples in the field using the kit. All the samples were brought to the central laboratory in Delhi, where iodine content was estimated using iodometric titration as a reference method. The agreement between the kit and titration values decreased as the number of observers increased. Although sensitivity was not much affected by the increase in the number of observers (93.3% for a single observer and 93.9% for multiple observers), specificity decreased sharply (90.4% for a single observer and 40.4% for multiple observers). Due to the low specificity and resulting high numbers of false-positives for the kit when used by multiple observers ("real-life situations"), kits were likely to consistently overestimate the availability of iodized salt. This overestimation could result in complacency. Therefore, we conclude that until a valid alternative is available, the titration method should be used for monitoring the iodine content of salt at all levels, from producer to consumer, to ensure effectiveness of the programme. PMID:10994281

Internal associations and dynamic expression of c-kit and nanog genes in ventricular remodelling induced by adriamycin.

PubMed

Liu, Zhen; Li, Shuo; Liu, Lingling; Guo, Zhikun; Wang, Pengfei

2016-09-01

The present study aimed to investigate the dynamic expression of the c-kit and nanog genes in rats with left ventricular remodelling induced by adriamycin (ADR), and explore its internal association and mechanism of action. Sprague-Dawley male rats were randomly divided into a normal control group and a heart failure model group. Heart failure was induced by a single intraperitoneal injection of ADR (4 mg/kg) weekly for six weeks. The normal control group was given the same amount of saline. At the eighth week, rat cardiac function was examined to demonstrate the formation of heart failure. The rat hearts were harvested frozen and sectioned, and the expression levels of the nanog and c-kit genes in the myocardial tissue samples were detected using immunohistochemistry, immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). Hematoxylin and eosin staining demonstrated various pathological changes in the myocardial cells in the heart failure model group, whereas myocardial infarction was not observed in the normal control group. Immunohistochemistry and immunofluorescence demonstrated that nanog-positive cells were predominantly expressed in the vascular endothelium, with a few myocardial cells and stem cells in normal myocardium. The expression levels of c-kit and nanog in the myocardium of the rats with heart failure decreased significantly. c-kit-positive cells clustered together in the epicardium and its vicinity, and c-kit expression significantly decreased in the myocardium of rats with heart failure, as compared with normal rats. In both groups, some cells co-expressed both the c-kit and nanog genes. The RT-PCR results demonstrated that the expression levels of the two genes in the heart failure model group were significantly lower compared with those in the normal control group (P<0.05). In conclusion, the c-kit- and nanog-positive stem cells decreased in the myocardium of the rats with left ventricular remodelling induced by ADR. Their abnormal expression was significantly correlated with left ventricular remodelling, thereby indicating an internal association (influences of two indexes in the experimental group and control group) between them.

C-MORE Science Kits: Putting Technology in the Hands of K-12 Teachers and Students

NASA Astrophysics Data System (ADS)

Achilles, K.; Weersing, K.; Daniels, C.; Puniwai, N.; Matsuzaki, J.; Bruno, B. C.

2008-12-01

The Center for Microbial Oceanography: Research and Education (C-MORE) is a NSF Science and Technology Center based at the University of Hawaii. The C-MORE education and outreach program offers a variety of resources and professional development opportunities for science educators, including online resources, participation in oceanography research cruises, teacher-training workshops, mini-grants to incorporate microbial oceanography-related content and activities into their classroom and, most recently, C- MORE science kits. C-MORE science kits provide hands-on classroom, field, and laboratory activities related to microbial oceanography for K-12 students. Each kit comes with complete materials and instructions, and is available free of charge to Hawaii's public school teachers. Several kits are available nationwide. C-MORE science kits cover a range of topics and technologies and are targeted at various grade levels. Here is a sampling of some available kits: 1) Marine Murder Mystery: The Case of the Missing Zooxanthellae. Students learn about the effect of climate change and other environmental threats on coral reef destruction through a murder-mystery experience. Participants also learn how to use DNA to identify a suspect. Grades levels: 3-8. 2) Statistical sampling. Students learn basic statistics through an exercise in random sampling, with applications to microbial oceanography. The laptops provided with this kit enable students to enter, analyze, and graph their data using EXCEL. Grades levels: 6-12. 3) Chlorophyll Lab. A research-quality fluorometer is used to measure the chlorophyll content in marine and freshwater systems. This enables students to compare biomass concentrations in samples collected from various locations. Grades levels: 9-12. 4) Conductivity-Temperature-Depth (CTD). Students predict how certain variables (e.g., temperature, pressure, chlorophyll, oxygen) vary with depth. A CTD, attached to a laptop computer, is deployed into deep water off a dock or a ship to collect real-time data and test their hypotheses. Grades levels: 9-12.

Emergence of CTNNB1 mutation at acquired resistance to KIT inhibitor in metastatic melanoma.

PubMed

Cho, J; Kim, S Y; Kim, Y J; Sim, M H; Kim, S T; Kim, N K D; Kim, K; Park, W; Kim, J H; Jang, K-T; Lee, J

2017-10-01

The KIT inhibitor, imatinib, has shown promising efficacy in patients with KIT-mutated melanoma; however, acquisition of resistance to imatinib occurs rapidly in the majority of patients. The mechanisms of acquired resistance to imatinib in melanoma remain unclear. We analyzed biopsy samples from paired baseline and post-treatment tumor lesions in one patient with KIT-mutated melanoma who had had an initial objective tumor regression in response to imatinib treatment followed by disease progression 8 months later. Targeted deep sequencing from post-treatment biopsy samples detected an additional mutation in CTNNB1 (S33C) with original KIT L576P mutation. We examined the functional role of the additional CTNNB1 S33C mutation in resistance to imatinib indirectly using the Ba/F3 cell model. Ba/F3 cell lines transfected with both the L576P KIT mutation and the CTNNB1 S33C mutation demonstrated no growth inhibition despite imatinib treatment, whereas growth inhibition was observed in the Ba/F3 cell line transfected with the L576 KIT mutation alone. We report the first identification of the emergence of a CTNNB1 mutation that can confer acquired resistance to imatinib. Further investigation into the causes of acquired resistance to imatinib will be essential to improve the prognosis for patients with KIT-mutated melanoma.

Stage-specific expression of DDX4 and c-kit at different developmental stages of the porcine testis.

PubMed

Lee, Ran; Lee, Won-Young; Park, Hyun-Jung; Ha, Woo-Tae; Woo, Jae-Seok; Chung, Hak-Jae; Lee, Ji-Heon; Hong, Kwonho; Song, Hyuk

2018-03-01

Spermatogenesis begins with spermatogonial stem cells (SSCs), which are located in the basement membrane of the adult testes. Previous studies have described specific biomarkers for undifferentiated porcine spermatogonia or SSCs; however, these markers are not sufficient to understand spermatogenesis at different developmental stages. The objective of this study was characterize the expression of DEAD-Box polypeptide 4 (DDX4, also known as VASA) and tyrosine-protein kinase kit (c-kit), as potential markers of male germ cells in the porcine testis. In porcine testis tissue at prepubertal stages (5, 30, and 60 days), DDX4 and c-kit protein expression was detected in the most undifferentiated spermatogonia, which also express protein gene product 9.5 (PGP9.5). However, in porcine testis tissues from pubertal and postpubertal stages (90, 120, and 150 days), DDX4 and c-kit were not detected in PGP9.5-positive undifferentiated spermatogonia. The DDX4 expression pattern was similar to that of c-kit in the porcine testis. In adult porcine testes, DDX4-expressing cells were located on the lumenal side, compared to synaptonemal complex protein 3-positive primary spermatocytes, but DDX-4 was not co-expressed with acrosin, a known acrosome marker. In addition, DDX4 was detected in PGP9.5-expressing porcine SSCs in culture. Based on our results, we suggest that DDX4 and c-kit are putative markers of undifferentiated spermatogonia in the prepubertal porcine testis. While in the postpubertal porcine testis, they are markers of differentiated spermatocytes. These findings may facilitate future studies of porcine spermatogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

KIT polymorphisms were associated with the risk for head and neck squamous carcinoma in Chinese population.

PubMed

Hang, Dong; Yuan, Hua; Liu, Li; Wang, Lihua; Miao, Limin; Zhu, Meng; Du, Jiangbo; Dai, Juncheng; Hu, Zhibin; Chen, Ning; Shen, Hongbing; Ma, Hongxia

2017-01-01

KITLG/KIT pathway plays a vital role in multiple types of human cancer including head and neck squamous cell carcinoma (HNSCC). Genetic variations in KITLG and KIT may affect the expression or function of these genes, thereby modifying cancer risk. In this study, we evaluated the association of KITLG and KIT polymorphisms with HNSCC risk among Chinese population. Twenty-two tagging SNPs in KITLG and KIT genes were genotyped in a case-control study with 576 HNSCC patients and 1552 healthy controls. Logistic regression analyses revealed that an upstream SNP rs6554198 [additive model: adjusted odds ratio (OR) = 0.85, 95% confidence interval (CI) = 0.74-0.97, P = 0.019] and two intron SNPs rs2237025 (additive model: adjusted OR = 0.82, 95%CI = 0.70-0.95, P = 0.007), and rs17084687 (additive model: adjusted OR = 0.85, 95%CI = 0.73-0.99, P = 0.042) of KIT were significantly associated with the decreased risk of HNSCC. Combined analysis of the three SNPs showed that subjects carrying the protective alleles had decreased risk of HNSCC in a dose-response manner (P trend  = 0.001). Furthermore, interaction analyses revealed a significant multiplicative interaction between rs17084687 and drinking on HNSCC risk (P = 0.012). Luciferase activity assay indicated that the allele A of potentially functional rs6554198 led to significantly lower transcription activity of KIT compared to the risk allele G. Summarily, our findings suggested that SNPs in KIT gene may play a role in genetic susceptibility to HNSCC, which may improve our understanding of the pathogenic mechanisms of this disease. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Retigeric acid B exerts antifungal effect through enhanced reactive oxygen species and decreased cAMP.

PubMed

Chang, Wen-Qiang; Wu, Xiu-Zhen; Cheng, Ai-Xia; Zhang, Li; Ji, Mei; Lou, Hong-Xiang

2011-05-01

Retigeric acid B (RAB), a triterpene acid isolated from Lobaria kurokawae exerts antifungal effect. The present study was designed to elucidate the underlying mechanisms by which RAB regulates the proliferation and cell death of Candida albicans. We measured the metabolic activity of C. albicans with WST1 Cell Proliferation and Cytotoxicity Assay Kit, analyzed the cell cycle by flow cytometry, visualized the ultrastructure by transmission electron microscopy (TEM) and investigated the apoptosis and necrosis induced by RAB using confocal microscopy. The reactive oxygen species (ROS) accumulation was determined by spectrophotometry, flow cytometry and fluorescent microscopy. The mtΔψ was detected using flow cytometry. And the levels of intracellular cAMP and ATP were measured with cAMP ELISA and ATP Assay Kits, respectively. The proliferation of the yeasts was blocked in G(2)/M phase by a low dose of RAB treatment and in G(1) phase at high concentration. When cultured in phosphate buffered saline (PBS) deprived of energy source, yeasts displayed the phenotype of death caused by accumulated ROS, mtΔψ hyperpolarization and dramatic decrease in ATP level in the presence of high dose of RAB. RAB inhibits the growth of C. albicans by stimulating ROS production and reducing intracellular cAMP. The ROS accumulation, mtΔψ hyperpolarization, ATP depletion and damaged plasma membrane integrity together mediate cell death of C. albicans induced by RAB. Our findings provide a novel molecular mechanism for exploring possible applications of lichen derived metabolites in fighting fungal infection in humans. Copyright © 2011 Elsevier B.V. All rights reserved.

KiT: a MATLAB package for kinetochore tracking.

PubMed

Armond, Jonathan W; Vladimirou, Elina; McAinsh, Andrew D; Burroughs, Nigel J

2016-06-15

During mitosis, chromosomes are attached to the mitotic spindle via large protein complexes called kinetochores. The motion of kinetochores throughout mitosis is intricate and automated quantitative tracking of their motion has already revealed many surprising facets of their behaviour. Here, we present 'KiT' (Kinetochore Tracking)-an easy-to-use, open-source software package for tracking kinetochores from live-cell fluorescent movies. KiT supports 2D, 3D and multi-colour movies, quantification of fluorescence, integrated deconvolution, parallel execution and multiple algorithms for particle localization. KiT is free, open-source software implemented in MATLAB and runs on all MATLAB supported platforms. KiT can be downloaded as a package from http://www.mechanochemistry.org/mcainsh/software.php The source repository is available at https://bitbucket.org/jarmond/kit and under continuing development. Supplementary data are available at Bioinformatics online. jonathan.armond@warwick.ac.uk. © The Author 2016. Published by Oxford University Press.

Mast cells regulate myofilament calcium sensitization and heart function after myocardial infarction

PubMed Central

Richart, Adèle; Vilar, Jose; Lemitre, Mathilde; Marck, Pauline; Branchereau, Maxime; Guerin, Coralie; Gautier, Gregory; Blank, Ulrich; Heymes, Christophe; Luche, Elodie; Cousin, Béatrice; Rodewald, Hans-Reimer

2016-01-01

Acute myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Inflammatory cells orchestrate postischemic cardiac remodeling after MI. Studies using mice with defective mast/stem cell growth factor receptor c-Kit have suggested key roles for mast cells (MCs) in postischemic cardiac remodeling. Because c-Kit mutations affect multiple cell types of both immune and nonimmune origin, we addressed the impact of MCs on cardiac function after MI, using the c-Kit–independent MC-deficient (Cpa3Cre/+) mice. In response to MI, MC progenitors originated primarily from white adipose tissue, infiltrated the heart, and differentiated into mature MCs. MC deficiency led to reduced postischemic cardiac function and depressed cardiomyocyte contractility caused by myofilament Ca2+ desensitization. This effect correlated with increased protein kinase A (PKA) activity and hyperphosphorylation of its targets, troponin I and myosin-binding protein C. MC-specific tryptase was identified to regulate PKA activity in cardiomyocytes via protease-activated receptor 2 proteolysis. This work reveals a novel function for cardiac MCs modulating cardiomyocyte contractility via alteration of PKA-regulated force–Ca2+ interactions in response to MI. Identification of this MC-cardiomyocyte cross-talk provides new insights on the cellular and molecular mechanisms regulating the cardiac contractile machinery and a novel platform for therapeutically addressable regulators. PMID:27353089

Unremitting Cell Proliferation in the Secretory Phase of Eutopic Endometriosis

PubMed Central

Franco-Murillo, Yanira; Miranda-Rodríguez, José Antonio; Rendón-Huerta, Erika; Montaño, Luis F.; Cornejo, Gerardo Velázquez; Gómez, Lucila Poblano; Valdez-Morales, Francisco Javier; Gonzalez-Sanchez, Ignacio

2014-01-01

Objective: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. Design: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3β), and phosphorylated glycogen synthase kinase 3 beta (pGSK3β), throughout the menstrual cycle. Results: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3β was identical in all samples and cycle phases, whereas pAkt and pGSK3β, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. Conclusion: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways. PMID:25194152

Remarkable effects of imatinib in a family with young onset gastrointestinal stromal tumors and cutaneous hyperpigmentation associated with a germline KIT-Trp557Arg mutation: case report and literature overview.

PubMed

Farag, S; van der Kolk, L E; van Boven, H H; van Akkooi, A C J; Beets, G L; Wilmink, J W; Steeghs, N

2018-04-01

Gastrointestinal stromal tumors (GISTs) occur mostly sporadically. GISTs associated with a familial syndrome are very rare and are mostly wild type for KIT and platelet-derived growth factor alpha (PDGFRA). To date 35 kindreds and 8 individuals have been described with GISTs associated with germline KIT mutations. This is the third family described with a germline p.Trp557Arg mutation in exon 11 of the KIT gene. The effect of imatinib in patients harboring a germline KIT mutation has been rarely described. Moreover, in some studies imatinib treatment was withheld considering the lack of evidence for efficacy of this treatment in GIST patients harboring a germline KIT mutation. This paper describes a 52-year old patient with a de novo germline p.Trp557Arg mutation with multiple GISTs throughout the gastrointestinal tract and cutaneous hyperpigmentation. Imatinib treatment showed long-term regression of the GISTs and evident pathological response was seen after resection. Remarkably, the hyperpigmentation of the skin also diminished during imatinib treatment. Genetic screening of the family revealed the same mutation in two daughters, both with similar cutaneous hyperpigmentation. One daughter, aged 23, was diagnosed with multiple small intestine GISTs, which were resected. She was treated with adjuvant imatinib which prompted rapid regression of the cutaneous hyperpigmentation. Imatinib treatment in GIST patients harboring a germline KIT mutation shows favorable and long-term responses in both the tumor and the phenotypical hyperpigmentation.

Investigating Associations Between Proliferation Indices, C-kit, and Lymph Node Stage in Canine Mast Cell Tumors.

PubMed

Krick, Erika Lauren; Kiupel, Matti; Durham, Amy C; Thaiwong, Tuddow; Brown, Dorothy C; Sorenmo, Karin U

Previous studies have evaluated cellular proliferation indices, KIT expression, and c-kit mutations to predict the clinical behavior of canine mast cell tumors (MCTs). The study purpose was to retrospectively compare mitotic index, argyrophilic nucleolar organizer regions (AgNORs)/nucleus, Ki-67 index, KIT labeling pattern, and internal tandem duplication mutations in c-KIT between stage I and stage II grade II MCTs. Medical records and tumor biopsy samples from dogs with Grade II MCTs with cytological or histopathological regional lymph node evaluation were included. Signalment, tumor location and stage, and presence of a recurrent versus de novo tumor were recorded. Mitotic index, AgNORs/nucleus, Ki-67, KIT staining pattern, and internal tandem duplication mutations in exon 11 of c-KIT were evaluated. Sixty-six tumors (51 stage I; 15 stage II) were included. Only AgNORs/nucleus and recurrent tumors were significantly associated with stage (odds ratio 2.8, 95% confidence interval [CI] 1.0-8.0, P = .049; odds ratio 8.8, 95% CI 1.1-69.5; P = .039). Receiver-operator characteristic analysis showed that the sensitivity and specificity of AgNORs/cell ≥ 1.87 were 93.3% and 27.4%, respectively, (area under the curve: 0.65) for predicting stage. Recurrent tumors and higher AgNORs/nucleus are associated with stage II grade II MCTs; however, an AgNOR cutoff value that reliably predicts lymph node metastasis was not determined.

Optimising the quantification of cytokines present at low concentrations in small human mucosal tissue samples using Luminex assays☆

PubMed Central

Staples, Emily; Ingram, Richard James Michael; Atherton, John Christopher; Robinson, Karen

2013-01-01

Sensitive measurement of multiple cytokine profiles from small mucosal tissue biopsies, for example human gastric biopsies obtained through an endoscope, is technically challenging. Multiplex methods such as Luminex assays offer an attractive solution but standard protocols are not available for tissue samples. We assessed the utility of three commercial Luminex kits (VersaMAP, Bio-Plex and MILLIPLEX) to measure interleukin-17A (IL-17) and interferon-gamma (IFNγ) concentrations in human gastric biopsies and we optimised preparation of mucosal samples for this application. First, we assessed the technical performance, limits of sensitivity and linear dynamic ranges for each kit. Next we spiked human gastric biopsies with recombinant IL-17 and IFNγ at a range of concentrations (1.5 to 1000 pg/mL) and assessed kit accuracy for spiked cytokine recovery and intra-assay precision. We also evaluated the impact of different tissue processing methods and extraction buffers on our results. Finally we assessed recovery of endogenous cytokines in unspiked samples. In terms of sensitivity, all of the kits performed well within the manufacturers' recommended standard curve ranges but the MILLIPLEX kit provided most consistent sensitivity for low cytokine concentrations. In the spiking experiments, the MILLIPLEX kit performed most consistently over the widest range of concentrations. For tissue processing, manual disruption provided significantly improved cytokine recovery over automated methods. Our selected kit and optimised protocol were further validated by measurement of relative cytokine levels in inflamed and uninflamed gastric mucosa using Luminex and real-time polymerase chain reaction. In summary, with proper optimisation Luminex kits (and for IL-17 and IFNγ the MILLIPLEX kit in particular) can be used for the sensitive detection of cytokines in mucosal biopsies. Our results should help other researchers seeking to quantify multiple low concentration cytokines in small tissue samples. PMID:23644159

Digital PCR: A Sensitive and Precise Method for KIT D816V Quantification in Mastocytosis.

PubMed

Greiner, Georg; Gurbisz, Michael; Ratzinger, Franz; Witzeneder, Nadine; Simonitsch-Klupp, Ingrid; Mitterbauer-Hohendanner, Gerlinde; Mayerhofer, Matthias; Müllauer, Leonhard; Sperr, Wolfgang R; Valent, Peter; Hoermann, Gregor

2018-03-01

The analytically sensitive detection of KIT D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KIT D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. We performed a validation study of dPCR for KIT D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR ( P < 0.001). Moreover, dPCR for KIT D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KIT D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KIT D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P < 0.001). Moreover, dPCR confirmed the prognostic significance of a high KIT D816V VAF regarding survival ( P < 0.001). dPCR for KIT D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KIT D816V allele burden measurement. Thus, dPCR is suitable as a new method for KIT D816V testing in patients with mastocytosis. © 2017 American Association for Clinical Chemistry.

Comparison of 3 kidney injury multiplex panels in rats.

PubMed

John-Baptiste, Annette; Vitsky, Allison; Sace, Frederick; Zong, Qing; Ko, Mira; Yafawi, Rolla; Liu, Ling

2012-01-01

Kidney injury biomarkers have been utilized by pharmaceutical companies as a means to assess the potential of candidate drugs to induce nephrotoxicity. Multiple platforms and assay methods exist, but the comparison of these methods has not been described. Millipore's Kidney Toxicity panel, EMD/Novagen's Widescreen Kidney Toxicity panel, and Meso Scales Kidney Injury panel were selected based on published information. Kidney injury molecule 1, cystatin C, clusterin, and osteopontin were the 4 biomarkers common among all kits tested and the focus of this study. Rats were treated with a low and high dose of para-aminophenol, a known nephrotoxicant, and urine samples were collected and analyzed on the Bio-Plex 200 or MSD's Sector Imager 6000, according to manufacturers specifications. Comparatively, of the 3 kits, Millipore was the most consistent in detecting elevations of 3 out of the 4 biomarkers at both dose levels and indicated time points.

The effect of test kit provision, and individual and family education on the uptake rates of fecal occult blood test in an Asian population: a randomized controlled trial.

PubMed

Ha, Tam Cam; Yong, Sook Kwin; Yeoh, Kheng-Wei; Kamberakis, Kay; Yeo, Richard Ming Chert; Koh, Gerald Choon-Huat

2014-11-01

The purpose of the study was to investigate whether fecal occult blood test (FOBT) home-delivery and individual education or combined with family education increases FOBT uptake rates in Singapore. This is a randomized controlled intervention study of Singaporean residents aged 50 years and above, conducted in May 2012 till May 2013. Eligible individuals in randomly selected households were screened, and one member was randomly selected and allocated to one of the four arms: Group A (individual and family education, FOBT kits provided), Group B (individual education only, FOBT kits provided), Group C (no education, FOBT kits provided) and Group D (no education or FOBT kits provided). Overall response rate was 74.7 %. The FOBT return rates for groups A, B, C and D were 24.5 % [CI 16.2-34.4 %], 25.3 % [CI 16.4-36.0 %], 10.7 % [CI 4.7-19.9 %] and 2.2 % [CI 0.3-7.7 %], respectively. Respondents who were provided education and home-delivered FOBT kits were 15 times more likely to return FOBT kits [Group A: OR 15.0 (3.4-66.2); Group B: OR 15.5 (3.5-68.8)] and those provided with home-delivered FOBT without education were five times more likely to return FOBT kits [Group C: OR 5.8 (1.2-28.3)] than those without education and FOBT kits (Group D). There was no significant difference in return of FOBT kits whether education was provided to subject with or without a family member. Home delivery of FOBT kits increased FOBT return rates and individual education combined with home-delivered FOBT increased FOBT return rates even further. However, additional combination with family education did not increase FOBT rates further.

DOE Office of Scientific and Technical Information (OSTI.GOV)

von Laszewski, G.; Gawor, J.; Lane, P.

In this paper we report on the features of the Java Commodity Grid Kit (Java CoG Kit). The Java CoG Kit provides middleware for accessing Grid functionality from the Java framework. Java CoG Kit middleware is general enough to design a variety of advanced Grid applications with quite different user requirements. Access to the Grid is established via Globus Toolkit protocols, allowing the Java CoG Kit to also communicate with the services distributed as part of the C Globus Toolkit reference implementation. Thus, the Java CoG Kit provides Grid developers with the ability to utilize the Grid, as well asmore » numerous additional libraries and frameworks developed by the Java community to enable network, Internet, enterprise and peer-to-peer computing. A variety of projects have successfully used the client libraries of the Java CoG Kit to access Grids driven by the C Globus Toolkit software. In this paper we also report on the efforts to develop serverside Java CoG Kit components. As part of this research we have implemented a prototype pure Java resource management system that enables one to run Grid jobs on platforms on which a Java virtual machine is supported, including Windows NT machines.« less

Use of the 22C3 anti-PD-L1 antibody to determine PD-L1 expression in multiple automated immunohistochemistry platforms.

PubMed

Ilie, Marius; Khambata-Ford, Shirin; Copie-Bergman, Christiane; Huang, Lingkang; Juco, Jonathan; Hofman, Veronique; Hofman, Paul

2017-01-01

For non-small cell lung cancer (NSCLC), treatment with pembrolizumab is limited to patients with tumours expressing PD-L1 assessed by immunohistochemistry (IHC) using the PD-L1 IHC 22C3 pharmDx (Dako, Inc.) companion diagnostic test, on the Dako Autostainer Link 48 (ASL48) platform. Optimised protocols are urgently needed for use of the 22C3 antibody concentrate to test PD-L1 expression on more widely available IHC autostainers. We evaluated PD-L1 expression using the 22C3 antibody concentrate in the three main commercially available autostainers Dako ASL48, BenchMark ULTRA (Ventana Medical Systems, Inc.), and Bond-III (Leica Biosystems) and compared the staining results with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Several technical conditions for laboratory-developed tests (LDTs) were evaluated in tonsil specimens and a training set of three NSCLC samples. Optimised protocols were then validated in 120 NSCLC specimens. Optimised protocols were obtained on both the VENTANA BenchMark ULTRA and Dako ASL48 platforms. Significant expression of PD-L1 was obtained on tissue controls with the Leica Bond-III autostainer when high concentrations of the 22C3 antibody were used. It therefore was not tested on the 120 NSCLC specimens. An almost 100% concordance rate for dichotomized tumour proportion score (TPS) results was observed between TPS ratings using the 22C3 antibody concentrate on the Dako ASL48 and VENTANA BenchMark ULTRA platforms relative to the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Interpathologist agreement was high on both LDTs and the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Availability of standardized protocols for determining PD-L1 expression using the 22C3 antibody concentrate on the widely available Dako ASL48 and VENTANA BenchMark ULTRA IHC platforms will expand the number of laboratories able to determine eligibility of patients with NSCLC for treatment with pembrolizumab in a reliable and concordant manner.

Multiple transport systems mediate virus-induced acquired resistance to oxidative stress

USDA-ARS?s Scientific Manuscript database

In this paper, we report the phenomenon of acquired cross-tolerance to oxidative (UV-C and H2O2) stress in Nicotiana benthamiana plants infected with Potato virus X (PVX) and investigate the functional expression of transport systems in mediating this phenomenon. By combining multiple approaches, we...

Thymus-autonomous T cell development in the absence of progenitor import.

PubMed

Martins, Vera C; Ruggiero, Eliana; Schlenner, Susan M; Madan, Vikas; Schmidt, Manfred; Fink, Pamela J; von Kalle, Christof; Rodewald, Hans-Reimer

2012-07-30

Thymus function is thought to depend on a steady supply of T cell progenitors from the bone marrow. The notion that the thymus lacks progenitors with self-renewal capacity is based on thymus transplantation experiments in which host-derived thymocytes replaced thymus-resident cells within 4 wk. Thymus grafting into T cell-deficient mice resulted in a wave of T cell export from the thymus, followed by colonization of the thymus by host-derived progenitors, and cessation of T cell development. Compound Rag2(-/-)γ(c)(-/-)Kit(W/Wv) mutants lack competitive hematopoietic stem cells (HSCs) and are devoid of T cell progenitors. In this study, using this strain as recipients for wild-type thymus grafts, we noticed thymus-autonomous T cell development lasting several months. However, we found no evidence for export of donor HSCs from thymus to bone marrow. A diverse T cell antigen receptor repertoire in progenitor-deprived thymus grafts implied that many thymocytes were capable of self-renewal. Although the process was most efficient in Rag2(-/-)γ(c)(-/-)Kit(W/Wv) hosts, γ(c)-mediated signals alone played a key role in the competition between thymus-resident and bone marrow-derived progenitors. Hence, the turnover of each generation of thymocytes is not only based on short life span but is also driven via expulsion of resident thymocytes by fresh progenitors entering the thymus.

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/Wv mutant mouse colon

PubMed Central

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice. PMID:26727725

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/W(v) mutant mouse colon.

PubMed

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/W(v) mice carrying W and W(v) mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/W(v) mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/W(v) mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/W(v) mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/W(v) mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/W(v) mutant mice.

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/Wv mutant mouse colon.

PubMed

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon.The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers,but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice.

DOE Office of Scientific and Technical Information (OSTI.GOV)

von Laszewski, G.; Foster, I.; Gawor, J.

In this paper we report on the features of the Java Commodity Grid Kit. The Java CoG Kit provides middleware for accessing Grid functionality from the Java framework. Java CoG Kit middleware is general enough to design a variety of advanced Grid applications with quite different user requirements. Access to the Grid is established via Globus protocols, allowing the Java CoG Kit to communicate also with the C Globus reference implementation. Thus, the Java CoG Kit provides Grid developers with the ability to utilize the Grid, as well as numerous additional libraries and frameworks developed by the Java community tomore » enable network, Internet, enterprise, and peer-to peer computing. A variety of projects have successfully used the client libraries of the Java CoG Kit to access Grids driven by the C Globus software. In this paper we also report on the efforts to develop server side Java CoG Kit components. As part of this research we have implemented a prototype pure Java resource management system that enables one to run Globus jobs on platforms on which a Java virtual machine is supported, including Windows NT machines.« less

The art of the chart note in clinical ethics consultation and bioethics mediation: conveying information that can be understood and evaluated.

PubMed

Dubler, Nancy Neveloff

2013-01-01

Unlike bioethics mediators who are employed by healthcare organizations as outside consultants, mediators who are embedded in an institution must be authorized to chronicle a clinical ethics consultation (CEC) or a mediation in a patient's medical chart. This is an important privilege, as the chart is a legal document. In this article I discuss this important part of a bioethics mediator's tool kit in my presentation of a case illustrating how bioethics mediation may proceed, and what this approach using both bioethics and mediation may add.

Coxiellosis in domestic livestock of Puducherry and Tamil Nadu: Detection of Coxiella burnetii DNA by polymerase chain reaction in slaughtered ruminants

PubMed Central

Pradeep, Jothimani; Stephen, Selvaraj; Pooja, Pratheesh; Akshayavardhini, Anbalagan; Sangeetha, Balakrishnan; Antony, Prabakar Xavier

2017-01-01

Background and Aim:: In the course of our Indian Council of Medical Research project on coxiellosis in Puducherry and Tamil Nadu, 5.64% goat, 1.85% sheep, 1.06% buffaloes, and 0.97% cattle were positive for Coxiella burnetii antibodies by enzyme linked immunosorbent assay kit (IDEXX, Liebefeld, Switzerland). In this preliminary study, we have proceeded to look for C. burnetii DNA in those antibody positive specimens employing an imported commercial C. burnetii polymerase chain reaction (PCR) kit. Materials and Methods:: Blood samples were collected during slaughtering. All 15 blood samples of antibody positive ruminants and three antibody negative samples were subjected to conventional Trans-PCR assay with a commercial PCR kit (Genekam Biotechnology AG, Duisburg, Germany). An in-house Trans-PCR was included in the study for comparison. Results:: A total of 15 antibody positive and three antibody-negative serum samples belonging to 11 goat, 4 sheep, 1 cattle, and 2 buffaloes were tested in duplicate for the presence of C. burnetii DNA by the commercial agar gel PCR kit and an in-house Trans-PCR. Only one buffalo serum sample was positive for C. burnetii with a band at 243 bp in in-house Trans-PCR. Discussion:: Seropositivity for C. burnetii need not necessarily translate into infectivity status of the animal. Conversely, seronegative ruminants can shed C. burnetii. Rapid disintegration of C. burnetii DNA during the storage period is an important impediment in QF-PCR research. This is the first time the performance of this commercial PCR kit is being validated in India. Conclusion:: Commercial PCR kit, Genekam did not identify any positive sample, probably because it targeted a larger amplicon of 687 bp. PMID:28717320

Hematopoietic stem cells found in lineage-positive subsets in the bone marrow of 5-fluorouracil-treated mice.

PubMed

Nishi, N; Osawa, M; Ishikawa, R; Nishikawa, M; Tsumura, H; Inoue, H; Sudo, T

1995-09-01

It is known that treatment of mice with 5-fluorouracil (5-FU, 150 mg/kg) confers radioprotection. To investigate this effect, we performed bone marrow transplantation (BMT) using C57BL/6-Ly5 congenic mice treated with 5-FU five days prior to experiments. The mononuclear cells (MNC) in 5-FU-treated bone marrow (BM) were 10 times more radioprotective than those in untreated BM. Moreover, the number of BM MNC expressing c-kit on their surface from 5-FU-treated mice was markedly decreased relative to those from untreated controls. These results showed that the surface characteristics of cells that contributed to this radio-protective effect differ from those of stem cells as reported recently. BM MNC of mice treated with 5-FU were separated on the basis of expression of the lineage-specific antigens (Lin), c-kit, and Ly6A/E. When injected into lethally irradiated mice, 1,000 Lin+ and Lin-c-kit+Ly6A/E+ cells showed radioprotective effects such that 100% and 60% survived, respectively. Flow cytometric analysis 165 days after BMT showed that 88.8% and 65.1% of peripheral blood (PB) in mice transplanted with Lin+ and Lin-c-kit+Ly6A/E+ was derived from donor mice, respectively. After six months, donor-derived Lin-c-kit+Ly6A/E+ cells which showed radioprotective effects on a secondary irradiated host were detected from mice transplanted with Lin+ cells from 5-FU-treated mice. Taken together, these findings demonstrated that stem cells expressing Lin+ present in the BM of mice treated with 5-FU other than Lin-c-kit+Ly6A/E+ cells and these Lin+ cells play an important role in the recovery of myeloablative mice.

An Oral Formulation of YK-4-279: Preclinical Efficacy and Acquired Resistance Patterns in Ewing Sarcoma.

PubMed

Lamhamedi-Cherradi, Salah-Eddine; Menegaz, Brian A; Ramamoorthy, Vandhana; Aiyer, Ramani A; Maywald, Rebecca L; Buford, Adrianna S; Doolittle, Dannette K; Culotta, Kirk S; O'Dorisio, James E; Ludwig, Joseph A

2015-07-01

Ewing sarcoma is a transcription factor-mediated pediatric bone tumor caused by a chromosomal translocation of the EWSR1 gene and one of several genes in the ETS family of transcription factors, typically FLI1 or ERG. Full activity of the resulting oncogenic fusion protein occurs only after binding RNA helicase A (RHA), and novel biologically targeted small molecules designed to interfere with that interaction have shown early promise in the preclinical setting. Herein, we demonstrate marked preclinical antineoplastic activity of an orally bioavailable formulation of YK-4-279 and identify mechanisms of acquired chemotherapy resistance that may be exploited to induce collateral sensitivity. Daily enteral administration of YK-4-279 led to significant delay in Ewing sarcoma tumor growth within a murine model. In advance of anticipated early-phase human clinical trials, we investigated both de novo and acquired mechanism(s) by which Ewing sarcoma cells evade YK-4-279-mediated cell death. Drug-resistant clones, formed by chronic in vitro exposure to steadily increased levels of YK-4-279, overexpressed c-Kit, cyclin D1, pStat3(Y705), and PKC isoforms. Interestingly, cross-resistance to imatinib and enzastaurin (selective inhibitors of c-Kit and PKC-β, respectively), was observed and the use of YK-4-279 with enzastaurin in vitro led to marked drug synergy, suggesting a potential role for combination therapies in the future. By advancing an oral formulation of YK-4-279 and identifying prominent mechanisms of resistance, this preclinical research takes us one step closer to a shared goal of curing adolescents and young adults afflicted by Ewing sarcoma. ©2015 American Association for Cancer Research.

Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes

PubMed Central

Jin, David K; Shido, Koji; Kopp, Hans-Georg; Petit, Isabelle; Shmelkov, Sergey V; Young, Lauren M; Hooper, Andrea T; Amano, Hideki; Avecilla, Scott T; Heissig, Beate; Hattori, Koichi; Zhang, Fan; Hicklin, Daniel J; Wu, Yan; Zhu, Zhenping; Dunn, Ashley; Salari, Hassan; Werb, Zena; Hackett, Neil R; Crystal, Ronald G; Lyden, David; Rafii, Shahin

2009-01-01

The mechanisms through which hematopoietic cytokines accelerate revascularization are unknown. Here, we show that the magnitude of cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4+VEGFR1+ hematopoietic progenitors, ‘hemangiocytes,’ constitute the major determinant of revascularization. Soluble Kit-ligand (sKitL), thrombopoietin (TPO, encoded by Thpo) and, to a lesser extent, erythropoietin (EPO) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induced the release of SDF-1 from platelets, enhancing neovascularization through mobilization of CXCR4+VEGFR1+ hemangiocytes. Although revascularization of ischemic hindlimbs was partially diminished in mice deficient in both GM-CSF and G-CSF (Csf2−/−Csf3−/−), profound impairment in neovascularization was detected in sKitL-deficient Mmp9−/− as well as thrombocytopenic Thpo−/− and TPO receptor–deficient (Mpl−/−) mice. SDF-1–mediated mobilization and incorporation of hemangiocytes into ischemic limbs were impaired in Thpo−/−, Mpl−/− and Mmp9−/− mice. Transplantation of CXCR4+VEGFR1+ hemangiocytes into Mmp9−/− mice restored revascularization, whereas inhibition of CXCR4 abrogated cytokine- and VEGF-A–mediated mobilization of CXCR4+VEGFR1+ cells and suppressed angiogenesis. In conclusion, hematopoietic cytokines, through graded deployment of SDF-1 from platelets, support mobilization and recruitment of CXCR4+VEGFR1+ hemangiocytes, whereas VEGFR1 is essential for their angiogenic competency for augmenting revascularization. Delivery of SDF-1 may be effective in restoring angiogenesis in individuals with vasculopathies. PMID:16648859

Causal Mediation Analysis of Survival Outcome with Multiple Mediators.

PubMed

Huang, Yen-Tsung; Yang, Hwai-I

2017-05-01

Mediation analyses have been a popular approach to investigate the effect of an exposure on an outcome through a mediator. Mediation models with multiple mediators have been proposed for continuous and dichotomous outcomes. However, development of multimediator models for survival outcomes is still limited. We present methods for multimediator analyses using three survival models: Aalen additive hazard models, Cox proportional hazard models, and semiparametric probit models. Effects through mediators can be characterized by path-specific effects, for which definitions and identifiability assumptions are provided. We derive closed-form expressions for path-specific effects for the three models, which are intuitively interpreted using a causal diagram. Mediation analyses using Cox models under the rare-outcome assumption and Aalen additive hazard models consider effects on log hazard ratio and hazard difference, respectively; analyses using semiparametric probit models consider effects on difference in transformed survival time and survival probability. The three models were applied to a hepatitis study where we investigated effects of hepatitis C on liver cancer incidence mediated through baseline and/or follow-up hepatitis B viral load. The three methods show consistent results on respective effect scales, which suggest an adverse estimated effect of hepatitis C on liver cancer not mediated through hepatitis B, and a protective estimated effect mediated through the baseline (and possibly follow-up) of hepatitis B viral load. Causal mediation analyses of survival outcome with multiple mediators are developed for additive hazard and proportional hazard and probit models with utility demonstrated in a hepatitis study.

C-Kit expression in the gallbladder of guinea pig with chronic calculous cholecystitis and the effect of Artemisia capillaris Thunb on interstitial cells of Cajal.

PubMed

Feng, Hua; Wang, Fang; Wang, Changmiao

2016-07-01

To study the c-Kit expression in the gallbladder of cholesterol lithogenic guinea pig model and the effect of Artemisia capillaris Thunb on interstitial cells of Cajal (ICCs). A total of 45 guinea pigs were randomly assigned into three groups: the control group (guinea pigs fed a standard diet, normal group); the model group (guinea pigs fed a cholesterol gallstone-inducing diet); and the Chinese medicine group (guinea pigs fed the cholesterol gallstone-inducing diet and treated with A. capillaris through intragastric administration, therapy group). Each group had 15 guinea pigs. The gallbladders of the guinea pigs were harvested after 8 weeks. C-Kit expression was detected using an immunohistochemistry staining, real-time PCR, and Western blot analyses. The effect of A. capillaris on ICCs was evaluated by muscle strip contraction experiments. C-Kit expression significantly decreased in the gallbladder of model group, but increased in the Chinese medicine group. The Contractility of guinea pig gallbladder muscle strip significantly improved in the Chinese medicine group. Our results indicated that A. capillaris improves gallbladder impairment by up-regulating c-Kit expression, and it also can improve the contractile response of in vitro guinea pig gallbladder muscle strips.

Promotion of ulcerative duodenitis in young ferrets by oral immunization with Helicobacter mustelae and muramyl dipeptide.

PubMed

Whary, M T; Palley, L S; Batchelder, M; Murphy, J C; Yan, L; Taylor, N S; Fox, J G

1997-06-01

The purpose of this study was to determine whether oral immunization of ferret kits with a whole-cell sonicate of Helicobacter mustelae lysate (Hml) and the adjuvant muramyl dipeptide (MDP) would reduce the incidence of natural colonization with H. mustelae and the extent of Helicobacter-associated gastritis by enhancing the host mucosal immune response. Between the ages of 4 and 11 weeks, 44 ferret kits were gavaged with Hml and various doses of MDP. The extent of gastritis and duodenitis and the immune response to H. mustelae were evaluated. All kits became colonized naturally with H. mustelae and the majority developed mild to severe gastritis and duodenitis. Kits that received Hml with MDP developed significantly greater inflammation of the gastric antrum and duodenum, as compared to kits vaccinated with Hml alone. Vaccination with Hml and 50 micrograms of MDP was associated with severe lesions in the proximal duodenum characterized by accumulation of mononuclear inflammatory cells, mucosal erosion, and ulceration. Although serum antibody specific for H. mustelae in 4-week-old kits was approximately 50% of adult levels, a finding attributable to passively acquired maternal antibody, both systemic and mucosal antibody levels became depressed over time despite oral vaccination. The humoral immune response was sufficiently low to prevent detection of any significant dose effect of MDP on antibody levels among experimental groups. Oral vaccination of young ferrets with Hml and 50 micrograms MDP increased the risk of Helicobacter-associated mucosal ulceration in the proximal duodenum, which was associated with low humoral (but significant cell-mediated) immune responses to H. mustelae. In retrospect, the frequency of vaccination may have suppressed the systemic humoral immune response, thereby promoting mucosal damage by H. mustelae. The 50-microgram dose of MDP enhanced the cell-mediated immune response, which indirectly contributed to development of severe lesions. The increased frequency of mucosal damage associated with this vaccination regimen enhances the value of the ferret model for studying duodenal ulceration secondary to Helicobacter infection.

Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

PubMed Central

Hayashi, Yujiro; Asuzu, David T.; Gibbons, Simon J.; Aarsvold, Kirsten H.; Bardsley, Michael R.; Lomberk, Gwen A.; Mathison, Angela J.; Kendrick, Michael L.; Shen, K. Robert; Taguchi, Takahiro; Gupta, Anu; Rubin, Brian P.; Fletcher, Jonathan A.; Farrugia, Gianrico; Urrutia, Raul A.; Ordog, Tamas

2013-01-01

Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes. PMID:24116170

Epidermal Growth Factor Receptor, C-kit, and Her2/neu Immunostaining in Advanced or Recurrent Thymic Epithelial Neoplasms Staged According to the 2004 World Health Organization in Patients Treated with Octreotide and Prednisone

PubMed Central

Aisner, Seena C.; Dahlberg, Suzanne; Hameed, Meera R.; Ettinger, David S.; Schiller, Joan H.; Johnson, David H.; Aisner, Joseph; Loehrer, Patrick J.

2011-01-01

Background Advanced or recurrent nonresectable thymic epithelial tumors show only a modest response to standard chemotherapy. A recent study using octreotide and prednisone in thymic tumors, Eastern Cooperative Oncology Group study E1C97, was conducted to verify the activity of octreotide for thymic tumors. The aim of this study was to determine whether epidermal growth factor receptor (EGFR) immunoreactivity correlated with outcomes and to identify new biologic markers for potential targeted therapy. Three markers, EGFR, C-kit, and Her2/neu, were selected for evaluation in patients with advanced thymic epithelial tumors treated on E1C97. Methods Of the 42 patients entered onto E1C97, 34 patients (World Health Organization [WHO] categories: type A = 1, type AB = 1, type B1 = 10, type B2 = 11 type B3 = 8, and type C = 3) had sufficient tissue available for immunohistologic study. Each tumor was assessed to have 0, 1+, 2+, or 3+ immunore-activity in the cytoplasm or membranes of the neoplastic cells for Her2/neu and EGFR and for the presence or absence of C-kit immunoreactivity. Results EGFR immunoreactivity of 2+ or 3+ was associated with more aggressive thymic tumors (WHO types B2 and B3). However, strong EGFR immunoreactivity was not consistently seen with thymic carcinoma. The presence of EGFR within cells was associated with a significantly improved progression-free survival (PFS) and a trend for overall survival (OS). Twelve patients demonstrated C-kit immunoreactivity; the lack of C-kit immunoreactivity was significantly associated with superior PFS but not OS. Her2/neu immunoreactivity was uniformly negative for all tumors evaluated. There was no association between response and biomarker status. Conclusions High EGFR immunoreactivity is seen in more aggressive thymic neoplasms as classified according to the 2004 WHO, but regardless of classification, the presence of EGFR in tumor cells (1+, 2+, and 3+) is associated with improved performance free survival (PFS) and a trend for better OS. In contrast, the absence of C-kit immunoreactivity was associated with improved PFS. These data suggest that EGFR and C-kit may be prognostic, and further studies of these markers in subcategories of thymic malignancies is warranted. PMID:20421818

A model "go-kit" for use at Strategic National Stockpile Points of Dispensing.

PubMed

May, Larissa; Cote, Timothy; Hardeman, Bernard; Gonzalez, Gabriela R; Adams, Sherry B; Blair, Roderick K; Pane, Gregg

2007-01-01

The Strategic National Stockpile (SNS) is a national repository of pharmaceuticals and other medical supplies forseeably needed during a medical disaster. In the event of SNS deployment, state and local public health authorities must be prepared to receive, distribute, and dispense the materials. We propose a cache of supplies, termed the "POD go-kit," prepared in advance and locally available prior to the establishment of Points of Dispensing (POD) for SNS material. Characteristics of the preassembled go-kit are its multiplicity of use, ease of storage and transportation, minimal redundancy with SNS material, and packaging in a manner consistent with POD function. The POD go-kit is assembled into 4 separate "subkits": administrative supplies, patient routing supplies, dispensing supplies, and POD staff protection supplies. Incorporating existing practices from the SNS Listserv, this article itemizes the contents of the POD go-kit and its subkits and provides a rationale for its packaging. The Division of Strategic National Stockpile (DSNS) has not certified the proposed "POD go-kit" as a standardized POD go-kit.

STAT3 Mediates Nilotinib Response in KIT-Altered Melanoma: A Phase II Multicenter Trial of the French Skin Cancer Network.

PubMed

Delyon, Julie; Chevret, Sylvie; Jouary, Thomas; Dalac, Sophie; Dalle, Stephane; Guillot, Bernard; Arnault, Jean-Philippe; Avril, Marie-Françoise; Bedane, Christophe; Bens, Guido; Pham-Ledard, Anne; Mansard, Sandrine; Grange, Florent; Machet, Laurent; Meyer, Nicolas; Legoupil, Delphine; Saiag, Philippe; Idir, Zakia; Renault, Victor; Deleuze, Jean-François; Hindie, Elif; Battistella, Maxime; Dumaz, Nicolas; Mourah, Samia; Lebbe, Celeste

2018-01-01

Mutated oncogenic KIT is a therapeutic target in melanoma. We conducted a multicenter phase II trial on the KIT inhibitor nilotinib in patients with unresectable melanoma harboring KIT alteration. The primary endpoint was the response rate (complete response or partial response following Response Evaluation Criteria in Solid Tumors criteria) at 6 months. Pharmacodynamic studies using KIT sequencing, qPCR array, and immunostaining of downstream KIT effectors were performed during treatment. Twenty-five patients were included and received 400 mg oral nilotinib twice daily. At 6 months, nilotinib induced tumor response in four patients. The best overall response rate was 20% and the disease control rate was 56%, limited to patients harboring exon 11 or 13 mutations. Four patients exhibited durable response, including three persisting (3.6 and 2.8 years for two patients with stage IIIC and 2.5 years for one with IVM1b melanoma). A reduction in signal transducer and activator of transcription (STAT) 3 phosphorylation and its effectors (BCL-2, MCL-1) in tumors during follow-up was significantly associated with clinical response. In the KIT-mutated melanoma cell line M230, nilotinib reduced STAT3 signaling and STAT inhibitors were as efficient as KIT inhibitors in reducing cell proliferation. Our study evidences a significant association between STAT3 inhibition and response to nilotinib, and provides a rationale for future research assessing STAT inhibitors in KIT-mutated melanoma. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Combined Targeting of BCL-2 and BCR-ABL Tyrosine Kinase Eradicates Chronic Myeloid Leukemia Stem Cells

PubMed Central

Mak, Po Yee; Mu, Hong; Zhou, Hongsheng; Mak, Duncan H.; Schober, Wendy; Leverson, Joel D.; Zhang, Bin; Bhatia, Ravi; Huang, Xuelin; Cortes, Jorge; Kantarjian, Hagop; Konopleva, Marina

2016-01-01

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein level in bone marrow cells, particularly in Lin−Sca-1+cKit+ cells of inducible CML in mice as determined by CyTOF mass cytometry. Further, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly decreased leukemic Lin−Sca-1+cKit+ cell numbers and long-term stem cell frequency, and prolonged survival in a murine CML model. Additionally, this combination effectively eradicated CD34+CD38−, CD34+CD38+, and quiescent stem/progenitor CD34+ cells from BC CML patient samples. Our results suggest that BCL-2 is a key survival factor for CML stem/progenitor cells and that combined inhibition of BCL-2 and BCR-ABL tyrosine kinase has the potential to significantly improve depth of response and cure rates of chronic phase and BC CML. PMID:27605552

In vivo competitive studies between normal and common gamma chain-defective bone marrow cells: implications for gene therapy.

PubMed

Otsu, M; Sugamura, K; Candotti, F

2000-09-20

Corrective gene transfer into hematopoietic stem cells (HSCs) is being investigated as therapy for X-linked severe combined immunodeficiency (XSCID) and it is hoped that selective advantage of gene-corrected HSCs will help in achieving full immune reconstitution after treatment. Lines of evidence from the results of allogeneic bone marrow transplantation in patients with XSCID support this hypothesis that, however, has not been rigorously tested in an experimental system. We studied the competition kinetics between normal and XSCID bone marrow (BM) cells using a murine bone marrow transplantation (BMT) model. For easy chimerism determination, we used genetic marking with retrovirus-mediated expression of the enhanced green fluorescent protein (EGFP). We found that XSCID BM cells were able to compete with normal BM cells for engraftment of myeloid lineages in a dose-dependent manner, whereas we observed selective repopulation of T, B, and NK cells deriving from normal BM cells. This was true despite the evidence of competitive engraftment of XSCID lineage marker-negative/c-Kit-positive (Lin-/c-Kit+) cells in the bone marrow of treated animals. From these results we extrapolate that genetic correction of XSCID HSCs will result in selective advantage of gene-corrected lymphoid lineages with consequent restoration of lymphocyte populations and high probability of clinical benefit.

Multiple gastrointestinal stromal tumors in type I neurofibromatosis: a pathologic and molecular study.

PubMed

Yantiss, Rhonda K; Rosenberg, Andrew E; Sarran, Lisa; Besmer, Peter; Antonescu, Cristina R

2005-04-01

Multiple gastrointestinal stromal tumors typically occur in familial form associated with KIT receptor tyrosine kinase or platelet-derived growth factor receptor-alpha (PDGFRA) germline mutations, but may also develop in the setting of type 1 neurofibromatosis. The molecular abnormalities of gastrointestinal stromal tumors arising in neurofibromatosis have not been extensively studied. We identified three patients with type 1 neuro-fibromatosis and multiple small intestinal stromal tumors. Immunostains for CD117, CD34, desmin, actins, S-100 protein, and keratins were performed on all of the tumors. DNA was extracted from representative paraffin blocks from separate tumor nodules in each case and subjected to a nested polymerase chain reaction, using primers for KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18, followed by direct sequencing. The mean patient age was 56 years (range: 37-86 years, male/female ratio: 2/1). One patient had three tumors, one had five, and one had greater than 10 tumor nodules, all of which demonstrated histologic features characteristic of gastrointestinal stromal tumors and stained strongly for CD117 and CD34. One patient died of disease at 35 months, one was disease free at 12 months and one was lost to follow-up. DNA extracts from 10 gastrointestinal stromal tumors (three from each of two patients and four from one patient) were subjected to polymerase chain reactions and assessed for mutations. All of the tumors were wild type for KIT exons 9, 13, and 17 and PDGFRA exons 12 and 18. Three tumors from one patient had identical point mutations in KIT exon 11, whereas the other tumors were wild type at this locus. We conclude that, although most patients with type 1 neurofibromatosis and gastrointestinal stromal tumors do not have KIT or PDGFRA mutations, KIT germline mutations might be implicated in the pathogenesis of gastrointestinal stromal tumors in some patients.

Repeated doses of cardiac mesenchymal cells are therapeutically superior to a single dose in mice with old myocardial infarction.

PubMed

Guo, Yiru; Wysoczynski, Marcin; Nong, Yibing; Tomlin, Alex; Zhu, Xiaoping; Gumpert, Anna M; Nasr, Marjan; Muthusamy, Senthikumar; Li, Hong; Book, Michael; Khan, Abdur; Hong, Kyung U; Li, Qianhong; Bolli, Roberto

2017-03-01

We have recently demonstrated that repeated administrations of c-kit POS cardiac progenitor cells (CPCs) have cumulative beneficial effects in rats with old myocardial infarction (MI), resulting in markedly greater improvement in left ventricular (LV) function compared with a single administration. To determine whether this paradigm applies to other species and cell types, mice with a 3-week-old MI received one or three doses of cardiac mesenchymal cells (CMCs), a novel cell type that we have recently described. CMCs or vehicle were infused percutaneously into the LV cavity, 14 days apart. Compared with vehicle-treated mice, the single-dose group exhibited improved LV ejection fraction (EF) after the 1st infusion (consisting of CMCs) but not after the 2nd and 3rd (vehicle). In contrast, in the multiple-dose group, LV EF improved after each CMC infusion, so that at the end of the study, LV EF averaged 35.5 ± 0.7% vs. 32.7 ± 0.6% in the single-dose group (P < 0.05). The multiple-dose group also exhibited less collagen in the non-infarcted region vs. the single-dose group. Engraftment and differentiation of CMCs were negligible in both groups, indicating paracrine effects. These results demonstrate that, in mice with ischemic cardiomyopathy, the beneficial effects of three doses of CMCs are significantly greater than those of one dose, supporting the concept that multiple treatments are necessary to properly evaluate the full therapeutic potential of cell therapy. Thus, the repeated-treatment paradigm is not limited to c-kit POS CPCs or to rats, but applies to other cell types and species. The generalizability of this concept dramatically augments its significance.

Modifying surface properties of KIT-6 zeolite with Ni and V for enhancing catalytic CO methanation

NASA Astrophysics Data System (ADS)

Cao, Hong-Xia; Zhang, Jun; Guo, Cheng-Long; Chen, Jingguang G.; Ren, Xiang-Kun

2017-12-01

The surface of the KIT-6 zeolite was modified with different amounts of Ni and V to promote the catalytic properties for CO methanation. A series of xNi-yV/KIT-6 with various Ni and V contents were prepared by the incipient-wetness impregnation method. The modified surfaces were characterized using N2 adsorption-desorption, Brunauer-Emmett-Teller (BET), X-ray diffraction (XRD), hydrogen temperature-programmed reduction (H2-TPR), Fourier transformed infrared spectroscopy (FT-IR), Raman, X-ray photoelectron spectroscopy (XPS), transmission electron microscope (TEM), and energy-dispersive X-ray spectroscopy (EDX), respectively. The characterization results illustrated that the modification of V species was able to significantly promote low-temperature catalytic performance below 350 °C compared to that of unmodified Ni/KIT-6, which was likely due to an increase in the H2 uptake accompanied by enhanced CO dissociation derived from stronger electron transfer from V species to Ni0. Correspondingly, the xNi-yV/KIT-6 catalysts exhibited a distinct enhancement in CO conversion, CH4 selectivity and CH4 yield over unmodified Ni/KIT-6. Among all catalysts, 20Ni-2V/KIT-6 showed the best catalytic performance, corresponding to nearly 100% CO conversion and 85% CH4 yield at a low temperature of 300 °C. Furthermore, 20Ni-2V/KIT-6 presented enhanced coking-resistant and anti-sintering properties during a 60h-lifetime test at 500 °C and 1 atm with a high weight hourly space velocity (WHSV) of 60000 ml/g/h.

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

PubMed Central

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-01-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure. PMID:26633465

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

PubMed

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-12-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

Evaluation of forensic genetic parameters of 12 STR loci in the Korean population using the InvestigatorⓇ HDplex kit.

PubMed

Jung, Ju Yeon; Kim, Eun Hye; Oh, Yu-Li; Park, Hyun-Chul; Hwang, Jung Ho; Lim, Si-Keun

2017-09-01

We genotyped and calculated the forensic parameters of 10 non-CODIS loci and 2 CODIS loci of 990 Korean individuals using the Investigator Ⓡ HDplex kit. No significant deviations from Hardy-Weinberg equilibrium (after Bonferroni correction for multiple testing) or genetic linkage disequilibrium were observed. The calculated matching probability and power of discrimination ranged from 0.0080 to 0.2014, and 0.7986 to 0.9920, respectively. We conclude that the markers of the kit are highly informative corroborative tools for forensic DNA analysis.

Hemostatic Function of Apheresis Platelets Stored at 4 deg C and 22 deg C

DTIC Science & Technology

2014-05-01

utilized. Thromboxane B2 (TxB2) enzyme immunoassay kits were purchased from Cayman Chemicals (Ann Arbor, MI), and human soluble CD40L (sCD40L) extra...sensitive platinum enzyme linked immunosorbent assay kits were pur chased from eBioscience (Vienna, Austria). CG4+ and CHEM8+ cartridges were purchased from...TruCount tubes (BD Biosciences). Enzyme linked immunosorbent assay Commercially available kits were used to assess sCD40L and TxB2 levels released into

Use of anoctamin 1 (ANO1) to evaluate interstitial cells of Cajal in Hirschsprung's disease.

PubMed

Coyle, David; Kelly, Danielle A M; O'Donnell, Anne Marie; Gillick, John; Puri, Prem

2016-02-01

Interstitial cells of Cajal (ICCs) are pacemaker cells involved in facilitating neurotransmission and the generation of slow electrical waves necessary for colonic peristalsis. Their distribution has been found to be abnormal in the aganglionic and ganglionic colon in Hirschsprung's disease (HSCR) using c-kit-labelling. Anoctamin-1 (ANO1) is a Ca(2+)-activated Cl(-) channel thought to be specifically expressed on ICCs. Unlike c-kit, it plays a key role in ICC pacemaker activity. We aimed to investigate the utility of ANO1 in evaluating the colonic ICC network in HSCR. We collected full-length pull-through specimens from children with HSCR (n = 10). Control colon specimens were collected at colostomy closure in children with anorectal malformation (n = 6). The distribution of ANO1 and c-kit expression was evaluated using immunofluorescence and confocal microscopy. ANO1 expression was quantified using Western blot analysis. ANO1 was not expressed on 23 % of c-kit immuno-positive cells in the circular muscle; however, 100 % of ANO1-positive ICCs were c-kit positive. The distribution of ANO1-positive ICCs was sparse in aganglionic colon, with a modest reduction in ICCs seen in the ganglionic colon in HSCR compared to controls (p = 0.044). ANO1 protein expression was reduced in aganglionic colon but similar in ganglionic colon relative to controls. ANO1 is preferential to c-kit in evaluating the ICC network in HSCR due to its specificity and functional importance. Abnormal distribution of ANO1-positive ICCs in the ganglionic colon in HSCR may contribute to persistent bowel symptoms in some patients after pull-through surgery.

Intraovarian transplantation of primordial follicles fails to rescue chemotherapy injured ovaries

PubMed Central

Park, Mi-Ryung; Choi, Yun-Jung; Kwon, Deug-Nam; Park, Chankyu; Bui, Hong-Thuy; Gurunathan, Sangiliyandi; Cho, Ssang-Goo; Song, Hyuk; Seo, Han Geuk; Min, Gyesik; Kim, Jin-Hoi

2013-01-01

Busulfan and cyclophosphamide (B/C)-treated mice exhibited a marked increase in apoptosis and a concomitant decrease in the ovarian weight. Histological and RT-PCR analysis indicate that the period of germ cell depletion in the B/C-treated ovaries coincides with decreased expression of genes Figla, Lhx8, Nobox, c-kit, and Sox3. However, depletion of the ovarian germ cells is mediated by autophagy-independent pathways that involve Fas/FasL-, TNF-, and/or p53-signalings. Treatment with B/C resulted in the cease of the reproductive function to produce their offspring during the 15th week post-treatment period in female mice. Furthermore, injection of the 3 × 106 GFP positive primordial follicles into the ovaries of the B/C treated mouse did not show apparent colonization of the transplanted follicles within the recipient ovaries. The present results suggest that B/C treatment is closely associated with an increased risk of premature ovarian failure. PMID:23463338

Inhibition of Oct 3/4 mitigates the cardiac progenitor-derived myocardial repair in infarcted myocardium.

PubMed

Zhao, Yu Tina; Du, Jianfeng; Chen, Youfang; Tang, Yaoliang; Qin, Gangjian; Lv, Guorong; Zhuang, Shougang; Zhao, Ting C

2015-12-24

Recent evidence has demonstrated that cardiac progenitor cells play an essential role in the induction of angiomyogenesis in infarcted myocardium. We and others have shown that engraftment of c-kit(+) cardiac stem cells (CSCs) into infarcted hearts led to myocardium regeneration and neovascularization, which was associated with an improvement of ventricular function. The purpose of this study is aimed at investigating the functional role of transcription factor (TF) Oct3/4 in facilitating CSCs to promote myocardium regeneration and preserve cardiac performance in the post-MI heart. c-kit(+) CSCs were isolated from adult hearts and re-introduced into the infarcted myocardium in which the mouse MI model was created by permanent ligation of the left anterior descending artery (LAD). The Oct3/4 of CSCs was inhibited by transfection of Oct3/4 siRNA, and transfection of CSCs with control siRNA serves as control groups. Myocardial functions were evaluated by echocardiographic measurement. Histological analysis was employed to assess newly formed cardiogenesis, neovascularization, and cell proliferations. Terminal deoxynucleotidyltransferase (TdT) nick-end labeling (TUNEL) was carried out to assess apoptotic cardiomyocytes. Real time polymerase chain reaction and Western blot were carried out to evaluate the level of Oct 3/4 in CSCs. Two weeks after engraftment, CSCs increased ventricular functional recovery as shown by a serial echocardiographic measurement, which is concomitant with the suppression of cardiac hypertrophy and attenuation of myocardial interstitial fibrosis. Suppression of Oct 3/4 of CSCs abrogated functional improvements and mitigated the hypertrophic response and cardiac remodeling. Transplantation of c-kit(+) CSCs into MI hearts promoted cardiac regeneration and neovascularization, which were abolished with the knockdown of Oct3/4. Additionally, suppression of Oct3/4 abrogated myocyte proliferation in the CSC-engrafted myocardium. Our results indicate that CSCs-derived cardiac regeneration improves the restoration of cardiac function and is mediated through Oct 3/4.

c-KIT receptor expression is strictly associated with the biological behaviour of thyroid nodules

PubMed Central

2012-01-01

Background A large amount of information has been collected on the molecular tumorigenesis of thyroid cancer. A low expression of c-KIT gene has been reported during the transformation of normal thyroid epithelium to papillary carcinoma suggesting a possible role of the gene in the differentiation of thyroid tissue rather than in the proliferation. The initial presentation of thyroid carcinoma is through a nodule and the best way nowadays to evaluate it is by fine-needle aspiration (FNA). However many thyroid FNAs are not definitively benign or malignant, yielding an indeterminate or suspicious diagnosis which ranges from 10 to 25% of FNAs. BRAF mutational analysis is commonly used to assess the malignancy of thyroid nodules but unfortunately it still leaves indeterminate diagnoses. The development of molecular initial diagnostic tests for evaluating a thyroid nodule is needed in order to define optimal surgical approach for patients with uncertain diagnosis pre- and intra-operatively. Methods In this study we extracted RNA from 82 FNA smears, 46 malignant and 36 benign at the histology, in order to evaluate by quantitative Real Time PCR the expression levels of c-KIT gene. Results We have found a highly preferential decrease rather than increase in transcript of c-KIT in malignant thyroid lesions compared to the benign ones. To explore the diagnostic utility of c-KIT expression in thyroid nodules, its expression values were divided in four arbitrarily defined classes, with class I characterized by the complete silencing of the gene. Class I and IV represented the two most informative groups, with 100% of the samples found malignant or benign respectively. The molecular analysis was proven by ROC (receiver operating characteristic) analysis to be highly specific and sensitive improving the cytological diagnostic accuracy of 15%. Conclusion We propose the use of BRAF test (after uncertain cytological diagnosis) to assess the malignancy of thyroid nodules at first, then the use of the c-KIT expression to ultimately assess the diagnosis of the nodules that otherwise would remain suspicious. The c-KIT expression-based classification is highly accurate and may provide a tool to overcome the difficulties in today's preoperative diagnosis of thyroid suspicious malignancies. PMID:22233760

Familial gastrointestinal stromal tumors, lentigines, and café-au-lait macules associated with germline c-kit mutation treated with imatinib.

PubMed

Gupta, Divya; Chandrashekar, Laxmisha; Larizza, Lidia; Colombo, Elisa A; Fontana, Laura; Gervasini, Cristina; Thappa, Devinder M; Rajappa, Medha; Rajendiran, Kalai Selvi; Sreenath, Gubbi Shamanna; Kate, Vikram

2017-02-01

Familial lentiginosis syndromes are characterized by a wide array of manifestations resulting from activation of molecular pathways which control growth, proliferation, and differentiation of a broad range of tissues. Familial gastrointestinal stromal tumors (GISTs) are often accompanied by additional features like hyperpigmentation, mastocytosis, and dysphagia. They have been described with mutations in c-kit (most commonly), platelet-derived growth factor receptor A, neurofibromatosis-1, and succinate dehydrogenase genes. We report on molecular characterization and tumor histopathology of two siblings in whom lentigines and café-au-lait macules were present along with multifocal GIST. Immuhistochemical analysis of CD34 and CD117 was performed on GIST biopsy samples from both siblings, while c-kit mutational analysis was done by PCR and direct sequencing on DNA from peripheral blood leukocytes of all family members and from paraffin-embedded gastric biopsy specimens of affected siblings. Histopathology revealed positive expression of CD117 and CD34. Mutational analysis showed the germline c.1676T>C mutation in c-kit exon 11, (p.(Val559Ala)), in the peripheral blood of both siblings and a second exon 11 mutation, c.1669T>A (p.(Trp557Arg)) in the tumor biopsy of one of them. Initiation of imatinib treatment resulted in striking resolution of their hyperpigmentation and a stable gastrointestinal disease in one of them. A c-kit mutational test in familial GISTs is indicated before initiation of imatinib therapy, as it can help predict tumor response to treatment. © 2017 The International Society of Dermatology.

Improved kit formulation for preparation of (99m)Tc-HYNIC-TOC: results of preliminary clinical evaluation in imaging patients with neuroendocrine tumors.

PubMed

Korde, Aruna; Mallia, Madhava; Shinto, Ajit; Sarma, H D; Samuel, Grace; Banerjee, Sharmila

2014-11-01

(99m)Tc-HYNIC-TOC is a cost-effective and logistically viable agent for scintigraphy of neuroendocrine tumors overexpressing somatostatin receptors as compared with [(111)In-DTPA-D-Phe(1)] Octreotide (Octreoscan(®)). Several studies have been reported, wherein the efficacy of this agent is demonstrated. In the present article, the authors report the preparation of a single-vial HYNIC-TOC kit suitable for the preparation of 4-5 patient doses (15 mCi/patient) of (99m)Tc-HYNIC-TOC. The kits were tested for sterility and bacterial endotoxins to assure safety of the product. A significant modification in this kit is the inclusion of buffer in the kit itself, unlike in commercially available kits where the buffer solution has to be added during preparation. (99m)Tc-HYNIC-TOC was prepared by adding 20-80 mCi (740-2960 MBq) of freshly eluted Na(99m)TcO4 in 1-3 mL of sterile saline directly into the kit vial and heating the vial in a water bath at 100°C for 20 minutes. The labeling yield and radiochemical purity of (99m)Tc-HYNIC-TOC, prepared using the lyophilized cold kit, were consistently >90%. The kits were evaluated over a period of 9 months and found to be stable when stored at -20°C. Limited clinical studies performed with the (99m)Tc-HYNIC-TOC, formulated using the kit, showed adequate sensitivity and specificity for the detection of gasteroenteropancreatic neuroendocrine tumors.

Comparison of Multiplex Suspension Array Large-Panel Kits for Profiling Cytokines and Chemokines in Rheumatoid Arthritis Patients

PubMed Central

Khan, Imran H.; Krishnan, V.V.; Ziman, Melanie; Janatpour, Kim; Wun, Ted; Luciw, Paul A.; Tuscano, Joseph

2015-01-01

Background Multiplex analysis allows measurements of a large number of analytes simultaneously in each sample. Based on the Luminex multiplex technology (xMAP), kits for measuring multiple cytokines and chemokines (immunomodulators) are commercially available and are useful in investigations on inflammatory diseases. This study evaluated four multiplex kits (Bio-Plex, LINCOplex, Fluorokine, and Beadlyte) that contained 27, 29, 20 and 22 analytes each, respectively, for the analysis of immunomodulators in plasma of rheumatoid arthritis (RA) patients who underwent treatment with antibody against CD20 (rituximab), a B-cell reductive therapy. Methods Multiplex kits were tested on serial plasma samples obtained from six RA patients at baseline and multiple time points (3, 6, and 9 months) post-treatment with rituximab. The RA patients included in this study had previously failed therapy with disease modifying anti-arthritis drugs (DMARD) and treatment with anti-TNFα antibody (infliximab). Results Computer modeling and hierarchical cluster analysis of the multiplex data allowed a comparison of the performance of multiplex assay kits and revealed profiles of immunomodulators in the RA patients. Conclusions In plasma of RA patients who appeared to have benefited from rituximab treatment the profile of significantly elevated immunomodulators by at least two of the three kits (BioPlex, LINCOplex, Beadlyte), is as follows: IL-12p70, Eotaxin, IL-4, TNFα, Il-9, IL-1β, IFNγ, IL-10, IL-6, and IL-13. Immunomodulator profiling by multiplex analysis may provide useful plasma biomarkers for monitoring response to B-cell reductive therapy in RA patients. PMID:18823005

Increasing Flight Software Reuse with OpenSatKit

NASA Technical Reports Server (NTRS)

McComas, David C.

2018-01-01

In January 2015 the NASA Goddard Space Flight Center (GSFC) released the Core Flight System (cFS) as open source under the NASA Open Source Agreement (NOSA) license. The cFS is based on flight software (FSW) developed for 12 spacecraft spanning nearly two decades of effort and it can provide about a third of the FSW functionality for a low-earth orbiting scientific spacecraft. The cFS is a FSW framework that is portable, configurable, and extendable using a product line deployment model. However, the components are maintained separately so the user must configure, integrate, and deploy them as a cohesive functional system. This can be very challenging especially for organizations such as universities building cubesats that have minimal experience developing FSW. Supporting universities was one of the primary motivators for releasing the cFS under NOSA. This paper describes the OpenSatKit that was developed to address the cFS deployment challenges and to serve as a cFS training platform for new users. It provides a fully functional out-of-the box software system that includes NASA's cFS, Ball Aerospace's command and control system COSMOS, and a NASA dynamic simulator called 42. The kit is freely available since all of the components have been released as open source. The kit runs on a Linux platform, includes 8 cFS applications, several kit-specific applications, and built in demos illustrating how to use key application features. It also includes the software necessary to port the cFS to a Raspberry Pi and instructions for configuring COSMOS to communicate with the target. All of the demos and test scripts can be rerun unchanged with the cFS running on the Raspberry Pi. The cFS uses a 3-tiered layered architecture including a platform abstraction layer, a Core Flight Executive (cFE) middle layer, and an application layer. Similar to smart phones, the cFS application layer is the key architectural feature for users to extend the FSW functionality to meet their mission-specific requirements. The platform abstraction layer and the cFE layers go a step further than smart phones by providing a platform-agnostic Application Programmer Interface (API) that allows applications to run unchanged on different platforms. OpenSatKit can serve two significant architectural roles that will further help the adoption of the cFS and help create a community of users that can share assets. First, the kit is being enhanced to automate the integration of applications with the goal of creating a virtual cFS "App Store".. Second, a platform certification test suite can be developed that would allow users to verify the port of the cFS to a new platform. This paper will describe the current state of these efforts and future plans.

Stromal and epithelial cells react differentially to c-kit in fibroepithelial tumors of the breast.

PubMed

Logullo, Angela F; Nonogaki, Suely; Do Socorro Maciel, Maria; Mourão-Neto, Mário; Soares, Fernando Augusto

2008-01-01

The CD117 protein is a tyrosine-kinase receptor encoded by the c-kit gene that frequently bears activating mutations in gastrointestinal tumors. Conflicting findings regarding CD117 expression in other stromal tumors, including phyllodes tumors (PTs), have been reported in the literature. The purpose of this study was to evaluate c-kit expression in the stroma and epithelia of fibroepithelial breast tumors and its correlation with clinical pathological variables. Ninety-six fibroepithelial tumors of the breast, including 14 fibroadenomas (FAs), 12 juvenile FAs and 70 PTs, were classified according to stromal cellularity, atypia, epithelial hyperplasia, mitosis and borders into 45 benign (PTB), 17 borderline (PTBL) and 8 malignant (PTM) tumors. CD117 expression was identified in the stromal component in only two cases of PTBL. Overall, 38 cases (39.6%) showed positive CD117 in the epithelial component, including 20 FAs (10 regular, 10 juvenile) and 18 PTs (11 PTBs and 8 PTBLs). Other cases, including all PTMs, 6 FAs (4 regular, 2 juvenile), 34 PTBs and 10 PTBLs, showed no positivity in the epithelial component. Expression of c-kit did not correlate with diagnosis or malignancy (p>0.05). In conclusion, c-kit is expressed more often in the epithelial than in the stromal component in fibroepithelial tumors of the breast, and is associated with benign lesions.

BRAF, NRAS and C-KIT Advanced Melanoma: Clinico-pathological Features, Targeted-Therapy Strategies and Survival.

PubMed

Ponti, Giovanni; Manfredini, Marco; Greco, Stefano; Pellacani, Giovanni; Depenni, Roberta; Tomasi, Aldo; Maccaferri, Monia; Cascinu, Stefano

2017-12-01

The mutational status of stage III and IV melanomas should be recognized in order to allow for targeted therapies. The aim of our study was the characterization of BRAF, NRAS and C-KIT melanoma patients, in order to define their optimal management. Between 1991 and 2015, 63 mutated melanoma patients were treated and monitored during their diagnostic and therapeutic management at a single institution. BRAF-mutated melanoma patients were the most common, representing 70% of the study population, while NRAS- and C-KIT-mutated melanoma represented 19% and 11% respectively. BRAF-mutated melanomas were mostly located at sites of intermittent sun exposure, and were associated with higher Breslow thickness and an increased number of mitosis. NRAS mutated melanoma were mainly observed in chronic sun-damaged areas and had a negative prognostic value, with shorter time to progression and a high incidence of central nervous system involvement. C-KIT mutated melanoma were located at acral and mucosal sites. Overall survival observed in the three groups of patients revealed wide differences. BRAF, NRAS and C-KIT melanomas constitute distinct clinico-pathological entities. BRAF-mutated melanoma benefit from both anti-BRAF and anti-MEK targeted therapies while triple-negative melanomas could benefit from novel anti-CTLA-4 and anti-PD-L1 immunotherapeutic approaches. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Molecular spectrum of c-KIT and PDGFRA gene mutations in gastro intestinal stromal tumor: determination of frequency, distribution pattern and identification of novel mutations in Indian patients.

PubMed

Ahmad, Firoz; Lad, Purnima; Bhatia, Simi; Das, Bibhu Ranjan

2015-01-01

KIT and PDGFRA gene mutations are the major genetic alterations seen in gastrointestinal stromal tumors (GISTs) and are being used clinically for predicting response to imatinib therapy. In the current study, we set out to explore the frequency and distribution pattern of c-KIT (exons 9, 11 and 13) and PDGFRA (exons 12 and 18) by direct sequencing in a series of 70 Indian GIST cases. Overall, 27 (38.5 %) and 4 (5.7 %) of the cases had c-KIT and PDGFRA mutations, respectively. Majority of KIT mutations involved exon 11 (85.7 %), followed by exon 9 (14.3 %), while none showed exon 13 mutation. Most exon 9 mutations showed Ala503-Tyr504 duplication, while one had novel point mutation at codon 476 (S476G). In contrast to exon 9 mutations, most exon 11 mutations were in-frame deletions (79 %, 19/24), predominantly at codons 550-560, while remaining exon 11 mutant cases were point mutations at codons 559, 560, 568, 573 and 575. Interestingly, P573T, Q556_V560delinsH, Q575H and Q575_P577 were novel variations observed in exon 11. The PDGFRA mutations were seen mostly in exon 18, which showed point mutation at codon 842 (D842V), while exon 12 showed a novel indel variation (V561_H570delinsT). No significant correlation between c-KIT/PDGFRA mutations and clinicopathological data was observed. In conclusion, this study highlights the frequency and distribution pattern of c-KIT/PDGFRA mutation in Indian cohort. The current study identified novel variations that added new insights into the genetic heterogeneity of GIST patients. Furthermore, this is the first study to report the presence of PDGFRA mutation from Indian subcontinent.

Effectiveness of a multimedia outreach kit for families of wounded veterans.

PubMed

Walker, David Ian; Cardin, Jean-François; Chawla, Neelu; Topp, David; Burton, Thomaseo; Macdermid Wadsworth, Shelley

2014-04-01

Young children in military families with a member who has a life changing injury can experience emotional difficulties and behavior changes. This study evaluated a Sesame Workshop multimedia kit called: Talk, Listen, Connect: Changes (TLC-II(C); 2008). The kit, which included video and print materials, aimed to help caregivers (i.e., at-home partner, at-home relative or family member of a current or discharged military member) assist young children as they adjusted to their parent's injury. We expected that the materials would be used and their quality evaluated. We hypothesized that use of the materials would produce improvements in caregiver and child outcomes as well as reductions in perceptions of disruption in the home. We also predicted that kit-use would have a positive impact on the family. One-hundred and fifty three families with children aged 2-8 years were randomly assigned to receive the kit being evaluated (TLC-II(C)) or a control kit (Healthy Habits for Life (HHL)), also developed by Sesame Workshop. Group outcomes were compared four weeks following receipt of the kits using multivariate analysis of variance. All materials were well used and highly rated. All caregivers reported less social isolation, less child aggression, and significantly less disruptive home environments after kit use. Test group caregivers reported significantly greater reductions in depressive symptoms and significant increases in children's social competence over time in comparison to the control group. These results signal important improvements among families as a consequence of using either test or control materials. As a preventative intervention designed for families with an injured member, TLC-II(C) was particularly effective at improving coping. Copyright © 2014 Elsevier Inc. All rights reserved.

The role of small molecule Kit protein-tyrosine kinase inhibitors in the treatment of neoplastic disorders.

PubMed

Roskoski, Robert

2018-04-25

The Kit proto-oncogene was found as a consequence of the discovery of the feline v-kit sarcoma oncogene. Stem cell factor (SCF) is the Kit ligand and it mediates Kit dimerization and activation. The Kit receptor contains an extracellular segment that is made up of five immunoglobulin-like domains (D1/2/3/4/5), a transmembrane segment, a juxtamembrane segment, a protein-tyrosine kinase domain that contains an insert of 77 amino acid residues, and a carboxyterminal tail. Activating somatic mutations in Kit have been documented in various neoplasms including gastrointestinal stromal tumors (GIST), mast cell overexpression (systemic mastocytosis), core-binding factor acute myeloid leukemias (AML), melanomas, and seminomas. In the case of gastrointestinal stromal tumors, most activating mutations occur in the juxtamembrane segment and these mutants are initially sensitive to imatinib. As with many targeted anticancer drugs, resistance to Kit antagonists occurs in about two years and is the result of secondary KIT mutations. An activation segment exon 17 D816V mutation is one of the more common resistance mutations in Kit and this mutant is resistant to imatinib and sorafenib. Type I protein kinase inhibitors interact with the active enzyme form with DFG-D of the proximal activation segment directed inward toward the active site (DFG-D in ). In contrast, type II inhibitors bind to their target with the DFG-D pointing away from the active site (DFG-D out ). Based upon the X-ray crystallographic structures, imatinib, sunitinib, and ponatinib are Type II Kit inhibitors. We used the Schrödinger induced fit docking protocol to model the interaction of midostaurin with Kit and the result indicates that it binds to the DFG-D in conformation of the receptor and is thus classified as type I inhibitor. This medication inhibits the notoriously resistant Kit D816V mutant and is approved for the treatment of systemic mastocytosis and is effective against tumors bearing the D816V activation/resistance mutation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Inflammatory Response in Preterm and Very Preterm Newborns with Sepsis

PubMed Central

Segura-Cervantes, Enrique; Mancilla-Ramírez, Javier; González-Canudas, Jorge; Alba, Erika; Santillán-Ballesteros, René; Morales-Barquet, Deneb; Sandoval-Plata, Gabriela

2016-01-01

The response of the adaptive immune system is usually less intense in premature neonates than term neonates. The primary objective of this study was to determine whether immunological parameters vary between preterm (PT) neonates (≥32 weeks of gestational age) and very preterm (VPT) neonates (<32 weeks of gestational age). A cross-sectional study was designed to prospectively follow PT and VPT neonates at risk of developing sepsis. Plasma concentrations of IFN-γ, TNF-α, IL-6, IL-4, and IL-10 were detected using flow cytometry. C-reactive protein (C-RP) and the complex SC5b-9 were detected in the plasma using commercial kits. A total of 83 patients were included. The laboratory results and clinical histories showed that 26 patients had sepsis; 14 were VPT, and 12 were PT. The levels of C-RP, SC5b-9 (innate immune response mediators), and IL-10 or IL-4 (anti-inflammatory cytokines) were elevated during sepsis in both groups. IFN-γ, TNF-α, and IL-6 (proinflammatory cytokines) were differentially elevated only in PT neonates. The VPT neonates with sepsis presented increases in C-RP, SC5b-9, and anti-inflammatory cytokines but not in proinflammatory cytokines, whereas PT neonates showed increases in all studied mediators of inflammation. PMID:27293317

Improved Quantification of Plasma Catecholamines by the Radioenzymic Kit Method.

DTIC Science & Technology

1982-11-01

Diagnostics under the tradename CAT -A-KIT. In the Up- john procedure (2) each "unknown" and control specimen is analyzed with and without addition of an...the purpose of this report includes the follow- ing: (a) to recount difficulties we initially encountered in the prescribed use of CAT -A-KITs for...with each CAT -A-KIT, (c) to describe our departure from the prescribed procedure, (d) to validate that departure through intermethod/ interlaboratory

Clinical evaluation of a new single-tube multiplex reverse transcription PCR assay for simultaneous detection of 11 respiratory viruses, Mycoplasma pneumoniae and Chlamydia in hospitalized children with acute respiratory infections.

PubMed

Zhao, Meng-Chuan; Li, Gui-Xia; Zhang, Dan; Zhou, Hang-Yu; Wang, Hao; Yang, Shuo; Wang, Le; Feng, Zhi-Shan; Ma, Xue-Jun

2017-06-01

Respiratory Pathogen 13 Detection Kit (13× kit) is able to simultaneously detect 11 respiratory viruses, Mycoplasma pneumoniae (MP) and Chlamydia in a single reaction. Using 572 Nasopharyngeal aspirates collected from hospitalized children, the clinical performance of 13× kit for detecting 11 respiratory viruses was evaluated in comparison with a routinely used 2-tube multiplex reverse transcription PCR assay (2-tube assay) at provincial Centers for Disease Control and Prevention in China. The clinical performance of 13× kit for detecting MP and Chlamydia was evaluated by commercial real-time quantitative PCR (qPCR) kits or sequencing. For tested viruses, the assay concordance was 95.98% and the kappa coefficient was 0.89. All the MP and Chlamydia positive samples detected by 13× kit were confirmed as true positives. The utilization of the 13× kit in clinical settings will be helpful for doctors to assess clinical outcome according to virus type or multiple infections, and to limit the use of antibiotics. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes

PubMed Central

Liu, Qiaozhen; Yang, Rui; Huang, Xiuzhen; Zhang, Hui; He, Lingjuan; Zhang, Libo; Tian, Xueying; Nie, Yu; Hu, Shengshou; Yan, Yan; Zhang, Li; Qiao, Zengyong; Wang, Qing-Dong; Lui, Kathy O; Zhou, Bin

2016-01-01

Cardiac cells marked by c-Kit or Kit, dubbed cardiac stem cells (CSCs), are in clinical trials to investigate their ability to stimulate cardiac regeneration and repair. These studies were initially motivated by the purported cardiogenic activity of these cells. Recent lineage tracing studies using Kit promoter to drive expression of the inducible Cre recombinase showed that these CSCs had highly limited cardiogenic activity, inadequate to support efficient cardiac repair. Here we reassess the lineage tracing data by investigating the identity of cells immediately after Cre labeling. Our instant lineage tracing approach identifies Kit-expressing cardiomyocytes, which are labeled immediately after tamoxifen induction. In combination with long-term lineage tracing experiments, these data reveal that the large majority of long-term labeled cardiomyocytes are pre-existing Kit-expressing cardiomyocytes rather than cardiomyocytes formed de novo from CSCs. This study presents a new interpretation for the contribution of Kit+ cells to cardiomyocytes and shows that Kit genetic lineage tracing over-estimates the cardiogenic activity of Kit+ CSCs. PMID:26634606

Determining the IgM and IgG antibody titer against CMV and helicobacter pylori in the serum of multiple sclerosis patients comparing to the control group in Hamadan.

PubMed

Salim, Masome Afiati; Eftekharian, Mohammad Mahdi; Taheri, Mohammad; Yousef Alikhani, Mohammad

2017-07-19

Multiple sclerosis (MS) is a chronic autoimmune disease that disables central nervous system (CNS) system. Cytomegalovirus (CMV) probably has an important role in the MS pathology. The infection with helicobacter pylori also is recognized as a protective agent against MS in female. Serum samples were isolated and frozen at -70∘C. The earlier mentioned anti-virus antibodies and antibacterial antibodies were quantified by Elisa kit. The results showed that IgG antibody average value against cytomegalovirus in the blood of multiple sclerosis patients not only decreased but also was significant statistically (p< 0.05). IgM and IgG antibodies average value in the blood of multiple sclerosis patients against helicobacter pylori shown a statistically significant decrease (p< 0.05). Therefore it may be considered that probably helicobacter pylori presence in the individuals especially in female can alleviate MS signs. CMV infection can intensify the symptoms in multiple sclerosis patients.

Kit signaling inhibits the sphingomyelin-ceramide pathway through PLC gamma 1: implication in stem cell factor radioprotective effect.

PubMed

Maddens, Stéphane; Charruyer, Alexandra; Plo, Isabelle; Dubreuil, Patrice; Berger, Stuart; Salles, Bernard; Laurent, Guy; Jaffrézou, Jean-Pierre

2002-08-15

Previous studies demonstrated that Kit activation confers radioprotection. However, the mechanism by which Kit signaling interferes with cellular response to ionizing radiation (IR) has not been firmly established. Based on the role of the sphingomyelin (SM) cycle apoptotic pathway in IR-induced apoptosis, we hypothesized that one of the Kit signaling components might inhibit IR-induced ceramide production or ceramide-induced apoptosis. Results show that, in both Ba/F3 and 32D murine cell lines transfected with wild-type c-kit, stem cell factor (SCF) stimulation resulted in a significant reduction of IR-induced apoptosis and cytotoxicity, whereas DNA repair remained unaffected. Moreover, SCF stimulation inhibited IR-induced neutral sphingomyelinase (N-SMase) stimulation and ceramide production. The SCF inhibitory effect on SM cycle was not influenced by wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor. The SCF protective effect was maintained in 32D-KitYF719 cells in which the PI3K/Akt signaling pathway is abolished due to mutation in Kit docking site for PI3K. In contrast, phospholipase C gamma (PLC gamma) inhibition by U73122 totally restored IR-induced N-SMase stimulation, ceramide production, and apoptosis in Kit-activated cells. Moreover, SCF did not protect 32D-KitYF728 cells (lacking a functional docking site for PLC gamma 1), from IR-induced SM cycle. Finally, SCF-induced radioprotection of human CD34(+) bone marrow cells was also inhibited by U73122. Altogether, these results suggest that SCF radioprotection is due to PLC gamma 1-dependent negative regulation of IR-induced N-SMase stimulation. Beyond the scope of Kit-expressing cells, it suggests that PLC gamma 1 status could greatly influence the post-DNA damage cellular response to IR, and perhaps, to other genotoxic agents.

Comparison of three feline leukaemia virus (FeLV) point-of-care antigen test kits using blood and saliva.

PubMed

Westman, Mark E; Malik, Richard; Hall, Evelyn; Sheehy, Paul A; Norris, Jacqueline M

2017-02-01

Feline leukaemia virus (FeLV) can be a challenging infection to diagnose due to a complex feline host-pathogen relationship and occasionally unreliable test results. This study compared the accuracy of three point-of-care (PoC) FeLV p27 antigen test kits commonly used in Australia and available commercially worldwide (SNAP FIV/FeLV Combo, Witness FeLV/FIV and Anigen Rapid FIV/FeLV), using detection of FeLV provirus by an in-house real-time polymerase chain reaction (qPCR) assay as the diagnostic gold standard. Blood (n=563) and saliva (n=419) specimens were collected from a population of cats determined to include 491 FeLV-uninfected and 72 FeLV-infected individuals (45 progressive infections [p27 and qPCR positive], 27 regressive infections [p27 negative, qPCR positive]). Sensitivity and specificity using whole blood was 63% and 94% for SNAP Combo, 57% and 98% for Witness, and 57% and 98% for Anigen Rapid, respectively. SNAP Combo had a significantly lower specificity using blood compared to the other two kits (P=0.004 compared to Witness, P=0.007 compared to Anigen Rapid). False-positive test results occurred with all three kits using blood, and although using any two kits in parallel increased specificity, no combination of kits completely eliminated the occurrence of false-positive results. We therefore recommend FeLV proviral PCR testing for any cat that tests positive with a PoC FeLV antigen kit, as well as for any cat that has been potentially exposed to FeLV but tests negative with a FeLV antigen kit, before final assignment of FeLV status can be made with confidence. For saliva testing, sensitivity and specificity was 54% and 100%, respectively, for all three test kits. The reduced sensitivity of saliva testing compared to blood testing, although not statistically significant, suggests saliva testing with the current generation of PoC FeLV antigen kits is unsuitable for screening large populations of cats, such as in shelters. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Role of the Stem Cell Factor/c-kit Complex in Neurofibrormatosis.

DTIC Science & Technology

1997-10-01

investigations of the Kit/ stem cell factor complex in hyperplasias of these cells. In the request for no-cost extension, several experiments were listed to be...soluble stem cell factor from the NF1 knockout Schwann cells. We also found that most neural tumors express mRNA for Kit, and also for stem cell factor.

Case report of a KIT-mutated melanoma patient with an excellent response to apatinib and temozolomide combination therapy.

PubMed

Luo, Cong; Shen, Jiayu; Ying, Jieer; Fang, Xianhua; Wang, Xiaohong; Fu, Zhixuan; Liu, Peng

2017-01-01

Malignant melanoma is one kind of malignant disease which has high rates of mortality, metastasis, and poor prognosis. The therapeutic landscape is rapidly changing with the development of novel agents in recent decades, such as anti-PD-1 agents, anti-CTLA-4 agents, and BRAF inhibitors. However, since most of these novel agents are very expensive, not all patients can afford them. Apatinib is a novel oral small-molecule tyrosine kinase inhibitor targeting the intracellular domain of vascular endothelial growth factor receptor 2 (VEGFR-2) and may also be effective on Ret, c-KIT, and c-src. Temozolomide (TMZ) is a second-generation alkylating agent and a cytotoxic drug for melanoma treatment. In this work, we reported a case of metastatic melanoma with an excellent response to apatinib/TMZ combination therapy with progression-free survival for more than one year. This patient showed high expression of CD117, VEGFR-3, and KIT mutation in exon 11, suggesting that apatinib may induce clinical response via inhibiting VEGFR and c-KIT. Apatinib/TMZ combination therapy could be a new option for the treatment of advanced melanoma with KIT mutation.

Relationship between pain and post-traumatic stress symptoms in palliative care.

PubMed

Roth, Maya L; St Cyr, Kate; Harle, Ingrid; Katz, Joel D

2013-08-01

Previous research suggests that patients receiving palliative care may simultaneously experience poorly managed pain and post-traumatic stress disorder (PTSD)-related symptoms as a result of their deteriorating health. To: 1) examine predictors of PTSD-related symptoms in patients requiring palliative care; 2) assess whether anxiety, depression, pain catastrophizing, and pain anxiety mediate the relationship between pain interference and PTSD-related symptoms; and 3) evaluate the impact of these variables on pain interference and PTSD-related symptoms. One hundred patients receiving palliative care at one of two palliative care sites in London, ON, Canada, completed the PTSD Checklist-Civilian version (PCL-C), the Hospital Anxiety and Depression Scale (HADS), the Pain Catastrophizing Scale (PCS), the Brief Pain Inventory-Short Form (BPI-SF), and the Pain Anxiety Symptoms Scale-20 (PASS-20). Hierarchical multiple regressions were used to examine HADS-Anxiety, HADS-Depression, PCS and PASS-20 scores as predictors of PCL-C scores; and mediation analyses were used to test the effect of HADS-Anxiety, HADS-Depression, PCS, and PASS-20 on the relationship between BPI-SF interference and PCL-C. Mediators that significantly affected this relationship in the individual mediator models were entered into a multiple mediator model. Only pain anxiety and pain catastrophizing emerged as significant mediators of the relationship between pain interference and PTSD-related symptoms. After being entered in a multiple mediator model, pain anxiety emerged as the strongest mediator. The findings of the present study reveal that pain and PTSD-related symptoms are important concerns in palliative care, and that pain must be addressed to best meet the needs of this population. Copyright © 2013 U.S. Cancer Pain Relief Committee. Published by Elsevier Inc. All rights reserved.

Japanese Reference Panel of Blood Specimens for Evaluation of Hepatitis C Virus RNA and Core Antigen Quantitative Assays

PubMed Central

Murayama, Asako; Sugiyama, Nao; Watashi, Koichi; Masaki, Takahiro; Suzuki, Ryosuke; Aizaki, Hideki; Mizuochi, Toshiaki; Wakita, Takaji

2012-01-01

An accurate and reliable quantitative assay for hepatitis C virus (HCV) is essential for measuring viral propagation and the efficacy of antiviral therapy. There is a growing need for domestic reference panels for evaluation of clinical assay kits because the performance of these kits may vary with region-specific genotypes or polymorphisms. In this study, we established a reference panel by selecting 80 donated blood specimens in Japan that tested positive for HCV. Using this panel, we quantified HCV viral loads using two HCV RNA kits and five core antigen (Ag) kits currently available in Japan. The data from the two HCV RNA assay kits showed excellent correlation. All RNA titers were distributed evenly across a range from 3 to 7 log IU/ml. Although the data from the five core Ag kits also correlated with RNA titers, the sensitivities of individual kits were not sufficient to quantify viral load in all samples. As calculated by the correlation with RNA titers, the theoretical lower limits of detection by these core Ag assays were higher than those for the detection of RNA. Moreover, in several samples in our panel, core Ag levels were underestimated compared to RNA titers. Sequence analysis in the HCV core region suggested that polymorphisms at amino acids 47 to 49 of the core Ag were responsible for this underestimation. The panel established in this study will be useful for estimating the quality of currently available and upcoming HCV assay kits; such quality control is essential for clinical usage of these kits. PMID:22495557

Ease fabrication of PCR modular chip for portable DNA detection kit

NASA Astrophysics Data System (ADS)

Whulanza, Yudan; Aditya, Rifky; Arvialido, Reyhan; Utomo, Muhammad S.; Bachtiar, Boy M.

2017-02-01

Engineering a lab-on-a-chip (LoC) to perform the DNA polymerase chain reaction (PCR) for malaria detection is the ultimate goal of this study. This paper investigates the ability to fabricate an LoC kit using conventional method to achieve the lowest production cost by using existing fabrication process. It has been known that majority of LoC was made of polydimethylsiloxane (PDMS) which in this study was realized through a contact mold process. CNC milling process was utilized to create channel features in the range of 150-250 µm on the mold. Characterization on the milling process was done to understand the shrinkage/contraction between mold to product, roughness and also angle of contact of PDMS surface. Ultimately, this paper also includes analysis on flow measurement and heat distribution of an assembled LoC PCR kit. The results show that the achieved dimension of microchannel is 227 µm wide with a roughness of 0.01 µm. The flow measurement indicates a deviation with simulation in the range of 10%. A heat distribution through the kit is achieved following the three temperature zones as desired.

Huntingtin-interacting protein 1: a Merkel cell carcinoma marker that interacts with c-Kit.

PubMed

Ames, Heather M; Bichakjian, Christopher K; Liu, Grace Y; Oravecz-Wilson, Katherine I; Fullen, Douglas R; Verhaegen, Monique E; Johnson, Timothy M; Dlugosz, Andrzej A; Ross, Theodora S

2011-10-01

Merkel cell carcinoma (MCC) is a neoplasm thought to originate from the neuroendocrine Merkel cells of the skin. Although the prevalence of MCC has been increasing, treatments for this disease remain limited because of a paucity of information regarding MCC biology. We have found that the endocytic oncoprotein Huntingtin-interacting protein 1 (HIP1) is expressed at high levels in ∼90% of MCC tumors and serves as a more reliable histological cytoplasmic stain than the gold standard, cytokeratin 20. Furthermore, high anti-HIP1 antibody reactivity in the sera of a cohort of MCC patients predicts the presence of metastases. Another protein that is frequently expressed at high levels in MCC tumors is the stem cell factor (SCF) receptor tyrosine kinase, c-Kit. In working toward an understanding of how HIP1 might contribute to MCC tumorigenesis, we have discovered that HIP1 interacts with SCF-activated c-Kit. These data not only identify HIP1 as a molecular marker for management of MCC patients but also show that HIP1 interacts with and slows the degradation of c-Kit.

MTHFR GENE C677T POLYMORPHISM AND LEVELS OF DNA METHYLTRASFERASES IN SUBCLINICAL HYPOTHYROIDISM.

PubMed

Kvaratskhelia, T; Kvaratskhelia, E; Kankava, K; Abzianidze, E

2017-04-01

The aim of our study was to investigate the link between MTHFR gene C677T polymorphism and DNMTs levels in patients with Subclinical Hypothyroidism (SCH). In this study 19 adult patients with subclinical hypothyroidism and 19 healthy controls (mean age 31±5.5 and 33±5.1 years respectively) were recruited. All patients were diagnosed based on serum levels of TSH, FT4, anti-TG and anti-TPO antibodies. Written informed consents were obtained from all study subjects. Genomic DNA was extracted using Quick-DNA Universal Kit (Zymo Research, USA). The MTHFR C677T polymorphism was genotyped by PCR-RFLP method. Levels of DNMT1 and 3a were measured in nuclear extracts of PBMC using DNMTs assay kits (Abcam). Our data indicates that the frequency of genotypes and alleles were different among the patient and the control group. There is a significant increase in CC genotype distribution in the control group when compared to the SCH patient group, while the CT as well as TT genotype distribution were not increased significantly in SCH group versus control group. However the C allele is significantly prevalent in the control group compared to the SCH group, while T allele is prevalent in patients compared to the control group with a statically significant difference. In addition, individuals with TT and CT genotypes and hypothyroidism showed elevated amount of DNMT3a in nuclear extracts of PBMC compared with controls, while no significant difference in DNMT1 levels was observed. This study indicates the MTHFR C677T variant may contribute in alteration of epigenetic regulation such as DNA methylation mediated by DNA methyltransferases in patients with subclinical hypothyroidism and also, carriers of the T allele might have an increasing risk of developing SCH.

cIMP synthesized by sGC as a mediator of hypoxic contraction of coronary arteries.

PubMed

Chen, Zhengju; Zhang, Xu; Ying, Lei; Dou, Dou; Li, Yanhui; Bai, Yun; Liu, Juan; Liu, Limei; Feng, Han; Yu, Xiaoxing; Leung, Susan Wai-Sum; Vanhoutte, Paul M; Gao, Yuansheng

2014-08-01

cGMP is considered the only mediator synthesized by soluble guanylyl cyclase (sGC) in response to nitric oxide (NO). However, purified sGC can synthesize several other cyclic nucleotides, including inosine 3',5'-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension. Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy. Rho kinase (ROCK) activity was determined by measuring the phosphorylation of myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by hypoxia in arteries with, but not in those without, endothelium [except if treated with diethylenetriamine (DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Hypoxia (Po2: 25-30 mmHg) augmented contractions of arteries with and without endothelium if treated with DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by inosine 5'-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr(853), which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK. Copyright © 2014 the American Physiological Society.

Functional TRPV2 and TRPV4 channels in human cardiac c-kit(+) progenitor cells.

PubMed

Che, Hui; Xiao, Guo-Sheng; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong

2016-06-01

The cellular physiology and biology of human cardiac c-kit(+) progenitor cells has not been extensively characterized and remains an area of active research. This study investigates the functional expression of transient receptor potential vanilloid (TRPV) and possible roles for this ion channel in regulating proliferation and migration of human cardiac c-kit(+) progenitor cells. We found that genes coding for TRPV2 and TRPV4 channels and their proteins are significantly expressed in human c-kit(+) cardiac stem cells. Probenecid, an activator of TRPV2, induced an increase in intracellular Ca(2+) (Ca(2+) i ), an effect that may be attenuated or abolished by the TRPV2 blocker ruthenium red. The TRPV4 channel activator 4α-phorbol 12-13-dicaprinate induced Ca(2+) i oscillations, which can be inhibited by the TRPV4 blocker RN-1734. The alteration of Ca(2+) i by probenecid or 4α-phorbol 12-13-dicprinate was dramatically inhibited in cells infected with TRPV2 short hairpin RNA (shRNA) or TRPV4 shRNA. Silencing TRPV2, but not TRPV4, significantly reduced cell proliferation by arresting cells at the G0/G1 boundary of the cell cycle. Cell migration was reduced by silencing TRPV2 or TRPV4. Western blot revealed that silencing TRPV2 decreased expression of cyclin D1, cyclin E, pERK1/2 and pAkt, whereas silencing TRPV4 only reduced pAkt expression. Our results demonstrate for the first time that functional TRPV2 and TRPV4 channels are abundantly expressed in human cardiac c-kit(+) progenitor cells. TRPV2 channels, but not TRPV4 channels, participate in regulating cell cycle progression; moreover, both TRPV2 and TRPV4 are involved in migration of human cardiac c-kit(+) progenitor cells. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Increasing Flight Software Reuse with OpenSatKit

NASA Technical Reports Server (NTRS)

McComas, David

2018-01-01

In January 2015 the NASA Goddard Space Flight Center (GSFC) released the Core Flight System (cFS) as open source under the NASA Open Source Agreement (NOSA) license. The cFS is based on flight software (FSW) developed for 12 spacecraft spanning nearly two decades of effort and it can provide about a third of the FSW functionality for a low-earth orbiting scientific spacecraft. The cFS is a FSW framework that is portable, configurable, and extendable using a product line deployment model. However, the components are maintained separately so the user must configure, integrate, and deploy them as a cohesive functional system. This can be very challenging especially for organizations such as universities building cubesats that have minimal experience developing FSW. Supporting universities was one of the primary motivators for releasing the cFS under NOSA. This paper describes the OpenSatKit that was developed to address the cFS deployment challenges and to serve as a cFS training platform for new users. It provides a fully functional out-of-the box software system that includes NASA's cFS, Ball Aerospaceâ€"TM"s command and control system COSMOS, and a NASA dynamic simulator called 42. The kit is freely available since all of the components have been released as open source. The kit runs on a Linux platform, includes 8 cFS applications, several kit-specific applications, and built in demos illustrating how to use key application features. It also includes the software necessary to port the cFS to a Raspberry Pi and instructions for configuring COSMOS to communicate with the target. All of the demos and test scripts can be rerun unchanged with the cFS running on the Raspberry Pi. The cFS uses a 3-tiered layered architecture including a platform abstraction layer, a Core Flight Executive (cFE) middle layer, and an application layer. Similar to smart phones, the cFS application layer is the key architectural feature for userâ€"TM"s to extend the FSW functionality to meet their mission-specific requirements. The platform abstraction layer and the cFE layers go a step further than smart phones by providing a platform-agnostic Application Programmer Interface (API) that allows applications to run unchanged on different platforms. OpenSatKit can serve two significant architectural roles that will further help the adoption of the cFS and help create a community of users that can share assets. First, the kit is being enhanced to automate the integration of applications with the goal of creating a virtual cFS 'App Store'. Second, a platform certification test suite can be developed that would allow users to verify the port of the cFS to a new platform. This paper will describe the current state of these efforts and future plans.

Changes in the interstitial cells of Cajal and neuronal nitric oxide synthase positive neuronal cells with aging in the esophagus of F344 rats.

PubMed

Kim, Hee Jin; Kim, Nayoung; Kim, Yong Sung; Nam, Ryoung Hee; Lee, Sun Min; Park, Ji Hyun; Choi, Daeun; Hwang, Young-Jae; Lee, Jongchan; Lee, Hye Seung; Kim, Min-Seob; Lee, Moon Young; Lee, Dong Ho

2017-01-01

The aging-associated cellular and molecular changes in esophagus have not been established, yet. Thus we evaluated histological structure, interstitial cells of Cajal (ICCs), neuronal nitric oxide synthase (nNOS)-positive cells, and contractility in the esophagus of Fischer 344 rat at different ages (6-, 31-, 74-weeks, and 2-years). The lamina propria thickness and endomysial area were calculated. The immunoreactivity of c-Kit, nNOS and protein gene product (PGP) 9.5 was counted after immunohistochemistry. Expression of c-Kit, stem cell factor (SCF), nNOS and PGP 9.5 mRNA was measured by real-time PCR, and expression of c-Kit and nNOS protein was detected by Western blot. Isovolumetric contractile force measurement and electrical field stimulation (EFS) were conducted. The lamina propria thickness increased (6 week vs 2 year, P = 0.005) and the endomysial area of longitudinal muscle decreased with aging (6 week vs 2 year, P<0.001), while endomysial area of circular muscle did not significantly decrease. The proportions of NOS-immunoreactive cells and c-Kit-immunoreactive areas declined with aging (6 week vs 2 year; P<0.001 and P = 0.004, respectively), but there was no significant change of PGP 9.5-immunopositiviy. The expressions of nNOS, c-Kit and SCF mRNA also reduced with aging (6 week vs 2 year; P = 0.006, P = 0.001 and P = 0.006, respectively), while the change of PGP 9.5 mRNA expression was not significant. Western blot showed the significant decreases of nNOS and c-Kit protein expression with aging (6 week vs 2 year; P = 0.008 and P = 0.012, respectively). The EFS-induced esophageal contractions significantly decreased in 2-yr-old rat compared with 6-wk-old rats, however, L-NG-Nitroarginine methylester did not significantly increase the spontaneous and EFS-induced contractions in the 6-wk- and 2-yr-old rat esophagus. In conclusion, an increase of lamina propria thickness, a decrease of endomysial area, c-Kit, SCF and NOS expression with preserved total enteric neurons, and contractility in aged rat esophagus may explain the aging-associated esophageal dysmotility.

Changes in the interstitial cells of Cajal and neuronal nitric oxide synthase positive neuronal cells with aging in the esophagus of F344 rats

PubMed Central

Kim, Hee Jin; Kim, Yong Sung; Nam, Ryoung Hee; Lee, Sun Min; Park, Ji Hyun; Choi, Daeun; Hwang, Young-Jae; Lee, Jongchan; Lee, Hye Seung; Kim, Min-Seob; Lee, Moon Young; Lee, Dong Ho

2017-01-01

The aging-associated cellular and molecular changes in esophagus have not been established, yet. Thus we evaluated histological structure, interstitial cells of Cajal (ICCs), neuronal nitric oxide synthase (nNOS)-positive cells, and contractility in the esophagus of Fischer 344 rat at different ages (6-, 31-, 74-weeks, and 2-years). The lamina propria thickness and endomysial area were calculated. The immunoreactivity of c-Kit, nNOS and protein gene product (PGP) 9.5 was counted after immunohistochemistry. Expression of c-Kit, stem cell factor (SCF), nNOS and PGP 9.5 mRNA was measured by real-time PCR, and expression of c-Kit and nNOS protein was detected by Western blot. Isovolumetric contractile force measurement and electrical field stimulation (EFS) were conducted. The lamina propria thickness increased (6 week vs 2 year, P = 0.005) and the endomysial area of longitudinal muscle decreased with aging (6 week vs 2 year, P<0.001), while endomysial area of circular muscle did not significantly decrease. The proportions of NOS-immunoreactive cells and c-Kit-immunoreactive areas declined with aging (6 week vs 2 year; P<0.001 and P = 0.004, respectively), but there was no significant change of PGP 9.5-immunopositiviy. The expressions of nNOS, c-Kit and SCF mRNA also reduced with aging (6 week vs 2 year; P = 0.006, P = 0.001 and P = 0.006, respectively), while the change of PGP 9.5 mRNA expression was not significant. Western blot showed the significant decreases of nNOS and c-Kit protein expression with aging (6 week vs 2 year; P = 0.008 and P = 0.012, respectively). The EFS-induced esophageal contractions significantly decreased in 2-yr-old rat compared with 6-wk-old rats, however, L-NG-Nitroarginine methylester did not significantly increase the spontaneous and EFS-induced contractions in the 6-wk- and 2-yr-old rat esophagus. In conclusion, an increase of lamina propria thickness, a decrease of endomysial area, c-Kit, SCF and NOS expression with preserved total enteric neurons, and contractility in aged rat esophagus may explain the aging-associated esophageal dysmotility. PMID:29182640

Mobile chemical detector (AP2C+SP4E) as an aid for medical decision making in the battlefield.

PubMed

Eisenkraft, Arik; Markel, Gal; Simovich, Shirley; Layish, Ido; Hoffman, Azik; Finkelstein, Arseny; Rotman, Eran; Dushnitsky, Tsvika; Krivoy, Amir

2007-09-01

The combination of the AP2C unit with the SP4E kit composes a lightweight mobile detector of chemical warfare agents (CWA), such as nerve and mustard agents, with both vapor- and liquid-sampling capabilities. This apparatus was recently introduced into our military medical units as an aid for detection of CWA on casualties. Importantly, critical information regarding the applicability in the battlefield was absent. In view of the serious consequences that might follow a proclamation of CWA recognition in battlefield, a high false-positive rate positions the utilization of this apparatus as a medical decision tool in question. We have therefore conducted a field experiment to test the false-positive rate as well as analyze possible factors leading to false-positive readings with this device. The experiment was carried out before and after a 4-day army field exercise, using a standard AP2C device, a SP4E surface sampling kit, and a specially designed medical sampling kit for casualties, intended for medical teams. Soldiers were examined at rest, after mild exercise, and after 4 days in the field. The readings with AP2C alone were compared to the combination of AP2C and SP4E and to the medical sampling kit. Various body fluids served as negative controls. Remarkably, we found a false-positive rate of 57% at rest and after mild exercise, and an even higher rate of 64% after the 4-day field exercise with the AP2C detector alone, as compared to almost no false-positive readings with the combination of AP2C and SP4E. Strikingly, the medical sampling kit has yielded numerous false-positive readings, even in normal body fluids such as blood, urine, and saliva. We therefore see no place for using the medical sampling kit due to an unaccepted high rate of false-positive readings. Finally, we have designed an algorithm that uses the entire apparatus of AP2C and SP4E as a reliable validation tool for medical triage in the setting of exposure to nerve agents in the battlefield.

Bent Bonds and Multiple Bonds.

ERIC Educational Resources Information Center

Robinson, Edward A.; Gillespie, Ronald J.

1980-01-01

Considers carbon-carbon multiple bonds in terms of Pauling's bent bond model, which allows direct calculation of double and triple bonds from the length of a CC single bond. Lengths of these multiple bonds are estimated from direct measurements on "bent-bond" models constructed of plastic tubing and standard kits. (CS)

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.

PubMed

Mahmoudi, Nagissa; Slater, Greg F; Fulthorpe, Roberta R

2011-08-01

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.

Expression and Mutational Analysis of c-kit in Ovarian Surface Epithelial Tumors

PubMed Central

Lee, Myung-Hoon; Park, Tae-In; Bae, Han-Ik

2006-01-01

Coexpression of Kit ligand and c-kit has been reported in some gynecologic tumors. To determine whether imatinib mesylate is useful in ovarian epithelial tumors, we performed immunohistochemical and mutational analysis. The cases consisted of 33 cases, which included 13 serous cystadenocarcinomas, 1 borderline serous tumor, 8 mucinous cystadenocarcinomas, 6 borderline mucinous tumors and 5 clear cell carcinomas. Five cases of serous cystadenoma and 5 cases of mucinous cystadenoma were also included. In the immunohistochemical study, 3 cases (3/6, 50%) of borderline mucinous cystic tumor and two cases (2/8, 25%) of mucinous cystadenocarcinoma show positive staining for KIT protein. Only one case (1/13, 7.7%) of serous cystadenocarcinoma had positive staining. On mutational analysis, no mutation was identified at exon 11. However, two cases of borderline mucinous tumors and one case of mucinous cystadenocarcinoma had mutations at exon 17. In these cases, the immunohistochemistry also shows focal positive staining at epithelial component. Although, KIT protein expression showed higher incidence in mucinous tumors than serous tumors, they lack KIT-activating mutations in exon 11. Thus, ovarian surface epithelial tumors are unlikely to respond to imatinib mesylate. PMID:16479070

Enteric colonization by staphylococcus delphini in four ferret kits with diarrhoea.

PubMed

Gary, J M; Langohr, I M; Lim, A; Bolin, S; Bolin, C; Moore, I; Kiupel, M

2014-11-01

Four, 1-to 4-week-old ferret kits were submitted to the Diagnostic Center for Population and Animal Health at Michigan State University for post-mortem examination. Grossly, multiple bowel loops in all ferret kits were distended by mucoid faecal material. Microscopically, there was no evidence of inflammation or notable alteration to the normal mucosal morphology. Gram-positive coccoid bacteria colonized variable segments of the small intestine. These bacteria were identified as Staphylococcus delphini by phenotypic and molecular analyses. Enzyme-linked immunosorbent assay for detection of Staphylococcus enterotoxins was positive and polymerase chain reaction detected the gene for Staphylococcus enterotoxin E in the isolates. The hypersecretory diarrhoea in these ferret kits may have been associated with colonization of the small intestine by S. delphini, cultures of which were shown in vitro to be potentially capable of producing enterotoxin E. The condition described in these ferrets is similar to 'sticky' kit syndrome in mink. Copyright © 2014 Elsevier Ltd. All rights reserved.

Accuracy of user-friendly blood typing kits tested under simulated military field conditions.

PubMed

Bienek, Diane R; Charlton, David G

2011-04-01

Rapid user-friendly ABO-Rh blood typing kits (Eldon Home Kit 2511, ABO-Rh Combination Blood Typing Experiment Kit) were evaluated to determine their accuracy when used under simulated military field conditions and after long-term storage at various temperatures and humidities. Rates of positive tests between control groups, experimental groups, and industry standards were measured and analyzed using the Fisher's exact chi-square method to identify significant differences (p < or = 0.05). When Eldon Home Kits 2511 were used in various operational conditions, the results were comparable to those obtained with the control group and with the industry standard. The performance of the ABO-Rh Combination Blood Typing Experiment Kit was adversely affected by prolonged storage in temperatures above 37 degrees C. The diagnostic performance of commercial blood typing kits varies according to product and environmental storage conditions.

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT

PubMed Central

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors’ sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib. PMID:27467080

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT.

PubMed

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib.

Mast cells and mastocytosis

PubMed Central

2008-01-01

Mast cells have been recognized for well over 100 years. With time, human mast cells have been documented to originate from CD34+ cells, and have been implicated in host responses in both innate and acquired immunity. In clinical immunology, they are recognized for their central role in IgE-mediated degranulation and allergic inflammation by virtue of their expression of the high-affinity receptor for IgE and release of potent proinflammatory mediators. In hematology, the clinical disease of mastocytosis is characterized by a pathologic increase of mast cells in tissues, often associated with mutations in KIT, the receptor for stem cell factor. More recently, and with increased understanding of how human mast cells are activated through receptors including the high-affinity receptor for IgE and KIT, specific tyrosine kinase inhibitors have been identified with the potential to interrupt signaling pathways and thus limit the proliferation of mast cells as well as their activation through immunoglobulin receptors. PMID:18684881

Augmentation of the anticancer activity of CYT997 in human prostate cancer by inhibiting Src activity.

PubMed

Teng, Yong; Cai, Yafei; Pi, Wenhu; Gao, Lixia; Shay, Chloe

2017-06-12

Abnormalities of tubulin polymerization and microtubule assembly are often seen in cancer, which make them very suitable targets for the development of therapeutic approach against rapidly dividing and aggressive cancer cells. CYT997 is a novel microtubule-disrupting agent with anticancer activity in multiple cancer types including prostate cancer. However, the molecular mechanisms of action of CYT997 in prostate cancer have not been well characterized. Src knockdown cells were achieved by lentiviral-mediated interference. The drug effects on cell proliferation were measured by MTS. The drug effects on cell viability and death were determined by Cell Titer-Glo® Luminescent cell viability kit and flow cytometry with Zombie Aqua™ staining. The drug effects on apoptosis were assessed by Cell Death Detection Elisa kit and Western blot with a cleaved PARP antibody. The drug effects on cell invasion were examined by Matrigel-coated Boyden chambers. Oxidative stress was detected by DCFH-DA staining and electrochemical biosensor. Mouse models generated by subcutaneous or intracardiac injection were used to investigate the in vivo drug efficacy in tumor growth and metastasis. CYT997 effectively inhibited proliferation, survival, and invasion of prostate cancer cells via blocking multiple oncogenic signaling cascades but not the Src pathway. Inhibition of Src expression by small hairpin RNA or inactivation of Src by dasatinib increased the CYT997-induced cytotoxicity of in vitro. Moreover, the combination of dasatinib and CYT997 exhibited a superior inhibitory effect on tumor growth and metastasis compared with either of the drugs alone. Our findings demonstrate that blockage of Src augments the anticancer effect of CYT997 on prostate cancer and suggest that co-treatment of dasatinib and CYT997 may represent an effective therapeutic regimen for limiting prostate cancer.

Comparison of the MUREX C. albicans, Albicans-Sure, and BactiCard Candida test kits with the germ tube test for presumptive identification of Candida albicans.

PubMed Central

Crist, A E; Dietz, T J; Kampschroer, K

1996-01-01

The MUREX C. albicans (MC)(Murex Diagnostics), Albicans-Sure (AS) (Clinical Standards Laboratories), and BactiCard Candida (BC) (Remel) test kits were compared with the germ tube (GT) test for the rapid, presumptive identification of Candida albicans. All three test kits detect the enzymes L-proline aminopeptidase and beta-galactosaminidase in yeast cells grown on culture media and are based on the principle that C. albicans produces both enzymes whereas other yeasts produce only one or neither of the enzymes. The organisms evaluated were fresh clinical isolates identified by methods routinely used in our laboratory (API 20C system and conventional methods) and included 303 C. albicans isolates, 153 Candida glabrata isolates, 70 Candida tropicalis isolates, 36 Candida parapsilosis isolates, 13 isolates of other Candida spp., 5 Cryptococcus neoformans isolates, and 3 Saccharomyces cerevisiae isolates. The MC, AS, BC, and GT tests detected 299 (98.7%), 300 (99.0%), 301 (99.3%), and 287 (94.7%) C. albicans isolates, respectively. There was one false-positive result with both the MC and BC kits and two false-positive results with the GT test. The enzymatic methods evaluated in this study provide rapid and accurate alternatives to the GT test for the presumptive identification of C. albicans. PMID:8880535

Effects of using multi-vide ruler kit in the acquisition of numeracy skills among PROTIM students

NASA Astrophysics Data System (ADS)

Arumugan, Hemalatha A./P.; Obeng, Sharifah Nasriah Wan; Talib, Corrienna Abdul; Bunyamin, Muhammad Abdul Hadi; Ali, Marlina; Ibrahim, Norhasniza; Zawadzki, Rainer

2017-08-01

One effective way to teach arithmetic more interestingly and make it easier to learn is through the use of instructional materials. These can help students master certain mathematical skills, particularly multiplication and division, often considered difficult amongst primary school pupils. Nevertheless, the insufficiency of appropriate instructional materials causes difficulty in understanding how to use the proper technique or apply the concept, especially in multiplication. With this in mind, this study investigated whether the innovative and creative instructional material designed to assist and enhance numeracy skills, namely the Multi-vide Ruler kit, could increase students' ability in solving multiplication and division questions and whether it affected their interest in solving numeracy problems. Participants in this study included ten PROTIM (Program Tiga M [Three M Program] - membaca [reading], menulis [writing] dan mengira [calculate]) students, 9-10 years old, who had difficulties in reading, writing and arithmetic. In order to get appropriate support for qualitative research, a pre and post-test containing ten basic mathematical operations, was implemented together with the Multi-vide Ruler Kit. The findings of the qualitative case study, with the pre and post-tests, showed significant differences in their achievement and interest in two-digit multiplication and division operations. The results suggest that this approach could improve PROTIM student's ability to solve basic mathematical operations. What was most encouraging was the increase in students' interest in solving numeracy problems.

Small-Scale Industries in Kenya: A Case Study of Economic Development. Toward a Better World Series, Learning Kit No. 3.

ERIC Educational Resources Information Center

Baldwin, Harriet; Ross-Larson, Bruce, Ed.

This World Bank (Washington, D.C.) kit is a case study designed to introduce secondary school social studies students to a project in Kenya established to strengthen small-scale industries. The kit contains a pamphlet, a booklet, a sound filmstrip, and a teacher's guide. The pamphlet, "Economic Summary: Kenya," points out the severe…

The Rajasthan Canal Project: A Case Study of Economic Development. Toward a Better World Series, Learning Kit No. 2.

ERIC Educational Resources Information Center

Baldwin, Harriet; Ross-Larson, Bruce, Ed.

This World Bank (Washington, D.C.) kit is designed to teach secondary school social studies students about the Rajasthan (India) Canal Project and the impact it has had on the state of Rajasthan and its population. The kit contains a pamphlet, a booklet, a sound filmstrip, and a teacher's guide. The pamphlet, "Economic Summary: India,"…

Method and platform standardization in MRM-based quantitative plasma proteomics.

PubMed

Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Jackson, Angela M; Domanski, Dominik; Burkhart, Julia; Sickmann, Albert; Borchers, Christoph H

2013-12-16

There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics. © 2013.

Characterization of CD34+ hematopoietic cells in systemic mastocytosis: Potential role in disease dissemination.

PubMed

Mayado, A; Teodosio, C; Dasilva-Freire, N; Jara-Acevedo, M; Garcia-Montero, A C; Álvarez-Twose, I; Sánchez-Muñoz, L; Matito, A; Caldas, C; Muñoz-González, J I; Henriques, A; Sánchez-Gallego, J I; Escribano, L; Orfao, A

2018-01-13

Recent studies show that most systemic mastocytosis (SM) patients, including indolent SM (ISM) with (ISMs+) and without skin lesions (ISMs-), carry the KIT D816V mutation in PB leukocytes. We investigated the potential association between the degree of involvement of BM hematopoiesis by the KIT D816V mutation and the distribution of different maturation-associated compartments of bone marrow (BM) and peripheral blood (PB) CD34 + hematopoietic precursors (HPC) in ISM and identified the specific PB cell compartments that carry this mutation. The distribution of different maturation-associated subsets of BM and PB CD34 + HPC from 64 newly diagnosed (KIT-mutated) ISM patients and 14 healthy controls was analyzed by flow cytometry. In 18 patients, distinct FACS-purified PB cell compartments were also investigated for the KIT mutation. ISM patients showed higher percentages of both BM and PB MC-committed CD34 + HPC vs controls, particularly among ISM cases with MC-restricted KIT mutation (ISM MC ); this was associated with progressive blockade of maturation of CD34 + HPC to the neutrophil lineage from ISM MC to multilineage KIT-mutated cases (ISM ML ). Regarding the frequency of KIT-mutated cases and cell populations in PB, variable patterns were observed, the percentage of KIT-mutated PB CD34 + HPC, eosinophils, neutrophils, monocytes and T cells increasing from ISMs- MC and ISMs+ MC to ISM ML patients. The presence of the KIT D816V mutation in PB of ISM patients is associated with (early) involvement of circulating CD34 + HPC and multiple myeloid cell subpopulations, KIT-mutated PB CD34 + HPC potentially contributing to early dissemination of the disease. © 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Ocular histiocytic sarcoma in a cat.

PubMed

Scurrell, Emma; Trott, Adele; Rozmanec, Michael; Belford, Chris J

2013-07-01

A 13-year-old male neutered British blue cat presented with uveitis, hyphema, and dyscoria in the right eye. Light microscopic examination revealed that the ciliary body, iris root, drainage angle, and adjacent choroid were infiltrated by sheets of large neoplastic mononuclear and multinucleate round to polygonal cells. Neoplastic cells stained immunopositive for CD18 and HLA-DR (MHC class II) and were immunonegative for CD3, CD79a, MUM-1, CD117 (c-Kit), and S100. These findings were consistent with a histiocytic sarcoma. The cat later developed multiple cutaneous masses composed of a similar neoplastic cell population to that seen in the eye. Eight months following enucleation, the cat developed respiratory distress and was euthanized. Postmortem examination revealed multiple pulmonary tumors associated with a pleural effusion. © 2013 American College of Veterinary Ophthalmologists.

Huntingtin Interacting Protein 1: a Merkel Cell Carcinoma Marker That Interacts with c-Kit

PubMed Central

Ames, Heather M.; Bichakjian, Christopher K.; Liu, Grace Y.; Oravecz-Wilson, Katherine I.; Fullen, Douglas R.; Verhaegen, Monique; Johnson, Timothy M.; Dlugosz, Andrzej A.; Ross, Theodora S.

2011-01-01

Merkel Cell Carcinoma (MCC) is a neoplasm thought to originate from the neuroendocrine Merkel cells of the skin. While the prevalence of MCC has been increasing, treatments for this disease remain limited due to a paucity of information regarding MCC biology. We have found that the endocytic oncoprotein Huntingtin interacting protein 1 (HIP1) is expressed at high levels in close to 90% of MCC tumors and serves as a more reliable histological cytoplasmic stain than the gold standard, cytokeratin 20 (CK20). Furthermore, high anti-HIP1 antibody reactivity in the sera of a cohort of MCC patients predicts the presence of metastases. Another protein that is frequently expressed at high levels in MCC tumors is the stem cell factor (SCF) receptor tyrosine kinase, c-Kit. In working towards an understanding of how HIP1 might contribute to MCC tumorigenesis, we have discovered that HIP1 interacts with SCF activated c-Kit. These data not only identify HIP1 as a molecular marker for management of MCC patients but also show that HIP1 interacts with and slows the degradation of c-Kit. PMID:21697888

Point of care diagnosis of multiple schistosome parasites: Species-specific DNA detection in urine by loop-mediated isothermal amplification (LAMP).

PubMed

Lodh, Nilanjan; Mikita, Kei; Bosompem, Kwabena M; Anyan, William K; Quartey, Joseph K; Otchere, Joseph; Shiff, Clive J

2017-09-01

Schistosomes are easily transmitted and high chance of repeat infection, so if control strategies based on targeted mass drug administration (MDA) are to succeed it is essential to have a test that is sensitive, accurate and simple to use. It is known and regularly demonstrated that praziquantel does not always eliminate an infection so in spite of the successes of control programs a residual of the reservoir survives to re-infect snails. The issue of diagnostic sensitivity becomes more critical in the assessment of program effectiveness. While serology, such as antigen capture tests might improve sensitivity, it has been shown that the presence of species-specific DNA fragments will indicate, most effectively, the presence of active parasites. Polymerase chain reaction (PCR) can amplify and detect DNA from urine residue captured on Whatman No. 3 filter paper that is dried after filtration. Previously we have detected S. mansoni and S. haematobium parasite-specific small repeat DNA fragment from filtered urine on filter paper by PCR. In the current study, we assessed the efficacy of detection of 86 urine samples for either or both schistosome parasites by PCR and loop-mediated isothermal amplification (LAMP) that were collected from a low to moderate transmission area in Ghana. Two different DNA extraction methods, standard extraction kit and field usable LAMP-PURE kit were also evaluated by PCR and LAMP amplification. With S. haematobium LAMP amplification for both extractions showed similar sensitivity and specificity when compared with PCR amplification (100%) verified by gel electrophoresis. For S. mansoni sensitivity was highest for LAMP amplification (100%) for standard extraction than PCR and LAMP with LAMP-PURE (99% and 94%). The LAMP-PURE extraction produced false negatives, which require further investigation for this field usable extraction kit. Overall high positive and negative predictive values (90% - 100%) for both species demonstrated a highly robust approach. The LAMP approach is close to point of care use and equally sensitive and specific to detection of species-specific DNA by PCR. LAMP can be an effective means to detect low intensity infection due to its simplicity and minimal DNA extraction requirement. This will enhance the effectiveness of surveillance and MDA control programs of schistosomiasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Current molecular profile of juvenile nasopharyngeal angiofibroma: First comprehensive study from India.

PubMed

Pandey, Praveen; Mishra, Anupam; Tripathi, Ashoak Mani; Verma, Veerendra; Trivedi, Ritu; Singh, Hitendra Prakash; Kumar, Sunil; Patel, Brijesh; Singh, Vinay; Pandey, Shivani; Pandey, Amita; Mishra, Subhash Chandra

2017-03-01

An attempt is made to analyze the molecular behavior of juvenile nasopharyngeal angiofibroma (JNA). Case Series METHODS: Quantification of mRNAs expression was undertaken through real-time polymerase chain reaction in JNA (9-24) samples for VEGF-A, basic fibroblast growth factor (b-FGF), platelet-derived growth factor PDGF-A, KIT proto-oncogene receptor tyrosine kinase (c-Kit), Avian myelomatosis viral oncogene homolog (c-Myc), Harvey rat sarcoma viral oncogene homolog (H-Ras), tumor suppressor gene TP53, and androgen receptor and interleukin 6 (IL-6). The β-catenin expression was evaluated by western blot in 16 samples. Nasal polyp was taken as control. A significantly increased (P < 0.01) expression of c-myc, VEGFA, bFGF, PDGFA, c-kit, and TP53 was seen, along with enhanced expression of β-catenin. A massive enhancement of H-Ras expression was seen for the first time. Androgen receptor expression was no different, whereas IL-6 despite showing upregulation trend was not significant. The enhanced expressions of various markers suggest their potential role in JNA. Although the biological significance of c-kit, c-myc, and one of the novel markers H-Ras has yet to be defined, their significant expression may have a therapeutic importance. NA. Laryngoscope, 127:E100-E106, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Protective Effect of Combined Caffeic Acid Phenethyl Ester and Bevacizumab Against Hydrogen Peroxide-Induced Oxidative Stress in Human RPE Cells.

PubMed

Dinc, Erdem; Ayaz, Lokman; Kurt, Akif Hakan

2017-12-01

This study aimed to evaluate the protective effects of caffeic acid phenethyl ester (CAPE) and combined CAPE-bevacizumab against oxidative stress induced by hydrogen peroxide (H 2 O 2 ) in human retinal pigment epithelium. ARPE-19 cells were pretreated with 5, 10, and 30 μM CAPE alone and in combination with bevacizumab for 3 h, then exposed to H 2 O 2 for 16 h. Cell viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Vascular endothelial growth factor (VEGF) protein levels in the medium were measured using a human VEGF ELISA kit. Total antioxidant status (TAS) and total oxidant status (TOS) were measured in ARPE-19 cells using the test kit from Rel Assay. Expression levels of VEGF, Bax, Bcl-2, cytochrome c, apoptotic protease activating factor-1 (apaf-1), and caspase-3 were determined using reverse transcription polymerase chain reaction. Pretreatment of ARPE-19 cells with 30 μM CAPE and combined CAPE-bevacizumab reduced H 2 O 2 mediated cell death. H 2 O 2 -induced oxidative stress increased TOS and VEGF production, which was significantly inhibited by CAPE and the CAPE-bevacizumab combination. VEGF, Bax, cytochrome c, apaf-1, and caspase-3 gene expressions were significantly decreased in cells pretreated with 5, 10, and 30 μM CAPE and combined CAPE-bevacizumab compared to the H 2 O 2 group. In addition, Bcl-2 expression was significantly increased in both the CAPE and CAPE-bevacizumab combination groups compared to the H 2 O 2 group. CAPE has a protective effect on ARPE-19 cells against oxidative stress, and VEGF protein level and expression can be decreased by incubation with different concentrations of CAPE. These results demonstrate that CAPE suppresses the mitochondria-mediated apoptosis in ARPE-19 cells under oxidative stress. In addition, the use of CAPE in combination with bevacizumab has an additive effect.

Decrease of cocaine, but not heroin, self-administration and relapse by the tyrosine kinase inhibitor masitinib in male Sprague Dawley rats.

PubMed

Belin-Rauscent, A; Lacoste, J; Hermine, O; Moussy, A; Everitt, B J; Belin, David

2018-05-01

Accumulating evidence shows that cocaine, and also heroin, influence several tyrosine kinases, expressed in neurons and in non-neuronal populations such as microglia, astrocytes and mast-cells. Drug-induced activation of mast cells both triggers inflammatory processes in the brain mediated by the glial cells they activate, and facilitates histamine release which may directly influence the dopamine system. Thus, by triggering the activation and degranulation of mast cells dependent on the tyrosine kinase c-kit and Fyn, the latter being also involved in NMDA-dependent synaptic plasticity, cocaine and heroin may indirectly influence the neural mechanisms that mediate their reinforcing properties. Masitinib, a novel tyrosine kinase inhibitor with high selectivity for c-Kit, Fyn and Lyn, may alter the aberrant consequences of the activation of these tyrosine kinases by cocaine and heroin. We investigated in rats the effect of a chronic oral treatment with masitinib (20 mg/kg) on the reinforcing and motivational properties of self-administered cocaine (250 μg/infusion) and heroin (40 μg/infusion). Three different cohorts of rats were trained instrumentally to respond for cocaine, heroin or food under continuous reinforcement. In each group, we assessed the influence of chronic daily treatment with masitinib on the maintenance of instrumental responding and intake and the motivation for the reinforcer. Thus, masitinib and vehicle-treated rats were challenged to adapt to high behavioural demand, to respond under a progressive ratio schedule of reinforcement and to reinstate instrumental responding after extinction and/or abstinence. Masitinib selectively decreased cocaine intake, the motivation for cocaine and the subsequent propensity to respond for cocaine under extinction, while having no effect on instrumental responding for heroin or food. The present findings suggest masitinib, a drug with proven efficacy in CNS disorders, could represent a novel treatment for cocaine addiction provided its influence on the reinforcing and incentive properties of the drug is confirmed.

Tackling Poverty in Rural Mexico: A Case Study of Economic Development. Toward a Better World Series, Learning Kit No. 4.

ERIC Educational Resources Information Center

Baldwin, Harriet; Ross-Larson, Bruce, Ed.

This World Bank (Washington, D.C.) kit is a case study designed to teach secondary school social studies students about an integrated rural development project in Mexico, and how it is helping to raise the standard of living for six million Mexicans in 131 microregions throughout Mexico. The kit contains a pamphlet, a booklet, a sound filmstrip,…

Stanniocalcin-1 Protects a Mouse Model from Renal Ischemia-Reperfusion Injury by Affecting ROS-Mediated Multiple Signaling Pathways.

PubMed

Liu, Dajun; Shang, Huiping; Liu, Ying

2016-07-12

Stanniocalcin-1 (STC-1) protects against renal ischemia-reperfusion injury (RIRI). However, the molecular mechanisms remain widely unknown. STC-1 inhibits reactive oxygen species (ROS), whereas most ROS-mediated pathways are associated with ischemic injury. Therefore, to explore the mechanism, the effects of STC-1 on ROS-medicated pathways were studied. Non-traumatic vascular clamps were used to establish RIRI mouse models. The serum levels of STC-1, interleukin-6 (IL-6), interferon (IFN) γ, P53, and capase-3 were measured by ELISA kits. Superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by fluorescence spectrofluorometer. All these molecules changed significantly in a RIRI model mouse when compared with those in a sham control. Kidney cells were isolated from sham and model mice. STC-1 was overexpressed or knockout in these kidney cells. The molecules in ROS-medicated pathways were measured by real-time quantitative PCR and Western blot. The results showed that STC-1 is an effective ROS scavenger. The serum levels of STC-1, MDA and SOD activity were increased while the serum levels of IL-6, iIFN-γ, P53, and capase-3 were decreased in a model group when compared with a sham control (p < 0.05). Furthermore, the levels of STC-1,p53, phosphorylated mitogen-activated protein kinase kinase (p-MEKK-1), c-Jun N-terminal kinase (p-JNK), extracellular signal-regulated kinase (p-ERK), IkB kinase (p-IKK), nuclear factor (NF) κB, apoptosis signal-regulating kinase 1 (ASK-1) and caspase-3 changed significantly in kidney cells isolated from a RIRI model when compared to those isolated from a sham control (p < 0.05). Meanwhile, STC-1 overexpression or silence caused significant changes of the levels of these ROS-mediated molecules. Therefore, STC-1 maybe improve anti-inflammation, anti-oxidant and anti-apoptosis activities by affecting ROS-mediated pathways, especially the phospho-modifications of the respective proteins, resulting in the increase of SOD and reduce of capase-3, p53, IL-6 and IFN-γ.

A silent allele in the locus D5S818 contained within the PowerPlex®21 PCR Amplification Kit.

PubMed

Chen, Ling; Tai, Yunchun; Qiu, Pingming; Du, Weian; Liu, Chao

2015-11-01

Three paternity tests cases were found with a single locus mismatch at the locus D5S818 with PowerPlex®21 PCR Amplification Kit (Promega). Forward and reverse primers were redesigned to type the samples again and to evaluate if there were alleles dropped out. The results showed the existence of a silent allele 12 in all the three families, due to a point mutation that changed cytosine to adenine at 90 nucleotides upstream from the 5' end of the AGAT repeat sequences in all the six individuals. A single locus mismatch due to a silent allele may occur in any locus using any kit. Therefore, we recommend using multiple kits to confirm the results in paternity testing cases with mismatches, especially when there is a single locus mismatch with homozygote involved. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Differential effects of c-fms and c-kit ligands on the lineage development of the lymphohematopoietic cell line EML C1.

PubMed

Tsai, S

1996-01-01

The lymphohematopoietic progenitors represent < 0.01% of nucleated marrow cells. We have shown that murine lymphohematopoietic progenitors can be immortalized by a recombinant retroviral vector harboring a dominant-negative retinoic acid (RA) receptor. The immortalized progenitors proliferate as a stem-cell factor-dependent clonal line designated EML C1. The EML C1 cell line spontaneously generates prepro-B-lymphocytes and erythroid and myeloid progenitors. Upon stimulation with interleukin 7 and marrow stromal cells, the prepro-B-lymphocytes express recombination-activating gene 1 (RAG-1) and undergo D-J rearrangements of the immunoglobulin heavy-chain genes. With erythropoietin, the erythroid progenitors proliferate and differentiate into red cells. Generation of the common progenitors for neutrophils and macrophages [colony-forming units-granulocyte-macrophage (CFU-GM)] is suppressed in EML C1 cells but is inducible by high concentrations of RA. An additional block in neutrophil differentiation occurs at the promyelocyte stage, but this can also be overcome by high concentrations of RA. Although c-fms is homologous to c-kit, which encodes the receptor for stem-cell factor (SCF), EML C1 cells neither express c-fms nor respond to macrophage colony-stimulating factor (M-CSF), the ligand for c-fms. Transduction and expression of c-fms cDNA in EML C1 cells confers responsiveness to M-CSF. This finding indicates that c-kit and c-fms share substantially overlapping signal-transduction pathways. However, c-fms-transduced EML C1 cells (EML C1/c-fms cells) exhibit different development patterns when stimulated by SCF alone or by M-CSF alone. When stimulated by SCF alone, EML C1/c-fms cells show mostly erythroid and B-lymphoid development. When stimulated by M-CSF alone, development switches to mostly myeloid (neutrophil and macrophage) development. This observation suggests that c-kit and c-fms must have unique signal-transduction pathways in addition to the common ones.

Development and Evaluation of User-Friendly Single Vial DOTA-Peptide Kit Formulations, Specifically Designed for Radiolabelling with 68Ga from a Tin Dioxide 68Ge/68Ga Generator.

PubMed

Prince, Deidré; Rossouw, Daniel; Davids, Claudia; Rubow, Sietske

2017-12-01

This study was aimed to develop single vial 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-peptide kits to be used with fractionated eluates from a SnO 2 -based 68 Ge/ 68 Ga generator. Kits were formulated with 35 μg DOTA-Tyr 3 -Thre 8 -octreotide, DOTA-[Tyr 3 ]-octreotide and DOTA-[NaI 3 ]-octreotide (DOTATATE, DOTATOC and DOTANOC) and sodium acetate powder, vacuum-dried and stored at -20 °C for up to 12 months. Labelling of the kits was carried out with 2 ml 68 Ga eluate. Comparative labelling was carried out using aqueous DOTA-peptide stock solutions kept frozen at -20 °C for up to 12 months. The quality of the kits was found to be suitable over a 1-year storage period (pH, sterility, endotoxin content, radiolabelling efficiency and radiochemical yields of 68 Ga-labelled DOTA-peptides). Radiochemical yields ranged from 73 to 83 %, while those obtained from stock solutions from 64 to 79 %. No significant decline in kit labelling yields was observed over a 12-month storage period. The single vial kit formulations met the quality release specifications for human administration and appear to be highly advantageous over using peptide stock solutions in terms of stability and user-friendliness.

Loop mediated isothermal amplification (LAMP) accurately detects malaria DNA from filter paper blood samples of low density parasitaemias.

PubMed

Aydin-Schmidt, Berit; Xu, Weiping; González, Iveth J; Polley, Spencer D; Bell, David; Shakely, Delér; Msellem, Mwinyi I; Björkman, Anders; Mårtensson, Andreas

2014-01-01

Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots. Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6-782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94-99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0-4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1-98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2-99.9) and 76.9% (95%CI 46.2-95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1-100) in both study groups. Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low-density malaria infections in pre-elimination settings.

Loop Mediated Isothermal Amplification (LAMP) Accurately Detects Malaria DNA from Filter Paper Blood Samples of Low Density Parasitaemias

PubMed Central

González, Iveth J.; Polley, Spencer D.; Bell, David; Shakely, Delér; Msellem, Mwinyi I.; Björkman, Anders; Mårtensson, Andreas

2014-01-01

Background Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots. Methods and Findings Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6–782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94–99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0–4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1–98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2–99.9) and 76.9% (95%CI 46.2–95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1–100) in both study groups. Conclusion Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low-density malaria infections in pre-elimination settings. PMID:25105591

KIT Mutations Are Common in Testicular Seminomas

PubMed Central

Kemmer, Kathleen; Corless, Christopher L.; Fletcher, Jonathan A.; McGreevey, Laura; Haley, Andrea; Griffith, Diana; Cummings, Oscar W.; Wait, Cecily; Town, Ajia; Heinrich, Michael C.

2004-01-01

Expression of KIT tyrosine kinase is critical for normal germ cell development and is observed in the majority of seminomas. Activating mutations in KIT are common in gastrointestinal stromal tumors and mastocytosis. In this study we examined the frequency and spectrum of KIT mutations in 54 testicular seminomas, 1 ovarian dysgerminoma and 37 non-seminomatous germ cell tumors (NSGCT). Fourteen seminomas (25.9%) contained exon 17 point mutations including D816V (6 cases), D816H (3 cases), Y823D (2 cases), and single examples of Y823C, N822K, and T801I. No KIT mutations were found in the ovarian dysgerminoma or the NSGCTs. In transient transfection assays, mutant isoforms D816V, D816H, Y823D, and N822K were constitutively phosphorylated in the absence of the natural ligand for KIT, stem cell factor (SCF). In contrast, activation of T801I and wild-type KIT required SCF. Mutants N822K and Y823D were inhibited by imatinib mesylate (Gleevec, previously STI571) whereas D816V and D816H were both resistant to imatinib mesylate. Biochemical evidence of KIT activation, as assessed by KIT phosphorylation and KIT association with phosphatidylinositol (PI) 3-kinase in tumor cell lysates, was largely confined to seminomas with a genomic KIT mutation. These findings suggest that activating KIT mutations may contribute to tumorigenesis in a subset of seminomas, but are not involved in NSGCT. PMID:14695343

Making an Adjustable C-Clamp. Kit No. 603. Instructor's Manual [and] Student Learning Activity Manual. [Revised.] T & I--Metalwork.

ERIC Educational Resources Information Center

White, Jim; Alexander, Larry

This student activity kit consists of a programmed, self-instructional learning guide and an accompanying instructor's manual for use in teaching trade and industrial education students how to make an adjustable C-clamp. The student guide contains step-by-step instructions in the following areas: basic layout principles; use of a hack saw, file,…

Validation of an improved anaplasma antibody cELISA kit for detection of anaplasma ovis antibody in domestic sheep at the U.S. Sheep Experiment Station in Dubois, ID

USDA-ARS?s Scientific Manuscript database

An accurate and simple-to-perform new version of a competitive ELISA (cELISA) kit that became commercially available in 2015 for testing of cattle for antibody to Anaplasma marginale was validated for detection of Anaplasma ovis antibody in domestic sheep. True positives and negatives were identifie...

Antiproliferation effect of imatinib mesylate on MCF7, T-47D tumorigenic and MCF 10A nontumorigenic breast cell lines via PDGFR-β, PDGF-BB, c-Kit and SCF genes

PubMed Central

Kadivar, Ali; Kamalidehghan, Behnam; Akbari Javar, Hamid; Karimi, Benyamin; Sedghi, Reihaneh; Noordin, Mohamed Ibrahim

2017-01-01

Recent cancer molecular therapies are targeting main functional molecules to control applicable process of cancer cells. Attractive targets are established by receptor tyrosine kinases, such as platelet-derived growth factor receptors (PDGFRs) and c-Kit as mostly irregular signaling, which is due to either over expression or mutation that is associated with tumorigenesis and cell proliferation. Imatinib mesylate is a selective inhibitor of receptor tyrosine kinase, including PDGFR-β and c-Kit. In this research, we studied how imatinib mesylate would exert effect on MCF7 and T-47D breast cancer and MCF 10A epithelial cell lines, the gene and protein expression of PDGFR-β, c-Kit and their relevant ligands platelet-derived growth factor (PDGF)-BB and stem cell factor (SCF). The MTS assay was conducted in therapeutic relevant concentration of 2–10 µM for 96, 120 and 144 h treatment. In addition, apoptosis induction and cytostatic activity of imatinib mesylate were investigated with the terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL and cell cycle assays, respectively, in a time-dependent manner. Comparative real-time PCR and Western blot analysis were conducted to evaluate the expression and regulation of imatinib target genes and proteins. Our finding revealed that imatinib mesylate antiproliferation effect, apoptosis induction and cytostatic activity were significantly higher in breast cancer cell lines compared to MCF 10A. This effect might be due to the expression of PDGFR-β, PDGF-BB, c-Kit and SCF, which was expressed by all examined cell lines, except the T-47D cell line which was not expressed c-Kit. However, examined gene and proteins expressed more in cancer cell lines. Therefore, imatinib mesylate was more effective on them. It is concluded that imatinib has at least two potential targets in both examined breast cancer cell lines and can be a promising drug for targeted therapy to treat breast cancer. PMID:28260860

Promotion of Cancer Stem-Like Cell Properties in Hepatitis C Virus-Infected Hepatocytes

PubMed Central

Kwon, Young-Chan; Bose, Sandip K.; Steele, Robert; Meyer, Keith; Di Bisceglie, Adrian M.; Ray, Ratna B.

2015-01-01

ABSTRACT We have previously reported that hepatitis C virus (HCV) infection of primary human hepatocytes (PHH) induces the epithelial mesenchymal transition (EMT) state and extends hepatocyte life span (S. K. Bose, K. Meyer, A. M. Di Bisceglie, R. B. Ray, and R. Ray, J Virol 86:13621–13628, 2012, http://dx.doi.org/10.1128/JVI.02016-12). These hepatocytes displayed sphere formation on ultralow binding plates and survived for more than 12 weeks. The sphere-forming hepatocytes expressed a number of cancer stem-like cell (CSC) markers, including high levels of the stem cell factor receptor c-Kit. The c-Kit receptor is regarded as one of the CSC markers in hepatocellular carcinoma (HCC). Analysis of c-Kit mRNA displayed a significant increase in the liver biopsy specimens of chronically HCV-infected patients. We also found c-Kit is highly expressed in transformed human hepatocytes (THH) infected in vitro with cell culture-grown HCV genotype 2a. Further studies suggested that HCV core protein significantly upregulates c-Kit expression at the transcriptional level. HCV infection of THH led to a significant increase in the number of spheres displayed on ultralow binding plates and in enhanced EMT and CSC markers and tumor growth in immunodeficient mice. The use of imatinib or dasatinib as a c-Kit inhibitor reduced the level of sphere-forming cells in culture. The sphere-forming cells were sensitive to treatment with sorafenib, a multikinase inhibitor, that is used for HCC treatment. Further, stattic, an inhibitor of the Stat3 molecule, induced sphere-forming cell death. A combination of sorafenib and stattic had a significantly stronger effect, leading to cell death. These results suggested that HCV infection potentiates CSC generation, and selected drugs can be targeted to efficiently inhibit cell growth. IMPORTANCE HCV infection may develop into HCC as an end-stage liver disease. We focused on understanding the mechanism for the risk of HCC from chronic HCV infection and identified targets for treatment. HCV-infected primary and transformed human hepatocytes (PHH or THH) generated CSC. HCV-induced spheres were highly sensitive to cell death from sorafenib and stattic treatment. Thus, our study is highly significant for HCV-associated HCC, with the potential for developing a target-specific strategy for improved therapies. PMID:26355082

From preclinical development to clinical application: Kit formulation for radiolabelling the minigastrin analogue CP04 with In-111 for a first-in-human clinical trial.

PubMed

Pawlak, Dariusz; Rangger, Christine; Kolenc Peitl, Petra; Garnuszek, Piotr; Maurin, Michał; Ihli, Laura; Kroselj, Marko; Maina, Theodosia; Maecke, Helmut; Erba, Paola; Kremser, Leopold; Hubalewska-Dydejczyk, Alicja; Mikołajczak, Renata; Decristoforo, Clemens

2016-03-31

A variety of radiolabelled minigastrin analogues targeting the cholecystokinin 2 (CCK2) receptor were developed and compared in a concerted preclinical testing to select the most promising radiotracer for diagnosis and treatment of medullary thyroid carcinoma (MTC). DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (CP04) after labelling with (111)In displayed excellent characteristics, such as high stability, receptor affinity, specific and persistent tumour uptake and low kidney retention in animal models. Therefore, it was selected for further clinical evaluation within the ERA-NET project GRAN-T-MTC. Here we report on the development of a pharmaceutical freeze-dried formulation of the precursor CP04 for a first multi-centre clinical trial with (111)In-CP04 in MTC patients. The kit formulation was optimised by adjustment of buffer, additives and radiolabelling conditions. Three clinical grade batches of a final kit formulation with two different amounts of peptide (10 or 50 μg) were prepared and radiolabelled with (111)In. Quality control and stability assays of both the kits and the resulting radiolabelled compound were performed by HPLC analysis. Use of ascorbic acid buffer (pH4.5) allowed freeze-drying of the kit formulation with satisfactory pellet-formation. Addition of methionine and gentisic acid as well as careful selection of radiolabelling temperature was required to avoid extensive oxidation of the Met(11)-residue. Trace metal contamination, in particular Zn, was found to be a major challenge during the pharmaceutical filling process in particular for the 10 μg formulation. The final formulations contained 10 or 50 μg CP04, 25mg ascorbic acid, 0.5mg gentisic acid and 5mg L-methionine. The radiolabelling performed by incubation of 200-250 MBq (111)InCl3 at 90 °C for 15 min resulted in reproducible radiochemical purity (RCP) >94%. Kit-stability was proven for >6 months at +5 °C and at +25 °C. The radiolabelled product was stable for >4h at +25 °C. A kit formulation to prepare (111)In-CP04 for clinical application was developed, showing high stability of the kit as well as high RCP of the final product. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

EVALUATION OF A DNA PROBE TEST KIT FOR DETECTION OF SALMONELLAE IN BIOSOLIDS

EPA Science Inventory

Aims: Current United States regulations (40 CFR 503) for "Class A" biosolids requires use of multiple-tube fermentation techniques for fecal coliform or multiple tube enrichment techniques for Salmonella spp. followed by isolation and biochemical and serological confirmation. T...

Improving Indonesia's Cities: A Case Study of Economic Development, Including a Teaching Guide and An Economic Summary of Indonesia. Toward a Better World Series, Learning Kit No. 5.

ERIC Educational Resources Information Center

Baldwin, Harriet, Ed.; Rosen, Carol, Ed.

This World Bank (Washington, D.C.) kit is designed to teach secondary school social studies students the impact of rapid urbanization on Jakarta, the capital city of Indonesia. The kit contains a pamphlet, a booklet, a filmstrip, and a teacher's guide. The pamphlet, "An Economic Summary of Indonesia" provides students with the structure,…

An In Silico Model of Endotoxic Shock Mediators (Briefing Charts)

DTIC Science & Technology

2012-03-12

complex probably because receptor activates multiple signaling pathways choline P O acetyl...calcium mobilization phospholipase C phospholipase D phospholipase A2 eicosanoids inositol metabolism protein kinase C COOH NH2 PAF

Primary Sinonasal Malignant Melanoma: Effect of Clinical and Histopathologic Prognostic Factors on Survival.

PubMed

Göde, Sercan; Turhal, Göksel; Tarhan, Ceyda; Yaman, Banu; Kandiloğlu, Gülşen; Öztürk, Kerem; Kaya, İsa; Midilli, Raşit; Karcı, Bülent

2017-05-05

Mucosal melanoma is a rare malignancy arising from melanocytes of the mucosal surfaces. The pattern and frequency of oncogenic mutations and histopathological biomarkers have a role on distinct tumour behaviour and survival. To assess the rate of C-KIT positivity and its effect on survival of surgically treated sinonasal malignant melanoma patients with other histopathological biomarkers and clinical features. Retrospective cross-sectional study. Seventeen sinonasal malignant melanoma patients with a mean age of 65.41 (39-86) years were included. Overall survival and disease-specific survival rates were calculated. The impact of age, gender, stage and extent of the disease, type of surgery, and adjuvant therapies were also taken into consideration. The effect of mitotic index, pigmentation, S100, HMB-45, Melan-A and C-KIT on survival were evaluated. Median tumour size was 20 mm (interquartile range=27.5 mm). Pigmentation was present in 7 (41.2%) cases. Median number of mitoses per millimetre squared was 11 (interquartile range=13). Melan A was positive in 7 (41.2%) patients, ulceration was present in 6 cases (35.3%), and necrosis was present in (47.1%) 8 cases. Six patients (35.3%) were positive for S100, 14 (82.4%) specimens stained positive for HMB-45 and C-KIT (CD117) was positive in 9 cases (52.9%). Three patients (16.7%) developed distant metastasis. Five year overall and disease free survival rates were 61.4% and 43.8%, respectively. Although C-KIT positive sinonasal malignant melanoma patients (52.9%) can be candidates for targeted tumour therapies, the studied clinical or histopathological features along with C-KIT seem to have no significant effect on survival in a small group of patients with sinonasal malignant melanoma.

Characterization and functionality of cardiac progenitor cells in congenital heart patients.

PubMed

Mishra, Rachana; Vijayan, Kalpana; Colletti, Evan J; Harrington, Daniel A; Matthiesen, Thomas S; Simpson, David; Goh, Saik Kia; Walker, Brandon L; Almeida-Porada, Graça; Wang, Deli; Backer, Carl L; Dudley, Samuel C; Wold, Loren E; Kaushal, Sunjay

2011-02-01

Human cardiac progenitor cells (hCPCs) may promote myocardial regeneration in adult ischemic myocardium. The regenerative capacity of hCPCs in young patients with nonischemic congenital heart defects for potential use in congenital heart defect repair warrants exploration. Human right atrial specimens were obtained during routine congenital cardiac surgery across 3 groups: neonates (age, <30 days), infants (age, 1 month to 2 years), and children (age, >2 to ≤13 years). C-kit(+) hCPCs were 3-fold higher in neonates than in children >2 years of age. hCPC proliferation was greatest during the neonatal period as evidenced by c-kit(+) Ki67(+) expression but decreased with age. hCPC differentiation capacity was also greatest in neonatal right atrium as evidenced by c-kit(+), NKX2-5(+), NOTCH1(+), and NUMB(+) expression. Despite the age-dependent decline in resident hCPCs, we isolated and expanded right atrium-derived CPCs from all patients (n=103) across all ages and diagnoses using the cardiosphere method. Intact cardiospheres contained a mix of heart-derived cell subpopulations that included cardiac progenitor cells expressing c-kit(+), Islet-1, and supporting cells. The number of c-kit(+)-expressing cells was highest in human cardiosphere-derived cells (hCDCs) grown from neonatal and infant right atrium. Furthermore, hCDCs could differentiate into diverse cardiovascular lineages by in vitro differentiation assays. Transplanted hCDCs promoted greater myocardial regeneration and functional improvement in infarcted myocardium than transplanted cardiac fibroblasts. Resident hCPCs are most abundant in the neonatal period and rapidly decrease over time. hCDCs can be reproducibly isolated and expanded from young human myocardial samples regardless of age or diagnosis. hCPCs are functional and have potential in congenital cardiac repair.

In-frame Val216-Ser217 deletion of KIT in mild piebaldism causes aberrant secretion and SCF response.

PubMed

Hattori, Mai; Ishikawa, Osamu; Oikawa, Daisuke; Amano, Hiroo; Yasuda, Masahito; Kaira, Kyoichi; Ishida-Yamamoto, Akemi; Nakano, Hajime; Sawamura, Daisuke; Terawaki, Shin-Ichi; Wakamatsu, Kaori; Tokunaga, Fuminori; Shimizu, Akira

2018-03-21

Piebaldism is a pigmentary disorder characterized by a white forelock and depigmented patches. Although the loss-of-function mutations in the KIT gene underlie the disease, the intracellular dynamics of the mutant KIT are largely unknown. We herein report a Japanese family with piebaldism in which the affected members showed a mild phenotype. The objective of this study is to investigate the functions and intracellular dynamics of the mutant KIT protein. We performed genetic analyses of the KIT gene using peripheral blood cells. We analyzed the intracellular localization of the mutant KIT protein in HEK293T cells transfected with wild-type (Wt) and/or mutant KIT genes. Immunoprecipitation analyses, immunoblotting and immunofluorescence studies were performed using antibodies against KIT and downstream signaling proteins. Glycosidase digestion analysis was performed to clarify the intracellular localization of KIT protein. A genetic analysis revealed a novel heterozygous mutation c.645_650delTGTGTC which results in the in-frame deletion of Val 216 and Ser 217 in the extracellular domain of KIT. Immunoprecipitation analyses confirmed that the wild and mutant KIT formed a heterodimer after treatment with stem cell factor (SCF); however, the phosphorylation of the downstream signaling factors was decreased. In an immunofluorescence study, the mutant KIT accumulated predominantly in the endoplasmic reticulum (ER) and was sparsely expressed on the cell surface. A glycosidase digestion study revealed that the mutant KIT is predominantly localized in the ER. These data reveal an aberrant function and intracellular localization of mutant KIT protein in piebaldism. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Mid-temperature deep removal of hydrogen sulfide on rare earth (RE = Ce, La, Sm, Gd) doped ZnO supported on KIT-6: Effect of RE dopants and interaction between active phase and support matrix

NASA Astrophysics Data System (ADS)

Li, Lu; Zhou, Pin; Zhang, Hongbo; Meng, Xianglong; Li, Juexiu; Sun, Tonghua

2017-06-01

Rare earth oxides (RE = Ce, La, Sm and Gd) doped ZnO supported on KIT-6 sorbents (RE-ZnO/KIT-6) were synthesized by sol-gel method and their performance for deep removal of H2S (bellow 0.1 ppmv) from gas stream at medium temperature was tested. The RE dopants (except Ce) significantly enhance the deep desulfurization capacity of ZnO/KIT-6 sorbent and maintained higher sulfur uptake capacities upon multiple cycles of regeneration by a simple thermal oxidation in 10 v% of O2 in N2 atmosphere. The results of SAXS, XRD, N2 physisorption, TEM, FIIR, and XPS implied that the KIT-6 structure of loading metal oxides remained intact. It was found that RE could hinder the ZnO crystal ripening during calcination resulted in smaller ZnO particles, enhance the interaction of ZnO and silica matrix to improve the dispersion of active phase on KIT-6. Furthermore, by increasing the outlayer electron density of Zn atom and oxygen transfer ability, the synergistic effect considered to be favorable for RE-ZnO/KIT-6 sulfidation. Even though the performance of improving ZnO dispersion was weaker than that of Sm and Gd, La-ZnO/KIT-6 performs the best deep desulfurizers by changing the surface chemical atmosphere for ZnO. Steam in the gas stream inhibited the capture of H2S by ZnO in the sorbents, in the case of La-ZnO/KIT-6, the steam content should control as lower as 5 v% to ensure the desulfurization efficiency and precision.

Clinical, biological, and molecular characteristics of clonal mast cell disorders presenting with systemic mast cell activation symptoms.

PubMed

Alvarez-Twose, Iván; González de Olano, David; Sánchez-Muñoz, Laura; Matito, Almudena; Esteban-López, Maria I; Vega, Arantza; Mateo, Maria Belén; Alonso Díaz de Durana, Maria D; de la Hoz, Belén; Del Pozo Gil, Maria D; Caballero, Teresa; Rosado, Ana; Sánchez Matas, Isabel; Teodósio, Cristina; Jara-Acevedo, María; Mollejo, Manuela; García-Montero, Andrés; Orfao, Alberto; Escribano, Luis

2010-06-01

Systemic mast cell activation disorders (MCADs) are characterized by severe and systemic mast cell (MC) mediators-related symptoms frequently associated with increased serum baseline tryptase (sBt). To analyze the clinical, biological, and molecular characteristics of adult patients presenting with systemic MC activation symptoms/anaphylaxis in the absence of skin mastocytosis who showed clonal (c) versus nonclonal (nc) MCs and to provide indication criteria for bone marrow (BM) studies. Eighty-three patients were studied. Patients showing clonal BM MCs were grouped into indolent systemic mastocytosis without skin lesions (ISMs(-); n = 48) and other c-MCADs (n = 3)-both with CD25(++) BM MCs and either positive mast/stem cell growth factor receptor gene (KIT) mutation or clonal human androgen receptor assay (HUMARA) tests-and nc-MCAD (CD25-negative BM MCs in the absence of KIT mutation; n = 32) and compared for their clinical, biological, and molecular characteristics. Most clonal patients (48/51; 94%) met the World Health Organization criteria for systemic mastocytosis and were classified as ISMs(-), whereas the other 3 c-MCAD and all nc-MCAD patients did not. In addition, although both patients with ISMs(-) and patients with nc-MCAD presented with idiopathic and allergen-induced anaphylaxis, the former showed a higher frequency of men, cardiovascular symptoms, and insect bite as a trigger, together with greater sBt. Based on a multivariate analysis, a highly efficient model to predict clonality before BM sampling was built that includes male sex (P = .01), presyncopal and/or syncopal episodes (P = .009) in the absence of urticaria and angioedema (P = .003), and sBt >25 microg/L (P = .006) as independent predictive factors. Patients with c-MCAD and ISMs(-) display unique clinical and laboratory features different from nc-MCAD patients. A significant percentage of c-MCAD patients can be considered as true ISMs(-) diagnosed at early phases of the disease. Copyright (c) 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Effect of processing on recovery and variability associated with immunochemical analytical methods for multiple allergens in a single matrix: sugar cookies.

PubMed

Khuda, Sefat; Slate, Andrew; Pereira, Marion; Al-Taher, Fadwa; Jackson, Lauren; Diaz-Amigo, Carmen; Bigley, Elmer C; Whitaker, Thomas; Williams, Kristina M

2012-05-02

Among the major food allergies, peanut, egg, and milk are the most common. The immunochemical detection of food allergens depends on various factors, such as the food matrix and processing method, which can affect allergen conformation and extractability. This study aimed to (1) develop matrix-specific incurred reference materials for allergen testing, (2) determine whether multiple allergens in the same model food can be simultaneously detected, and (3) establish the effect of processing on reference material stability and allergen detection. Defatted peanut flour, whole egg powder, and spray-dried milk were added to cookie dough at seven incurred levels before baking. Allergens were measured using five commercial enzyme-linked immunosorbent assay (ELISA) kits. All kits showed decreased recovery of all allergens after baking. Analytical coefficients of variation for most kits increased with baking time, but decreased with incurred allergen level. Thus, food processing negatively affects the recovery and variability of peanut, egg, and milk detection in a sugar cookie matrix when using immunochemical methods.

Association of Piebaldism, multiple café-au-lait macules, and intertriginous freckling: clinical evidence of a common pathway between KIT and sprouty-related, ena/vasodilator-stimulated phosphoprotein homology-1 domain containing protein 1 (SPRED1).

PubMed

Chiu, Yvonne E; Dugan, Stefanie; Basel, Donald; Siegel, Dawn H

2013-01-01

Piebaldism is a rare genodermatosis caused by KIT mutations. We report the case of a 5-year-old boy who had the white forelock and leukoderma of piebaldism, but the presence of many café-au-lait macules and axillary and inguinal freckling complicated the diagnosis. Patients with similar cutaneous findings have been previously reported, and their disorder has been attributed to an overlap of piebaldism and neurofibromatosis type 1. Legius syndrome is a recently described syndrome caused by Sprouty-related, Ena/vasodilator-stimulated phosphoprotein homology-1 domain containing protein 1 (SPRED1) mutations that also has multiple café-au-lait macules and intertriginous freckling. Based on our current understanding of KIT and SPRED1 protein interactions, we propose that café-au-lait macules and freckling may be seen in some patients with piebaldism and does not necessarily represent coexistence of neurofibromatosis type 1. © 2012 Wiley Periodicals, Inc.

Decreased number of interstitial cells of Cajal play an important role in the declined intestinal transit during cholesterol gallstone formation in guinea pigs fed on high cholesterol diet

PubMed Central

Fan, Ying; Wu, Shuo-Dong; Fu, Bei-Bei; Weng, Chao; Wang, Xin-Peng

2014-01-01

To study the changes of interstitial cells of Cajal (ICCs) and expression of c-kt and scf mRNA in terminal ileum tissue during cholesterol gallstone formation in guinea pigs fed on high cholesterol diet, forty guinea pigs were divided into the gallstone group and the control group. The animals in the gallstone group were fed on a high cholesterol diet (HCD), while those in the control group fed on a standard diet (StD). The guinea pigs were sacrificed at the 8th week. The expression of c-kit and scf in terminal ileum were determined by RT-PCR and the morphological characteristics and number of ICCs were observed and calculated by using immunohistochemistry. RT-PCR showed that, compared with the control group, the c-kit and scf mRNA expression levels in the gallstone group were significantly declined. In the animal assay, the decreased number of ICCs was present obviously in the gallstone group. We concluded from the study that decreased number of ICCs, decreased expression of c-kit and scf in terminal ileum are present in guinea pigs fed on high cholesterol diet. The c-kit/scf pathway inhibition might be involved in the decline of intestinal transit function during cholesterol gallstone formation. PMID:24995081

Emerging Agents for the Treatment of Advanced, Imatinib-Resistant Gastrointestinal Stromal Tumors: Current Status and Future Directions.

PubMed

Bauer, Sebastian; Joensuu, Heikki

2015-08-01

Imatinib is strongly positioned as the recommended first-line agent for most patients with advanced gastrointestinal stromal tumor (GIST) due to its good efficacy and tolerability. Imatinib-resistant advanced GIST continues to pose a therapeutic challenge, likely due to the frequent presence of multiple mutations that confer drug resistance. Sunitinib and regorafenib are approved as second- and third-line agents, respectively, for patients whose GIST does not respond to imatinib or who do not tolerate imatinib, and their use is supported by large randomized trials. ATP-mimetic tyrosine kinase inhibitors provide clinical benefit even in heavily pretreated GIST suggesting that oncogenic dependency on KIT frequently persists. Several potentially useful tyrosine kinase inhibitors with distinct inhibitory profiles against both KIT ATP-binding domain and activation loop mutations have not yet been fully evaluated. Agents that have been found promising in preclinical models and early clinical trials include small molecule KIT and PDGFRA mutation-specific inhibitors, heat shock protein inhibitors, histone deacetylase inhibitors, allosteric KIT inhibitors, KIT and PDGFRA signaling pathway inhibitors, and immunological approaches including antibody-drug conjugates. Concomitant or sequential administration of tyrosine kinase inhibitors with KIT signaling pathway inhibitors require further evaluation, as well as rotation of tyrosine kinase inhibitors as a means to suppress drug-resistant cell clones.

Effects of storage temperature on the quantity and integrity of genomic DNA extracted from mice tissues: A comparison of recovery methods

PubMed Central

Al-Griw, Huda H.; Zraba, Zena A.; Al-Muntaser, Salsabiel K.; Draid, Marwan M.; Zaidi, Aisha M.; Tabagh, Refaat M.; Al-Griw, Mohamed A.

2017-01-01

Efficient extraction of genomic DNA (gDNA) from biological materials found in harsh environments is the first step for successful forensic DNA profiling. This study aimed to evaluate two methods for DNA recovery from animal tissues (livers, muscles), focusing on the best storage temperature for DNA yield in term of quality, quantity, and integrity for use in several downstream molecular techniques. Six male Swiss albino mice were sacrificed, liver and muscle tissues (n=32) were then harvested and stored for one week in different temperatures, -20°C, 4°C, 25°C and 40°C. The conditioned animal tissues were used for DNA extraction by Chelex-100 method or NucleoSpinC Blood and Tissue kit. The extracted gDNA was visualized on 1.5% agarose gel electrophoresis to determine the quality of gDNA and analysed spectrophotometrically to determine the DNA concentration and the purity. Both methods, Chelex-100 and NucleoSpin Blood and Tissue kit found to be appropriate for yielding high quantity of gDNA, with the Chelex 100 method yielding a greater quantity (P < 0.045) than the kit. At -20°C, 4°C, and 25°C temperatures, the concentration of DNA yield was numerically lower than at 40°C. The NucleoSpinC Blood and Tissue kit produced a higher (P=0.031) purity product than the Chelex-100 method, particularly for muscle tissues. The Chelex-100 method is cheap, fast, effective, and is a crucial tool for yielding DNA from animal tissues (livers, muscles) exposed to harsh environment with little limitations. PMID:28884076

OpenSatKit Enables Quick Startup for CubeSat Missions

NASA Technical Reports Server (NTRS)

McComas, David; Melton, Ryan

2017-01-01

The software required to develop, integrate, and operate a spacecraft is substantial regardless of whether its a large or small satellite. Even getting started can be a monumental task. To solve this problem, NASAs Core Flight System (cFS), NASA's 42 spacecraft dynamics simulator, and Ball Aerospaces COSMOS ground system have been integrated together into a kit called OpenSatKit that provides a complete and open source software solution for starting a new satellite mission. Users can have a working system with flight software, dynamics simulation, and a ground command and control system up and running within hours.Every satellite mission requires three primary categories of software to function. The first is Flight Software (FSW) which provides the onboard control of the satellites and its payload(s). NASA's cFS provides a great platform for developing this software. Second, while developing a satellite on earth, it is necessary to simulate the satellites orbit, attitude, and actuators, to ensure that the systems that control these aspects will work correctly in the real environment. NASAs 42 simulator provides these functionalities. Finally, the ground has to be able to communicate with the satellite, monitor its performance and health, and display its data. Additionally, test scripts have to be written to verify the system on the ground. Ball Aerospace's COSMOS command and control system provides this functionality. Once the OpenSatKit is up and running, the next step is to customize the platform and get it running on the end target. Starting from a fully working system makes porting the cFS from Linux to a users platform much easier. An example Raspberry Pi target is included in the kit so users can gain experience working with a low cost hardware target. All users can benefit from OpenSatKit but the greatest impact and benefits will be to SmallSat missions with constrained budgets and small software teams. This paper describes OpenSatKits system design, the steps necessary to run the system to target the Raspberry Pi, and future plans. OpenSatKit is a free fully functional spacecraft software system that we hope will greatly benefit the SmallSat community.

Efficacy of a chairside diagnostic test kit for estimation of C-reactive protein levels in periodontal disease.

PubMed

Nagarale, Girish; Ravindra, S; Thakur, Srinath; Setty, Swati

2010-10-01

C-reactive protein [CRP] levels increase to hundreds of mg/mL within hours following infection. Studies have shown that serum CRP levels were elevated in periodontal disease. However, in all the previous studies, CRP levels were measured by using high-sensitivity CRP assay kits with minimal detection limits of 0.1 to 3 mg/L, which was much below the normal value of 10 mg/L. These high-sensitivity CRP assays need a proper laboratory setup, and these methods cannot be used as a routine chair-side test in the dental office. The purpose of this study was to investigate the serum CRP levels in subjects with periodontal disease by using a rapid chair-side diagnostic test kit with a lower detection limit of 6 mg/L and to compare the CRP levels before and after periodontal therapy. A total of 45 systemically healthy subjects were selected for the study. Subjects were divided into three groups: group A: healthy controls, group B: gingivitis, group C: periodontitis. Serum levels of CRP were determined by using a latex slide agglutination method with commercially available kit with lower detection limit of 6 mg/L. CRP was negative in all the 15 subjects in groups A and B at baseline, 7th and 30th day. CRP was positive only in 2 subjects in Group C at baseline and 7th day. Estimation of serum CRP by using a rapid chair-side diagnostic test kit is not of any significance in subjects with periodontitis.

Efficacy of a chairside diagnostic test kit for estimation of C-reactive protein levels in periodontal disease

PubMed Central

Nagarale, Girish; Ravindra, S.; Thakur, Srinath; Setty, Swati

2010-01-01

Background: C-reactive protein [CRP] levels increase to hundreds of mg/mL within hours following infection. Studies have shown that serum CRP levels were elevated in periodontal disease. However, in all the previous studies, CRP levels were measured by using high-sensitivity CRP assay kits with minimal detection limits of 0.1 to 3 mg/L, which was much below the normal value of 10 mg/L. These high-sensitivity CRP assays need a proper laboratory setup, and these methods cannot be used as a routine chair-side test in the dental office. Aim: The purpose of this study was to investigate the serum CRP levels in subjects with periodontal disease by using a rapid chair-side diagnostic test kit with a lower detection limit of 6 mg/L and to compare the CRP levels before and after periodontal therapy. Materials and Methods: A total of 45 systemically healthy subjects were selected for the study. Subjects were divided into three groups: group A: healthy controls, group B: gingivitis, group C: periodontitis. Serum levels of CRP were determined by using a latex slide agglutination method with commercially available kit with lower detection limit of 6 mg/L. Results: CRP was negative in all the 15 subjects in groups A and B at baseline, 7th and 30th day. CRP was positive only in 2 subjects in Group C at baseline and 7th day. Conclusion: Estimation of serum CRP by using a rapid chair-side diagnostic test kit is not of any significance in subjects with periodontitis. PMID:21731244

Immunohistochemistry of a choroidal melanoma: nestin, CD34 and CD117÷c-kit labeling.

PubMed

Vrapciu, Alexandra Diana; Rusu, Mugurel Constantin; Voinea, Liliana Mary

2014-01-01

In a case of choroidal melanoma (CM) in a 70-year-old male patient, was firstly aimed at studying the processes of angiogenesis by use of nestin and CD34 antibodies. Anti-CD117÷c-kit antibodies were further considered for their progenitor cells specificity. Choroidal melanoma was histopathologically confirmed. Nestin-positive endothelia were found in the CM and the adjacent retina, but not in endothelia elsewhere in that eye. Nestin-positive non-pigmentary cells were found within the CM. Filopodia-projecting endothelial tip cells (ETCs), nestin- and CD34-positive were found in the CM. CD34-positive ETCs were also found in the iridial stroma. There were found two different immune patterns of the retinal Müller cells (MCs). They were nestin-positive in the retina adjacent to the tumor, but negative in any other part of retina. On the other hand, CD117÷c-kit antibodies labeled MCs as follows: (a) discontinuously, or continuously, in the retina adjacent to the CM; (b) only the inner segments of the MCs were labeled in the retina unrelated to the CM. While nestin could be a reliable marker for retinal damage, the CD117÷c-kit phenotype of MCs still needs further investigations. Antiangiogenic therapy appears as a good choice for tumor therapy.

Limits of diagnostic accuracy of anti-hepatitis C virus antibodies detection by ELISA and immunoblot assay.

PubMed

Suslov, Anatoly P; Kuzin, Stanislav N; Golosova, Tatiana V; Shalunova, Nina V; Malyshev, Nikolai A; Sadikova, Natalia V; Vavilova, Lubov M; Somova, Anna V; Musina, Elena E; Ivanova, Maria V; Kipor, Tatiana T; Timonin, Igor M; Kuzina, Lubov E; Godkov, Mihail A; Bajenov, Alexei I; Nesterenko, Vladimir G

2002-07-01

When human sera samples are tested for anti-hepatitis C virus (HCV) antibodies using different ELISA kits as well as immunoblot assay kits discrepant results often occur. As a result the diagnostics of HCV infection in such sera remains unclear. The purpose of this investigation is to define the limits of HCV serodiagnostics. Overall 7 different test kits of domestic and foreign manufacturers were used for the sampled sera testing. Preliminary comparative study, using seroconversion panels PHV905, PHV907, PHV908 was performed and reference kit was chosen (Murex anti-HCV version 4) as the most sensitive kit on the base of this study results. Overall 1640 sera samples have been screened using different anti-HCV ELISA kits and 667 of them gave discrepant results in at least two kits. These sera were then tested using three anti-HCV ELISA kits (first set of 377 samples) or four anti-HCV ELISA kits (second set of 290 samples) at the conditions of reference laboratory. In the first set 17.2% samples remained discrepant and in the second set - 13.4%. "Discrepant" sera were further tested in RIBA 3.0 and INNO-LIA immunoblot confirmatory assays, but approximately 5-7% of them remained undetermined after all the tests. For the samples with signal-to-cutoff ratio higher than 3.0 high rate of result consistency by reference, ELISA routing and INNO-LIA immunoblot assay was observed. On the other hand the results of tests 27 "problematic" sera in RIBA 3.0 and INNO-LIA were consistent only in 55.5% cases. Analysis of the antigen spectrum reactive with antibodies in "problematic" sera, demonstrated predominance of Core, NS3 and NS4 antigens for sera, positive in RIBA 3.0 and Core and NS3 antigens for sera, positive in INNO-LIA. To overcome the problem of undetermined sera, methods based on other principles, as well as alternative criteria of HCV infection diagnostics are discussed.

Acupuncture Ameliorates Postoperative Ileus via IL-6-miR-19a-KIT Axis to Protect Interstitial Cells of Cajal.

PubMed

Deng, Jingjing; Yang, Shu; Yuan, Qing; Chen, Yuzhong; Li, Dandan; Sun, Haimei; Tan, Xinghua; Zhang, Fuchun; Zhou, Deshan

2017-01-01

Acupuncture is a therapy effective in treating postoperative ileus (POI); its underlying mechanisms remain unclear. MicroRNAs (miRNAs) participate in inflammation and injury to the interstitial cells of Cajal (ICCs), both of which are considered to be contributors to POI. C-kit, encoding KIT, a specific marker of ICCs, is predicted to be targeted by miR-19a, an inflammation-related miRNA. Therefore, we investigated a possible link between inflammation, miR-19a, and ICCs in POI, as well as the mechanism by which these factors are affected by acupuncture. The effects of acupuncture on POI were assessed in patients after colorectal resection and in colocolic anastomosis mice. Immunofluorescence staining demonstrated that KIT[Formula: see text]/ano1[Formula: see text] ICCs dramatically decreased around the colonic incision in mice, which was negatively correlated with the pronounced increase in macrophage. However, this decrease was not due to apoptosis. IL-6R was expressed in ICCs, and IL-6 level was significantly increased, as measured by ELISA, in accompaniment with high miR-19a expression. The increase in IL-6 and miR-19a levels was negatively correlated with the decrease in KIT[Formula: see text]/ano1[Formula: see text] ICCs. A luciferase reporter assay demonstrated that miR-19a directly targeted C-kit, indicating that miR-19a caused ICC damage. Interestingly, acupuncture inhibited macrophage activation, IL-6 release, and miR-19a upregulation, while promoting KIT and ano1 restoration in ICCs. High serum miR-19a level in patients after colorectal resection was also reduced by acupuncture. Conclusively, the IL-6 released by macrophages during gastrointestinal surgery upregulated miR-19a, which downregulated KIT in ICCs and finally resulted in POI. Acupuncture can interfere with the "IL-6-miR-19a-KIT" axis, suggesting that it may be a therapeutic mechanism that works against POI.

Clinical Evaluation of a Loop-Mediated Amplification Kit for Diagnosis of Imported Malaria

PubMed Central

Polley, Spencer D.; González, Iveth J.; Mohamed, Deqa; Daly, Rosemarie; Bowers, Kathy; Watson, Julie; Mewse, Emma; Armstrong, Margaret; Gray, Christen; Perkins, Mark D.; Bell, David; Kanda, Hidetoshi; Tomita, Norihiro; Kubota, Yutaka; Mori, Yasuyoshi; Chiodini, Peter L.; Sutherland, Colin J.

2013-01-01

Background. Diagnosis of malaria relies on parasite detection by microscopy or antigen detection; both fail to detect low-density infections. New tests providing rapid, sensitive diagnosis with minimal need for training would enhance both malaria diagnosis and malaria control activities. We determined the diagnostic accuracy of a new loop-mediated amplification (LAMP) kit in febrile returned travelers. Methods. The kit was evaluated in sequential blood samples from returned travelers sent for pathogen testing to a specialist parasitology laboratory. Microscopy was performed, and then malaria LAMP was performed using Plasmodium genus and Plasmodium falciparum–specific tests in parallel. Nested polymerase chain reaction (PCR) was performed on all samples as the reference standard. Primary outcome measures for diagnostic accuracy were sensitivity and specificity of LAMP results, compared with those of nested PCR. Results. A total of 705 samples were tested in the primary analysis. Sensitivity and specificity were 98.4% and 98.1%, respectively, for the LAMP P. falciparum primers and 97.0% and 99.2%, respectively, for the Plasmodium genus primers. Post hoc repeat PCR analysis of all 15 tests with discrepant results resolved 4 results in favor of LAMP, suggesting that the primary analysis had underestimated diagnostic accuracy. Conclusions. Malaria LAMP had a diagnostic accuracy similar to that of nested PCR, with a greatly reduced time to result, and was superior to expert microscopy. PMID:23633403

Contact Lens-Induced Discomfort and Inflammatory Mediator Changes in Tears.

PubMed

Masoudi, Simin; Zhao, Zhenjun; Stapleton, Fiona; Willcox, Mark

2017-01-01

Studies indicate that contact lens (CL) discontinuation mostly occurs because of dryness and discomfort symptoms. This study aimed to investigate relationships between changes in the concentration of tear inflammatory mediators with subjective comfort ratings with CL wear and no contact lens wear between morning and evening. Forty-five subjects collected tears twice daily in the morning and in the evening with or without lenses. Comfort was rated subjectively on a scale from 1 to 100 (where 100 was extremely comfortable) just before each tear collection. Tear samples were assayed for complement components (C3 and C3a), leukotriene B4 (LTB4), secretory phospholipase A2 (sPLA2), secretory immunoglobulin A (sIgA), and bradykinin using commercially available immuno-based assay kits. Comfort ratings showed a statistically significant decline from morning to evening both with CL (89.0±10.1 AM vs. 76.7±15.2 PM; P<0.001) and without CL (89.1±10.2 AM vs. 84.2±12.6 PM; P<0.005) wear. The decline was steeper with lens wear (P<0.001). Bradykinin and sPLA2 levels did not change between morning and evening or with CL wear (P>0.05). Leukotriene B4 levels were slightly higher in CL (CL 43.4±12.6 pg/ml vs. No CL 39.4±13.4 pg/mL; P=0.034), whereas the concentration of LTB4, C3, C3a, and sIgA dropped by the end of the day in the presence or absence of lens wear (P<0.001). For most mediators, tear levels were not correlated with comfort ratings in any of the conditions. Leukotriene B4 had a higher concentration in the evening, and when measured as a ratio to sIgA, there was a trend for increased concentration of this mediator during CL wear. Although specific mediators showed changes from morning to evening with and without lens wear, most of these were not correlated with subjective comfort ratings in lens wear. The only mediator that showed an increase in concentration during the day and during lens wear was LTB4, and further studies on this mediator are warranted.

Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method

PubMed Central

Salwe, Sukeshani; Kothari, Sweta; Chowdhary, Abhay; Deshmukh, Ranjana A.

2018-01-01

12–14 days of culturing of bone marrow (BM) cells containing various growth factors is widely used method for generating dendritic cells (DCs) from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs' purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P < 0.05) between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population. PMID:29682352

Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method.

PubMed

Gosavi, Rahul Ashok; Salwe, Sukeshani; Mukherjee, Sandeepan; Dahake, Ritwik; Kothari, Sweta; Patel, Vainav; Chowdhary, Abhay; Deshmukh, Ranjana A

2018-01-01

12-14 days of culturing of bone marrow (BM) cells containing various growth factors is widely used method for generating dendritic cells (DCs) from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs' purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference ( P < 0.05) between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.

Mechanism-related circulating proteins as biomarkers for clinical outcome in patients with unresectable hepatocellular carcinoma receiving sunitinib

PubMed Central

2011-01-01

Background Several proteins that promote angiogenesis are overexpressed in hepatocellular carcinoma (HCC) and have been implicated in disease pathogenesis. Sunitinib has antiangiogenic activity and is an oral multitargeted inhibitor of vascular endothelial growth factor receptors (VEGFRs)-1, -2, and -3, platelet-derived growth factor receptors (PDGFRs)-α and -β, stem-cell factor receptor (KIT), and other tyrosine kinases. In a phase II study of sunitinib in advanced HCC, we evaluated the plasma pharmacodynamics of five proteins related to the mechanism of action of sunitinib and explored potential correlations with clinical outcome. Methods Patients with advanced HCC received a starting dose of sunitinib 50 mg/day administered orally for 4 weeks on treatment, followed by 2 weeks off treatment. Plasma samples from 37 patients were obtained at baseline and during treatment and were analyzed for vascular endothelial growth factor (VEGF)-A, VEGF-C, soluble VEGFR-2 (sVEGFR-2), soluble VEGFR-3 (sVEGFR-3), and soluble KIT (sKIT). Results At the end of the first sunitinib treatment cycle, plasma VEGF-A levels were significantly increased relative to baseline, while levels of plasma VEGF-C, sVEGFR-2, sVEGFR-3, and sKIT were significantly decreased. Changes from baseline in VEGF-A, sVEGFR-2, and sVEGFR-3, but not VEGF-C or sKIT, were partially or completely reversed during the first 2-week off-treatment period. High levels of VEGF-C at baseline were significantly associated with Response Evaluation Criteria in Solid Tumors (RECIST)-defined disease control, prolonged time to tumor progression (TTP), and prolonged overall survival (OS). Baseline VEGF-C levels were an independent predictor of TTP by multivariate analysis. Changes from baseline in VEGF-A and sKIT at cycle 1 day 14 or cycle 2 day 28, and change in VEGF-C at the end of the first off-treatment period, were significantly associated with both TTP and OS, while change in sVEGFR-2 at cycle 1 day 28 was an independent predictor of OS. Conclusions Baseline plasma VEGF-C levels predicted disease control (based on RECIST) and were positively associated with both TTP and OS in this exploratory analysis, suggesting that this VEGF family member may have utility in predicting clinical outcome in patients with HCC who receive sunitinib. Trial registration ClinicalTrials.gov: NCT00247676 PMID:21787417

Mast cell-deficient Kit(W-sh) "Sash" mutant mice display aberrant myelopoiesis leading to the accumulation of splenocytes that act as myeloid-derived suppressor cells.

PubMed

Michel, Anastasija; Schüler, Andrea; Friedrich, Pamela; Döner, Fatma; Bopp, Tobias; Radsak, Markus; Hoffmann, Markus; Relle, Manfred; Distler, Ute; Kuharev, Jörg; Tenzer, Stefan; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Schild, Hansjörg; Schmitt, Edgar; Becker, Marc; Stassen, Michael

2013-06-01

Mast cell-deficient Kit(W-sh) "sash" mice are widely used to investigate mast cell functions. However, mutations of c-Kit also affect additional cells of hematopoietic and nonimmune origin. In this study, we demonstrate that Kit(W-sh) causes aberrant extramedullary myelopoiesis characterized by the expansion of immature lineage-negative cells, common myeloid progenitors, and granulocyte/macrophage progenitors in the spleen. A consistent feature shared by these cell types is the reduced expression of c-Kit. Populations expressing intermediate and high levels of Ly6G, a component of the myeloid differentiation Ag Gr-1, are also highly expanded in the spleen of sash mice. These cells are able to suppress T cell responses in vitro and phenotypically and functionally resemble myeloid-derived suppressor cells (MDSC). MDSC typically accumulate in tumor-bearing hosts and are able to dampen immune responses. Consequently, transfer of MDSC from naive sash mice into line 1 alveolar cell carcinoma tumor-bearing wild-type littermates leads to enhanced tumor progression. However, although it can also be observed in sash mice, accelerated growth of transplanted line 1 alveolar cell carcinoma tumors is a mast cell-independent phenomenon. Thus, the Kit(W-sh) mutation broadly affects key steps in myelopoiesis that may have an impact on mast cell research.

The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

PubMed Central

Diccianni, M B; Imagawa, M; Muramatsu, M

1992-01-01

Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lack of a role for AP1 in GPEI mediated trans-activation in F9 cells, although endogenously present AP1 can influence GPEI in HeLa cells. Co-transfection of delta fosB with c-jun, which forms an inactive c-Jun/delta FosB heterodimer that binds TRE sequences, inhibits GPEI-mediated transcription in AP1-lacking F9 cells as well as AP1-containing HeLa cells. These data suggest novel factor(s) other than AP1 are influencing GPEI. Binding studies reveal multiple nucleoproteins bind to GPEI. These factors are likely responsible for the high level of GPEI-mediated transcription observed in the absence of AP1 and during hepatocarcinogenesis. Images PMID:1408831

The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

PubMed

Diccianni, M B; Imagawa, M; Muramatsu, M

1992-10-11

Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lack of a role for AP1 in GPEI mediated trans-activation in F9 cells, although endogenously present AP1 can influence GPEI in HeLa cells. Co-transfection of delta fosB with c-jun, which forms an inactive c-Jun/delta FosB heterodimer that binds TRE sequences, inhibits GPEI-mediated transcription in AP1-lacking F9 cells as well as AP1-containing HeLa cells. These data suggest novel factor(s) other than AP1 are influencing GPEI. Binding studies reveal multiple nucleoproteins bind to GPEI. These factors are likely responsible for the high level of GPEI-mediated transcription observed in the absence of AP1 and during hepatocarcinogenesis.

Combined Spectroscopic and Calorimetric Studies to Reveal Absorption Mechanisms and Conformational Changes of Protein on Nanoporous Biomaterials

PubMed Central

Ahmadi, Saharnaz; Farokhi, Maryam; Padidar, Parisa; Falahati, Mojtaba

2015-01-01

In this study the effect of surface modification of mesoporous silica nanoparticles (MSNs) on its adsorption capacities and protein stability after immobilization of beta-lactoglobulin B (BLG-B) was investigated. For this purpose, non-functionalized (KIT-6) and aminopropyl-functionalized cubic Ia3d mesoporous silica ([n-PrNH2-KIT-6]) nanoparticles were used as nanoporous supports. Aminopropyl-functionalized mesoporous nanoparticles exhibited more potential candidates for BLG-B adsorption and minimum BLG leaching than non-functionalized nanoparticles. It was observed that the amount of adsorbed BLG is dependent on the initial BLG concentration for both KIT-6 and [n-PrNH2-KIT-6] mesoporous nanoparticles. Also larger amounts of BLG-B on KIT-6 was immobilized upon raising the temperature of the medium from 4 to 55 °C while such increase was undetectable in the case of immobilization of BLG-B on the [n-PrNH2-KIT-6]. At temperatures above 55 °C the amounts of adsorbed BLG on both studied nanomaterials decreased significantly. By Differential scanning calorimetry or DSC analysis the heterogeneity of the protein solution and increase in Tm may indicate that immobilization of BLG-B onto the modified KIT-6 results in higher thermal stability compared to unmodified one. The obtained results provide several crucial factors in determining the mechanism(s) of protein adsorption and stability on the nanostructured solid supports and the development of engineered nano-biomaterials for controlled drug-delivery systems and biomimetic interfaces for the immobilization of living cells. PMID:26230687

C3-PRO: Connecting ResearchKit to the Health System Using i2b2 and FHIR.

PubMed

Pfiffner, Pascal B; Pinyol, Isaac; Natter, Marc D; Mandl, Kenneth D

2016-01-01

A renewed interest by consumer information technology giants in the healthcare domain is focused on transforming smartphones into personal health data storage devices. With the introduction of the open source ResearchKit, Apple provides a framework for researchers to inform and consent research subjects, and to readily collect personal health data and patient reported outcomes (PRO) from distributed populations. However, being research backend agnostic, ResearchKit does not provide data transmission facilities, leaving research apps disconnected from the health system. Personal health data and PROs are of the most value when presented in context along with health system data. Our aim was to build a toolchain that allows easy and secure integration of personal health and PRO data into an open source platform widely adopted across 140 academic medical centers. We present C3-PRO: the Consent, Contact, and Community framework for Patient Reported Outcomes. This open source toolchain connects, in a standards-compliant fashion, any ResearchKit app to the widely-used clinical research infrastructure Informatics for Integrating Biology and the Bedside (i2b2). C3-PRO leverages the emerging health data standard Fast Healthcare Interoperability Resources (FHIR).

C3-PRO: Connecting ResearchKit to the Health System Using i2b2 and FHIR

PubMed Central

Pfiffner, Pascal B.; Pinyol, Isaac; Natter, Marc D.; Mandl, Kenneth D.

2016-01-01

A renewed interest by consumer information technology giants in the healthcare domain is focused on transforming smartphones into personal health data storage devices. With the introduction of the open source ResearchKit, Apple provides a framework for researchers to inform and consent research subjects, and to readily collect personal health data and patient reported outcomes (PRO) from distributed populations. However, being research backend agnostic, ResearchKit does not provide data transmission facilities, leaving research apps disconnected from the health system. Personal health data and PROs are of the most value when presented in context along with health system data. Our aim was to build a toolchain that allows easy and secure integration of personal health and PRO data into an open source platform widely adopted across 140 academic medical centers. We present C3-PRO: the Consent, Contact, and Community framework for Patient Reported Outcomes. This open source toolchain connects, in a standards-compliant fashion, any ResearchKit app to the widely-used clinical research infrastructure Informatics for Integrating Biology and the Bedside (i2b2). C3-PRO leverages the emerging health data standard Fast Healthcare Interoperability Resources (FHIR). PMID:27031856

An Application of Cost-Effectiveness Analysis in a Major Defense Acquisition Program: the Decision by the U.S. Department of Defense to Retain the C-17 Transport Aircraft

DTIC Science & Technology

2010-12-01

adding hush kits to dampen the sound of the engines and adding extra material to the fuselage. The cost to restart the C-5 line was estimated at $750...include adding hush kits to dampen the sound of the engines and extra material to the fuselage. A recently completed SAB had recommended that the weep

[Effects of naloxone on the expression of stem cell factor and C-kit receptor in combined oxygen-glucose deprivation of primary cultured human embryonic neuron in vitro].

PubMed

Zhu, Bo; Li, Lan-ying; Lü, Guo-yi; Xue, Yu-liang; Ye, Tie-hu

2010-04-01

To explore the effects of naloxone on the expression of c-kit receptor (c-kit R) and its ligand stem cell factor (SCF) in human embryo neuronal hypoxic injury. Serum-free cerebral cortical cultures prepared from embryonic human brains were deprived of both oxygen and glucose which would set up an environment more likely with that of in vivo ischemic injury. Neurons in 24-well culture plates were randomly divided into four groups: control group, hypoxia group, naloxone 0.5 microg/ml group and naloxone 10 microg/ml group. MTT assay and biological analysis were performed to study the cell death and the changes of extracellular concentrations of lactate dehydrogenase (LDH) after combined oxygen-glucose deprivation. Neurons in 25 ml culture flasks were also randomly allocated into four groups as previously described. Intracellular total RNA were extracted at different time points: pre-hypoxia, immediately after hypoxia, and 3, 6, 12, and 24 hours after reoxygenation. The changes of SCF/c-kit R mRNA expression in hypoxic neurons treated with different concentrations of naloxone pre and post oxygen-glucose deprivation were determined with RT-PCR. The cell vitality detected by MTT assay decreased significantly in hypoxia group and naloxone 0.5 microg/ml group when compared with control group (P<0.01), while no significant difference was found between naloxone 0.5 microg/ml group and hypoxia group or between naloxone 10 microg/ml group and control group. Extracellular concentration of LDH significantly increased in hypoxia group (P<0.05), while no difference was found between naloxone 0.5 microg/ml group and control group, between naloxone 0.5 microg/ml and hypoxia group, or between naloxone 10 microg/ml and control group (all P>0.05). Immediately after oxygen-glucose deprivation, the expression of SCF/c-kit R mRNA increased significantly (P<0.01). Among those the expression of SCF presented a distribution of double-peak value within 24 hours. After treated with different concentrations of naloxone, the peak value of each group were delayed to appear and went down with the increasing of naloxone concentration. The peak values in all treated groups were significantly different from that in control group (P<0.01). The expression of SCF/c-kit R mRNA increases at the early stage after combined oxygen-glucose deprivation. Naloxone 0.5 microg/ml can attenuate cell injuries and regulate the expression of SCF/c-kit R. Naloxone may protect neurons by modulating the expressions of some cytokines.

Design of an NF-kB Activation-Coupled Apoptotic Molecule for Prostate Cancer Therapy

DTIC Science & Technology

2008-07-31

p65-LS) hetero-dimer. We used this immunocomplex for caspase activity assay using a colorimetric caspase activity assay kit ( Biovision ). The...by a Caspase-3 colorimetric assay kit ( BioVision ). The purified Caspase-3 (10 ng) was used as a positive control in the assay. As shown in Figure...caspase-3 activity assay with a caspase-3 activity assay kit ( BioVision ). The activity of caspase-3 is in an arbitrary unit. 16 c), co-expressed

VPAC1 Targeted 64Cu-TP3805 kit preparation and its evaluation.

PubMed

Tripathi, Sushil K; Kumar, Pardeep; Trabulsi, Edouard J; Kim, Sung; McCue, Peter A; Intenzo, Charles; Berger, Adam; Gomella, Leonard; Thakur, Mathew L

2017-08-01

Previously, our laboratory has shown that 64 Cu-TP3805 can specifically target VPAC1 receptors and be used for positron emission tomography (PET) imaging of breast (BC) and prostate cancer (PC) in humans. Present work is aimed at the formulation of a freeze-dried diaminedithiol-peptide (N 2 S 2 -TP3805) kit and it's evaluation for the preparation of 64 Cu labeled TP3805. Parameters such as pH, temperature and incubation time were examined that influenced the radiolabeling efficiency and stability of the product. Kits were prepared under different conditions and radiolabeling efficiency of TP3805 kit was evaluated for a range of pH3.5-8.5, after addition of 64 Cu in 30μl, 0.1M HCl. Incubation temperature (37-90°C) and time (30-120min.) were also investigated. Kits were stored at -10°C and their long term stability was determined as a function of their radiolabeling efficiency. Further, stability of 64 Cu-TP3805 complex was evaluated in presence of fetal bovine serum and bovine serum albumin by using SDS polyacrylamide gel electrophoresis. Kits were then used for PET imaging of BC and PC following eIND (101550) and institutional approvals. Specificity of 64 Cu-TP3805 for VPAC1 was examined with digital autoradiography (DAR) of prostate tissues obtained after prostatectomy, benign prostatic hyperplasia (BPH) tissue, and benign and malignant lymph nodes. Results were compared with corresponding tissue histology. Radiolabeling efficiency was ≥95% at final pH ~7.2 when incubated at 50°C for 90min. Kits were stable up to 18months when stored at -10°C, and 64 Cu-TP3805 complex exhibited excellent stability for up to 4h at room temperature. 64 Cu-TP3805 complex did not show any transchelation even after 2h incubation at 37°C in 10% FBS as well as in BSA as determined by SDS PAGE analysis. DAR identified ≥95% of malignant lesions 11 new PC lesions, 20 high grade prostatic intraepithelial neoplasia, 2/2 ejaculatory ducts and 5/5 urethra verumontanum not previously identified The malignant lymph nodes were correctly identified by DAR and for 3/3 BPH patients, and 5/5 cysts, DAR was negative. In human BC (n=19) and PC (n=26) were imaged with 100% sensitivity. Availability of ready to use N 2 S 2 -peptide kits for 64 Cu labeling is convenient and eliminates possible day to day variation during its routine preparation for clinical use. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanical Complication with Broviac Repair Kit in a 4-Year-Old Boy with MEN 2a.

PubMed

Sesia, Sergio B; Haecker, Frank-Martin; Mayr, Johannes

2009-01-01

Background. Mechanical complications in the use of indwelling central venous catheters (CVCs) such as the Broviac catheter (BC) include kinking, occlusion, dislocation or leaking. We report on a mechanical complication after using a repair kit for the BC. Method. A 4-year old boy, suffering from multiple endocrine neoplasia type 2a (MEN 2a), intestinal aganglionosis (Hirschsprung's disease), and short bowel syndrome, required a BC for home parenteral nutrition. Result. Due to recurrent leakage of the BC, 5 subsequent repairs were necessary within seven months. During one repair a metallic tube belonging to the repair kit was found to have migrated proximally to the skin entrance level within the BC and requiring surgical removal. Conclusion. To our knowledge, this is the first report focusing on such a serious complication using a BC and its repair kit. The proximal migration of this metallic tube constitutes a distinct theoretical risk of endothoracic foreign body embolization.

Shuttle orbiter TPS flight repair kit development

NASA Technical Reports Server (NTRS)

1979-01-01

The design and application of a TPS repair kit is presented. The repair kit is designed for on orbit use by a crew member working in the manned maneuvering unit (MMU). The kit includes the necessary equipment and materials to accomplish the repair tasks which include the following: HRSI emittance coating repair, damaged tile repair, missing tile repair, and multiple tile repair. Two types of repair materials required to do the small area repair and the large area repair are described. The materials area cure in place, silicone base ablator for small damaged areas and precured ablator tile for repair of larger damaged areas is examined. The cure in place ablator is also used as an adhesive to bond the precured tiles in place. An applicator for the cure in place ablator, designed to contain a two-part silicon compound, mix the two components at correct ratio, and dispense the materials at rates compatible with mission timelines established for the EVA is described.

Comparison of Vitek Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Versus Conventional Methods in Candida Identification.

PubMed

Keçeli, Sema Aşkın; Dündar, Devrim; Tamer, Gülden Sönmez

2016-02-01

Candida species are generally identified by conventional methods such as germ tube or morphological appearance on corn meal agar, biochemical methods using API kits and molecular biological methods. Alternative to these methods, rapid and accurate identification methods of microorganisms called matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDİ-TOF MS) has recently been described. In this study, Candida identification results by API Candida kit, API 20C AUX kit and identifications on corn meal agar (CMA) are compared with the results obtained on Vitek-MS. All results were confirmed by sequencing internal transcribed spacer (ITS) regions of rDNA. Totally, 97 Candida strains were identified by germ tube test, CMA, API and Vitek-MS. Vitek-MS results were compatible with 74.2 % of API 20C AUX and 81.4 % of CMA results. The difference between the results of API Candida and API 20C AUX was detected. The ratio of discrepancy between Vitek-MS and API 20C AUX was 25.8 %. Candida species mostly identified as C. famata or C. tropicalis by and not compatible with API kits were identified as C. albicans by Vitek-MS. Sixteen Candida species having discrepant results with Vitek-MS, API or CMA were randomly chosen, and ITS sequence analysis was performed. The results of sequencing were compatible 56.2 % with API 20C AUX, 50 % with CMA and 93.7 % with Vitek-MS. When compared with conventional identification methods, MS results are more reliable and rapid for Candida identification. MS system may be used as routine identification method in clinical microbiology laboratories.

Procurement tool kit.

DOT National Transportation Integrated Search

2009-09-30

Todays 9-1-1 emergency communications professionals face challenges on multiple frontsa : fundamental lack of resources, aging equipment operating past its intended lifecycle, emerging : consumer technology that has outpaced public safety answe...

Magnolol pretreatment prevents sepsis-induced intestinal dysmotility by maintaining functional interstitial cells of Cajal.

PubMed

Miao, Bin; Zhang, Shuwen; Wang, Hong; Yang, Tiecheng; Zhou, Deshan; Wang, Bao-en

2013-08-01

The purpose of this study was to investigate the mechanism by which magnolol treatment prevents lipopolysaccharide (LPS)-induced septic dysmotility in mice. Sepsis was induced by intravenous tail vein injection of LPS (4 mg/kg body weight). Animals were divided into three groups: the magnolol-treated septic group, the placebo-treated septic group, and the control group. Intestinal transit and circular smooth muscle contraction were measured 12 h after LPS injection, and immunocytochemisty was performed to study the morphology of interstitial cells of Cajal (ICCs). Stem cell factor (SCF) expression and c-kit phosphorylation were determined by Western blot analysis, and the mRNA levels of inducible NO synthase (iNOS) were determined by RT-PCR. Nitric oxide (NO) content, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration were detected using commercial kits. Intestinal transit and muscular contractility were significantly lower in the LPS-treated group than in the control group. Immunocytochemical experiments showed that the total number of ICCs, and the total and average lengths of the ICC processes were significantly decreased in the LPS-treated group compared with those in the control group. In LPS-treated animals, magnolol pretreatment significantly accelerated intestinal transit, increased circular muscle contraction, and prevented ICC morphology changes. Phosphorylation of c-kit and expression of SCF were significantly downregulated in LPS-treated animals compared with control animals. Magnolol pretreatment prevented sepsis-induced decreases in c-kit phosphorylation and SCF expression in LPS-treated animals. Magnolol pretreatment prevented the sepsis-induced increase in NO concentration, iNOS expression, and MDA concentration, and decrease in SOD activity in LPS-treated animals. Our results suggest that magnolol treatment prevents sepsis-induced intestinal dysmotility by regulating SCF/c-kit and NO signaling to maintain functional ICCs.

Increased Cardiac Myocyte Progenitors in Failing Human Hearts

PubMed Central

Kubo, Hajime; Jaleel, Naser; Kumarapeli, Asangi; Berretta, Remus M.; Bratinov, George; Shan, Xiaoyin; Wang, Hongmei; Houser, Steven R.; Margulies, Kenneth B.

2009-01-01

Background Increasing evidence, derived mainly from animal models, supports the existence of endogenous cardiac renewal and repair mechanisms in adult mammalian hearts that could contribute to normal homeostasis and the responses to pathological insults. Methods and Results Translating these results, we isolated small c-kit+ cells from 36 of 37 human hearts using primary cell isolation techniques and magnetic cell sorting techniques. The abundance of these cardiac progenitor cells was increased nearly 4-fold in patients with heart failure requiring transplantation compared with nonfailing controls. Polychromatic flow cytometry of primary cell isolates (<30 μm) without antecedent c-kit enrichment confirmed the increased abundance of c-kit+ cells in failing hearts and demonstrated frequent coexpression of CD45 in these cells. Immunocytochemical characterization of freshly isolated, c-kit–enriched human cardiac progenitor cells confirmed frequent coexpression of c-kit and CD45. Primary cardiac progenitor cells formed new human cardiac myocytes at a relatively high frequency after coculture with neonatal rat ventricular myocytes. These contracting new cardiac myocytes exhibited an immature phenotype and frequent electric coupling with the rat myocytes that induced their myogenic differentiation. Conclusions Despite the increased abundance and cardiac myogenic capacity of cardiac progenitor cells in failing human hearts, the need to replace these organs via transplantation implies that adverse features of the local myocardial environment overwhelm endogenous cardiac repair capacity. Developing strategies to improve the success of endogenous cardiac regenerative processes may permit therapeutic myocardial repair without cell delivery per se. PMID:18645055

Targeting siRNA Missiles to Her2+ Breast Cancer

DTIC Science & Technology

2009-06-01

that HerPBK10 protects siRNA from serum nuclease-mediated degradation, T7 transcribed siRNA is more cytotoxic than synthetic siRNA when delivered to...nuclease-mediated degradation, T7 transcribed siRNA is more cytotoxic than synthetic siRNA when delivered to HER2+ breast cancer cells by HerPBK10...produced either synthetically by a commercial vendor (Dharmacon), or from a T7 transcription kit (Ambion), and shRNA, which is reportedly a more effective

Novel somatic and germline mutations in intracranial germ cell tumours.

PubMed

Wang, Linghua; Yamaguchi, Shigeru; Burstein, Matthew D; Terashima, Keita; Chang, Kyle; Ng, Ho-Keung; Nakamura, Hideo; He, Zongxiao; Doddapaneni, Harshavardhan; Lewis, Lora; Wang, Mark; Suzuki, Tomonari; Nishikawa, Ryo; Natsume, Atsushi; Terasaka, Shunsuke; Dauser, Robert; Whitehead, William; Adekunle, Adesina; Sun, Jiayi; Qiao, Yi; Marth, Gábor; Muzny, Donna M; Gibbs, Richard A; Leal, Suzanne M; Wheeler, David A; Lau, Ching C

2014-07-10

Intracranial germ cell tumours (IGCTs) are a group of rare heterogeneous brain tumours that are clinically and histologically similar to the more common gonadal GCTs. IGCTs show great variation in their geographical and gender distribution, histological composition and treatment outcomes. The incidence of IGCTs is historically five- to eightfold greater in Japan and other East Asian countries than in Western countries, with peak incidence near the time of puberty. About half of the tumours are located in the pineal region. The male-to-female incidence ratio is approximately 3-4:1 overall, but is even higher for tumours located in the pineal region. Owing to the scarcity of tumour specimens available for research, little is currently known about this rare disease. Here we report the analysis of 62 cases by next-generation sequencing, single nucleotide polymorphism array and expression array. We find the KIT/RAS signalling pathway frequently mutated in more than 50% of IGCTs, including novel recurrent somatic mutations in KIT, its downstream mediators KRAS and NRAS, and its negative regulator CBL. Novel somatic alterations in the AKT/mTOR pathway included copy number gains of the AKT1 locus at 14q32.33 in 19% of patients, with corresponding upregulation of AKT1 expression. We identified loss-of-function mutations in BCORL1, a transcriptional co-repressor and tumour suppressor. We report significant enrichment of novel and rare germline variants in JMJD1C, which codes for a histone demethylase and is a coactivator of the androgen receptor, among Japanese IGCT patients. This study establishes a molecular foundation for understanding the biology of IGCTs and suggests potentially promising therapeutic strategies focusing on the inhibition of KIT/RAS activation and the AKT1/mTOR pathway.

Novel somatic and germline mutations in intracranial germ cell tumors

PubMed Central

Wang, Linghua; Yamaguchi, Shigeru; Burstein, Matthew D.; Terashima, Keita; Chang, Kyle; Ng, Ho-Keung; Nakamura, Hideo; He, Zongxiao; Doddapaneni, Harshavardhan; Lewis, Lora; Wang, Mark; Suzuki, Tomonari; Nishikawa, Ryo; Natsume, Atsushi; Terasaka, Shunsuke; Dauser, Robert; Whitehead, William; Adekunle, Adesina; Sun, Jiayi; Qiao, Yi; Marth, Gábor; Muzny, Donna M.; Gibbs, Richard A.; Leal, Suzanne M.; Wheeler, David A.; Lau, Ching C.

2015-01-01

Intracranial germ cell tumors (IGCTs) are a group of rare heterogeneous brain tumors which are clinically and histologically similar to the more common gonadal GCTs. IGCTs show great variation in their geographic and gender distribution, histological composition and treatment outcomes. The incidence of IGCTs is historically 5–8 fold greater in Japan and other East Asian countries than in Western countries1 with peak incidence near the time of puberty2. About half of the tumors are located in the pineal region. The male-to-female incidence ratio is approximately 3–4:1 overall but even higher for tumors located in the pineal region3. Due to the scarcity of tumor specimens available for research, little is currently known about this rare disease. Here we report the analysis of 62 cases by next generation sequencing, SNP array and expression array. We find the KIT/RAS signaling pathway frequently mutated in over 50% of IGCTs including novel recurrent somatic mutations in KIT, its downstream mediators KRAS and NRAS, and its negative regulator CBL. Novel somatic alterations in the AKT/mTOR pathway included copy number gain of the AKT1 locus at 14q32.33 in 19% of patients, with corresponding upregulation of AKT1 expression. We identified loss-of-function mutations in BCORL1, a transcriptional corepressor and tumor suppressor. We report significant enrichment of novel and rare germline variants in JMJD1C, a histone demethylase and coactivator of the androgen receptor, among Japanese IGCT patients. This study establishes a molecular foundation for understanding the biology of IGCTs and suggests potentially promising therapeutic strategies focusing on the inhibition of KIT/RAS activation and the AKT1/mTOR pathway. PMID:24896186

A new LAMP-based assay for the molecular diagnosis of toxoplasmosis: comparison with a proficient PCR assay.

PubMed

Varlet-Marie, Emmanuelle; Sterkers, Yvon; Perrotte, Marina; Bastien, Patrick

2018-05-01

Toxoplasmosis is generally a benign infection caused by the protozoan parasite Toxoplasma gondii but can have severe consequences in fetuses of mothers infected during pregnancy (congenital toxoplasmosis) and immunocompromised individuals. PCR-based diagnostic tests have become crucial for its diagnosis. However, this molecular diagnosis essentially relies upon laboratory-developed methods and suffers from a lack of standardization, leading to great variation in methods and performance among laboratories. With the need for accreditation of clinical microbiological laboratories, the use of commercial PCR kits has become an attractive alternative; but thorough evaluation of newly commercialized kits by proficient groups is necessary before any recommendation can be made to parasitology laboratories by health authorities or learned societies. Here, we compared the performance of an original commercial method, the Iam TOXO Q-LAMP (DiaSorin®), using Loop-mediated isothermal amplification (LAMP) technology, with our reference laboratory-developed method using real-time PCR. The kit was first tested using amniotic fluid (AF) and plasma samples (either negative or spiked with live T. gondii tachyzoites at different concentrations (from 7 to 10 5  tachyzoites/mL)). It was then assessed using a cohort of 11 AF, five placental and 32 blood clinical samples preserved at -20 °C. For the processing of placental/blood samples, a pretreatment step was used, which did not strictly follow the manufacturer's recommendations. The practical ease of use and compliance with good laboratory practices were also evaluated. Although the LAMP assay was less sensitive than the laboratory-developed method at very low parasite concentrations (0.1 T. gondii genome equivalents/mL), the two methods yielded identical results qualitatively and, in some instances, quantitatively, particularly for AF samples. Copyright © 2018. Published by Elsevier Ltd.

The In Vitro Differentiation of GDNF Gene-Engineered Amniotic Fluid-Derived Stem Cells into Renal Tubular Epithelial-Like Cells.

PubMed

Lu, Ying; Wang, Zhuojun; Chen, Lu; Wang, Jia; Li, Shulin; Liu, Caixia; Sun, Dong

2018-05-01

Amniotic fluid is an alternative source of stem cells, and human amniotic fluid-derived stem cells (AFSCs) obtained from a small amount of amniotic fluid collected during the second trimester represent a novel source for use in regenerative medicine. These AFSCs are characterized by lower diversity, a higher proliferation rate, and a wider differentiation capability than adult mesenchymal stem cells. AFSCs are selected based on the cell surface marker c-kit receptor (CD117) using immunomagnetic sorting. Glial cell line-derived neurotrophic factor (GDNF) is expressed during early kidney development and regulates the proliferation and differentiation of stem cells in vitro. In this study, c-kit-sorted AFSCs were induced toward osteogenic or adipogenic differentiation. AFSCs engineered via the insertion of GDNF were cocultured with mouse renal tubular epithelial cells (mRTECs), which were preconditioned by hypoxia-reoxygenation in vitro. After coculture for 8 days, AFSCs differentiation into epithelial-like cells was evaluated by performing immunofluorescence, flow cytometry, and quantitative real-time polymerase chain reaction to identify cells expressing the renal epithelial markers, cytokeratin 18 (CK18), E-cadherin, aquaporin-1 (AQP1), and paired box 2 gene (Pax2). The GDNF gene enhanced AFSCs differentiation into RTECs. AFSCs possess self-renewal ability and multiple differentiation potential and thus represent a new source of stem cells.

Gonadoblastoma: evidence for a stepwise progression to dysgerminoma in a dysgenetic ovary.

PubMed

Pauls, Katharina; Franke, Folker E; Büttner, Reinhard; Zhou, Hui

2005-09-01

Gonadoblastomas are neoplasms of dysgenetic gonads which may undergo regression or become overgrown by malignant germ cell tumors (mGCTs). Since little is known about their relationship to normal gonadal development and mGCTs, we studied the phenotype and antigenic profile of gonadoblastomas in comparison with adjacent dysgerminomas and fetal gonads. Three cases of gonadoblastomas and fetal gonads of both sexes were analyzed using oncofetal markers to M2A-antigen (M2A), germ cell alkaline phosphatase (PLAP/GCAP), receptor tyrosine kinase c-kit (c-kit), and somatic angiotensin converting enzyme (sACE) as well as the proliferation marker MIB-1. Morphologically, microfollicular pattern of gonadoblastomas showed a fetal germ cell organization reminiscent of oocytic clusters of fetal ovaries. They contained both cell types, similar to oocytes (M2A-, GCAP-, c-kit+/-, sACE-) and oogonia (M2A+, GCAP+, c-kit+, sACE+). The percentage of germ cells immunoreactive for oncofetal markers and the proliferation index increased from microfollicular over coronary patterns to adjacent dysgerminomas. Supportive cells of gonadoblastomas showed a uniform phenotype (CK18+, vimentin+, sACE+, alpha-inhibin+, M2A-) but in contrast to fetal germ cells lacked a clear equivalence to fetal tissues. Our results show that gonadoblastomas mimic female fetal ovary and exhibit a stepwise progression from follicular pattern to coronary pattern and finally to dysgerminomas.

A phase 2, open-label, multi-center study of amuvatinib in combination with platinum etoposide chemotherapy in platinum-refractory small cell lung cancer patients

PubMed Central

Byers, Lauren Averett; Horn, Leora; Ghandi, Jitendra; Kloecker, Goetz; Owonikoko, Taofeek; Waqar, Saiama Naheed; Krzakowski, Maciej; Cardnell, Robert J.; Fujimoto, Junya; Taverna, Pietro; Azab, Mohammad; Camidge, David Ross

2017-01-01

Background Amuvatinib (MP-470) is a multi-targeted kinase inhibitor with potent activity against c-Kit, synergistic with DNA-damaging agents. We evaluated amuvatinib in combination with platinum-etoposide (EP) chemotherapy by objective response rate, survival, and tolerability in platinum-refractory small cell lung cancer (SCLC) patients. Methods This study used a Simon 2-stage design requiring ≥3 centrally confirmed responses in the first 21 subjects. Subjects received EP with 300 mg amuvatinib orally three times daily in cycles of 21 days. A three-day amuvatinib run-in period before EP occurred in Cycle 1. Subjects received the same EP chemotherapy regimen given prior to progression/relapse. Results Among 23 subjects treated, we observed four PRs (17.4%) per RECIST 1.1, only two of which were centrally confirmed (8.7%, response duration 119, 151 days). Three subjects (13%) had confirmed stable disease. c-Kit H-score was ≥100 in two subjects whose respective durations of disease control were 151 and 256 days. Conclusions The addition of amuvatinib to EP chemotherapy in unselected, platinum-refractory SCLC did not meet the primary endpoint of ≥3 confirmed responses in stage 1. However, high c-Kit expression in two subjects with durable disease control suggests the potential for further study of amuvatinib in SCLC patients with high c-Kit expression. PMID:29113403

A phase 2, open-label, multi-center study of amuvatinib in combination with platinum etoposide chemotherapy in platinum-refractory small cell lung cancer patients.

PubMed

Byers, Lauren Averett; Horn, Leora; Ghandi, Jitendra; Kloecker, Goetz; Owonikoko, Taofeek; Waqar, Saiama Naheed; Krzakowski, Maciej; Cardnell, Robert J; Fujimoto, Junya; Taverna, Pietro; Azab, Mohammad; Camidge, David Ross

2017-10-06

Amuvatinib (MP-470) is a multi-targeted kinase inhibitor with potent activity against c-Kit, synergistic with DNA-damaging agents. We evaluated amuvatinib in combination with platinum-etoposide (EP) chemotherapy by objective response rate, survival, and tolerability in platinum-refractory small cell lung cancer (SCLC) patients. This study used a Simon 2-stage design requiring ≥3 centrally confirmed responses in the first 21 subjects. Subjects received EP with 300 mg amuvatinib orally three times daily in cycles of 21 days. A three-day amuvatinib run-in period before EP occurred in Cycle 1. Subjects received the same EP chemotherapy regimen given prior to progression/relapse. Among 23 subjects treated, we observed four PRs (17.4%) per RECIST 1.1, only two of which were centrally confirmed (8.7%, response duration 119, 151 days). Three subjects (13%) had confirmed stable disease. c-Kit H-score was ≥100 in two subjects whose respective durations of disease control were 151 and 256 days. The addition of amuvatinib to EP chemotherapy in unselected, platinum-refractory SCLC did not meet the primary endpoint of ≥3 confirmed responses in stage 1. However, high c-Kit expression in two subjects with durable disease control suggests the potential for further study of amuvatinib in SCLC patients with high c-Kit expression.

Mechanisms of Thrombocytopenia During Septic Shock: A Multiplex Cluster Analysis of Endogenous Sepsis Mediators.

PubMed

Bedet, Alexandre; Razazi, Keyvan; Boissier, Florence; Surenaud, Mathieu; Hue, Sophie; Giraudier, Stéphane; Brun-Buisson, Christian; Mekontso Dessap, Armand

2018-06-01

Thrombocytopenia is a common feature of sepsis and may involve various mechanisms often related to the inflammatory response. This study aimed at evaluating factors associated with thrombocytopenia during human septic shock. In particular, we used a multiplex analysis to assess the role of endogenous sepsis mediators. Prospective, observational study. Thrombocytopenia was defined as an absolute platelet count <100 G/L or a 50% relative decrease in platelet count during the first week of septic shock. Plasma concentrations of 27 endogenous mediators involved in sepsis and platelet pathophysiology were assessed at day-1 using a multi-analyte Milliplex human cytokine kit. Patients with underlying diseases at risk of thrombocytopenia (hematological malignancies, chemotherapy, cirrhosis, and chronic heart failure) were excluded. Thrombocytopenia occurred in 33 (55%) of 60 patients assessed. Patients with thrombocytopenia were more prone to present with extrapulmonary infections and bacteremia. Disseminated intravascular coagulation was frequent (81%) in these patients. Unbiased hierarchical clustering identified five different clusters of sepsis mediators, including one with markers of platelet activation (e.g., thrombospondin-1) positively associated with platelet count, one with markers of inflammation (e.g., tumor necrosis factor alpha and heat shock protein 70), and endothelial dysfunction (e.g., intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) negatively associated with platelet count, and another involving growth factors of thrombopoiesis (e.g., thrombopoietin), also negatively associated with platelet count. Surrogates of hemodilution (e.g., hypoprotidemia and higher fluid balance) were also associated with thrombocytopenia. Multiple mechanisms seemed involved in thrombocytopenia during septic shock, including endothelial dysfunction/coagulopathy, hemodilution, and altered thrombopoiesis.

Validation testing of a portable kit for measuring an active soil carbon fraction

EPA Science Inventory

Increasing demands exist for information about properties related to soil quality and human-induced soil change, particularly soil C. To help address this need, the USDA-NRCS Soil Survey Laboratory (SSL) developed a portable kit for rapid and relatively accurate assessment of soi...

Exclusion of EDNRB and KIT as the basis for white spotting in Border Collies.

PubMed

Metallinos, D; Rine, J

2000-01-01

White spotting patterns in mammals can be caused by mutations in the genes for the endothelin B receptor and c-Kit, whose protein products are necessary for proper migration, differentiation or survival of the melanoblast population of cells. Although there are many different dog breeds that segregate white spotting patterns, no genes have been identified that are linked to these phenotypes. An intercross was generated from a female Newfoundland and a male Border Collie and the white spotting phenotypes of the intercross progeny were evaluated by measuring percentage surface area of white in the puppies. The Border Collie markings segregated as a simple autosomal recessive (7/25 intercross progeny had the phenotype). Two candidate genes, for the endothelin B receptor (EDNRB) and c-Kit (KIT), were evaluated for segregation with the white spotting pattern. Polymorphisms between the Border Collie and Newfoundland were identified for EDNRB using Southern analysis after a portion of the canine gene had been cloned. Polymorphisms for KIT were identified using a microsatellite developed from a bacterial artificial chromosome containing the canine gene. Both EDNRB and KIT were excluded as a cause of the white spotting pattern in at least two of the intercross progeny. Although these genes have been implicated in white spotting in other mammals, including horses, pigs, cows, mice and rats, they do not appear to be responsible for the white spotting pattern found in the Border Collie breed of dog.

Exclusion of EDNRB and KIT as the basis for white spotting in Border Collies

PubMed Central

Metallinos, Danika; Rine, Jasper

2000-01-01

Background: White spotting patterns in mammals can be caused by mutations in the genes for the endothelin B receptor and c-Kit, whose protein products are necessary for proper migration, differentiation or survival of the melanoblast population of cells. Although there are many different dog breeds that segregate white spotting patterns, no genes have been identified that are linked to these phenotypes. Results: An intercross was generated from a female Newfoundland and a male Border Collie and the white spotting phenotypes of the intercross progeny were evaluated by measuring percentage surface area of white in the puppies. The Border Collie markings segregated as a simple autosomal recessive (7/25 intercross progeny had the phenotype). Two candidate genes, for the endothelin B receptor (EDNRB) and c-Kit (KIT), were evaluated for segregation with the white spotting pattern. Polymorphisms between the Border Collie and Newfoundland were identified for EDNRB using Southern analysis after a portion of the canine gene had been cloned. Polymorphisms for KIT were identified using a microsatellite developed from a bacterial artificial chromosome containing the canine gene. Conclusions: Both EDNRB and KIT were excluded as a cause of the white spotting pattern in at least two of the intercross progeny. Although these genes have been implicated in white spotting in other mammals, including horses, pigs, cows, mice and rats, they do not appear to be responsible for the white spotting pattern found in the Border Collie breed of dog. PMID:11178229

Transforming and differentiation-inducing potential of constitutively activated c-kit mutant genes in the IC-2 murine interleukin-3-dependent mast cell line.

PubMed Central

Hashimoto, K.; Tsujimura, T.; Moriyama, Y.; Yamatodani, A.; Kimura, M.; Tohya, K.; Morimoto, M.; Kitayama, H.; Kanakura, Y.; Kitamura, Y.

1996-01-01

Two mutations of c-kit receptor tyrosine kinase (KIT), valine-559 to glycine (G559) and aspartic acid-814 to valine (V814), resulted in its constitutive activation. To examine the transforming and differentiation-inducing potential of the mutant KIT, we used the murine interleukin-3-dependent IC-2 mast cell line as a transfectant. The IC-2 cells contained few basophilic granules and did not express KIT on the surface. The KITG559 or KITV814 gene was introduced into IC-2 cells using a retroviral vector. KITG559 and KITV814 expressed in IC-2 cells were constitutively phosphorylated on tyrosine and demonstrated kinase activity in the absence of stem cell factor, which is a ligand for KIT. IC-2 cells expressing either KITG559 or KITV814 (IC-2G559 or IC-2V814 cells) showed factor-independent growth in suspension culture and produced tumors in nude athymic mice. In addition, IC-2G559 and IC-2V814 cells showed a more mature phenotype compared with the phenotype of the original IC-2 cells, especially after transplantation into nude mice. The number of basophilic granules and the content of histamine increased remarkably. KITG559 and KITV814 also influenced the transcriptional phenotype of mouse mast cell proteases (MMCP) in IC-2 cells. The expression of MMCP-2, MMCP-4, and MMCP-6 was much greater in IC-2G559 and IC-2V814 cells than in the original IC-2 cells. The results indicated that constitutively activated KIT had not only oncogenic activity but also differentiation-inducing activity in mast cells. Images Figure 1 Figure 4 Figure 5 Figure 6 PMID:8546206

Effects of grain species and cultivar, thermal processing, and enzymatic hydrolysis on gluten quantitation.

PubMed

Pahlavan, Autusa; Sharma, Girdhari M; Pereira, Marion; Williams, Kristina M

2016-10-01

Gluten from wheat, rye, and barley can trigger IgE-mediated allergy or Celiac disease in sensitive individuals. Gluten-free labeled foods are available as a safe alternative. Immunoassays such as the enzyme-linked immunosorbent assay (ELISA) are commonly used to quantify gluten in foods. However, various non-assay related factors can affect gluten quantitation. The effect of gluten-containing grain cultivars, thermal processing, and enzymatic hydrolysis on gluten quantitation by various ELISA kits was evaluated. The ELISA kits exhibited variations in gluten quantitation depending on the gluten-containing grain and their cultivars. Acceptable gluten recoveries were obtained in 200mg/kg wheat, rye, and barley-spiked corn flour thermally processed at various conditions. However, depending on the enzyme, gluten grain source, and ELISA kit used, measured gluten content was significantly reduced in corn flour spiked with 200mg/kg hydrolyzed wheat, rye, and barley flour. Thus, the gluten grain source and processing conditions should be considered for accurate gluten analysis. Published by Elsevier Ltd.

High levels of macrolide-resistant Mycoplasma genitalium in Queensland, Australia.

PubMed

Trembizki, Ella; Buckley, Cameron; Bletchly, Cheryl; Nimmo, Graeme R; Whiley, David M

2017-10-01

The macrolide azithromycin is recommended for treatment of Mycoplasma genitalium infection; however, M. genitalium strains possessing macrolide resistance-mediating mutations (MRMMs) are increasingly being reported. Here, we used the SpeeDx ResistancePlus MG kit, which provides simultaneous detection of M. genitalium and MRMMs, to assess MRMM carriage among M. genitalium infections in Queensland, Australia. Performance characteristics of the ResistancePlus MG kit for M. genitalium detection were compared to in-house PCR. Available M. genitalium PCR-positive (n=67) and negative (n=281) samples from the years 2011 to 2017 were tested using the SpeeDx ResistancePlus MG kit. In total, 63.6 % M. genitalium-positive samples were indicated to harbour MRMMs. The ResistancePlus MG method provided sensitivity and specificity of 97 and 99.6 % respectively compared to in-house PCR for M. genitalium detection. Such high levels of macrolide-resistant M. genitalium raise further concerns over future use of azithromycin for treatment of M. genitalium infection.

Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

NASA Astrophysics Data System (ADS)

Lu, Ying-Ying; Chen, Tong-Sheng; Wang, Xiao-Ping; Li, Li

2010-07-01

Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

Analysis of direct-to-consumer marketed Chlamydia trachomatis diagnostic tests in Norway.

PubMed

Reinton, Nils; Hjelmevoll, Stig Ove; Håheim, Håkon; Garstad, Kjersti; Mørch-Reiersen, Lisa Therese; Moghaddam, Amir

2015-08-01

Background In 2014, and for the first time in Norway, a pharmacy chain started selling home sampling kits for Chlamydia trachomatis (C. trachomatis) detection. Direct-to-consumer diagnostic kits for C. trachomatis have been available in Norway from an Internet company since 2005. There has been little assessment of persons who purchase direct-to-consumer diagnostic tests for sexually transmissible infections (STIs) detection and if low-risk populations are being unnecessarily encouraged to buy these tests. The prevalence of C. trachomatis in customers who purchased home sampling kits from the pharmacy chain and from the commercial Internet Co. were compared to that of patients attending STI clinics and other free primary healthcare services. Prevalences of other STIs in pharmacy and Internet customers were also determined. The prevalence of C. trachomatis among pharmacy customers was 11%, almost identical to the prevalence among Internet customers (12%). In comparison, the prevalence among patients attending STI clinics in Oslo was 7.2%, which is similar to the prevalence among patients who have been tested through primary healthcare services. The prevalence of Mycoplasma genitalium was two-fold less than that of C. trachomatis in the STI and primary physician population, and significantly less in the Internet and the pharmacy population. Neisseria gonorrhoeae was not detected in urine samples from pharmacy customers or from Internet customers. Both pharmacy and Internet C. trachomatis home-sampling kits seem to be purchased by the right risk population. Marketing of direct-to-consumer N. gonorrhoeae tests and possibly M. genitalium tests cannot be justified in Norway. Direct-to-consumer diagnostic tests should be actively utilised as part of national programs in preventing the spread of C. trachomatis.

Genomics of mucoepidermoid and adenoid cystic carcinomas.

PubMed

Yan, Kenneth; Yesensky, Jessica; Hasina, Rifat; Agrawal, Nishant

2018-02-01

To report on the current state of the literature on the genetics of mucoepidermoid and adenoid cystic carcinomas of the salivary glands with a focus on genomic screens and recently discovered genetic translocations. A PubMed based literature review was performed to query for genetics related basic science and preclinical studies about mucoepidermoid and adenoid cystic carcinomas of the salivary glands. Genetic translocations between CRTC1 and MAML2 in mucoepidermoid carcinoma and between MYB and NFIB in adenoid cystic carcinoma have been recently discovered and have therapeutic implications. Key signaling pathways such as the EGFR pathway in mucoepidermoid carcinoma and the Notch pathway, chromatin regulation, and c-kit mediated epithelial-mesenchymal transitions in adenoid cystic carcinoma have recently been elucidated, pointing to possible therapeutic targets in both cancers.

Pure versus combined Merkel cell carcinomas: immunohistochemical evaluation of cellular proteins (p53, Bcl-2, and c-kit) reveals significant overexpression of p53 in combined tumors.

PubMed

Lai, Jonathan H; Fleming, Kirsten E; Ly, Thai Yen; Pasternak, Sylvia; Godlewski, Marek; Doucette, Steve; Walsh, Noreen M

2015-09-01

Merkel cell polyomavirus is of oncogenic significance in approximately 80% of Merkel cell carcinomas. Morphological subcategories of the tumor differ in regard to viral status, the rare combined type being uniformly virus negative and the predominant pure type being mainly virus positive. Indications that different biological subsets of the tumor exist led us to explore this diversity. In an Eastern Canadian cohort of cases (75 patients; mean age, 76 years [range, 43-91]; male/female ratio, 43:32; 51 [68%] pure and 24 [34%] combined tumors), we semiquantitatively compared the immunohistochemical expression of 3 cellular proteins (p53, Bcl-2, and c-kit) in pure versus combined groups. Viral status was known in a subset of cases. The significant overexpression of p53 in the combined group (mean [SD], 153.8 [117.8] versus 121.6 [77.9]; P = .01) and the increased epidermal expression of this protein (p53 patches) in the same group lend credence to a primary etiologic role for sun damage in these cases. Expression of Bcl-2 and c-kit did not differ significantly between the 2 morphological groups. A relative increase in c-kit expression was significantly associated with a virus-negative status (median [interquartile range], 100 [60-115] versus 70 [0-100]; P = .03). Emerging data reveal divergent biological pathways in Merkel cell carcinoma, each with a characteristic immunohistochemical profile. Virus-positive tumors (all pure) exhibit high retinoblastoma protein and low p53 expression, whereas virus-negative cases (few pure and all combined) show high p53 and relatively high c-kit expression. The potential biological implications of this dichotomy call for consistent stratification of these tumors in future studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Randomised trial of three approaches for marketing smoking cessation programmes to Australian general practitioners.

PubMed

Cockburn, J; Ruth, D; Silagy, C; Dobbin, M; Reid, Y; Scollo, M; Naccarella, L

1992-03-14

To compare three approaches for marketing a quit smoking intervention kit to general practitioners. Randomised trial of (a) personal delivery and presentation by an educational facilitator with a follow up visit six weeks later; (b) delivery to the receptionist by a friendly volunteer courier with a follow up phone call six weeks later, or (c) postal delivery with a follow up letter six weeks later. Melbourne, Australia. 264 randomly selected general practitioners. A research assistant visited each doctor four months after delivery and measured use of components of the kit. A questionnaire measuring perceptions of aspects of the kit and its delivery was completed by doctors. Costs of each approach were calculated. Doctors receiving the educational facilitator approach were significantly more likely than those receiving the other two approaches to have seen the kit, to rate the method of delivery as engendering motivation to try the kit, to have used one of the "intensive intervention" components from the kit, to report that they found the kit less complicated, and to report greater knowledge of how to use the kit. There were no significant differences in use of "minimal intervention" components of the kit, ratings of overall acceptability of delivery, perceptions of cultural and structural barriers to using the kit, and ratings of the overall acceptability of the kit. The cost of the educational facilitator approach ($A142/doctor) was 24 times that of the mailed approach. The volunteer courier approach ($A14) was twice the cost of the mailed approach. Educational facilitators and volunteer couriers do not seem to be cost effective strategies for distributing smoking interventions.

Failure to generate bone marrow adipocytes does not protect mice from ovariectomy-induced osteopenia.

PubMed

Iwaniec, Urszula T; Turner, Russell T

2013-03-01

A reciprocal association between bone marrow fat and bone mass has been reported in ovariectomized rodents, suggesting that bone marrow adipogenesis has a negative effect on bone growth and turnover balance. Mice with loss of function mutations in kit receptor (kit(W/W-v)) have no bone marrow adipocytes in tibia or lumbar vertebra. We therefore tested the hypothesis that marrow fat contributes to the development of osteopenia by comparing the skeletal response to ovariectomy (ovx) in growing wild type (WT) and bone marrow adipocyte-deficient kit(W/W-v) mice. Mice were ovx at 4 weeks of age and sacrificed 4 or 10 weeks post-surgery. Body composition was measured at necropsy by dual-energy X-ray absorptiometry. Cortical (tibia) and cancellous (tibia and lumbar vertebra) bone architecture were evaluated by microcomputed tomography. Bone marrow adipocyte size and density, osteoblast- and osteoclast-lined bone perimeters, and bone formation were determined by histomorphometry. Ovx resulted in an increase in total body fat mass at 10 weeks post-ovx in both genotypes, but the response was attenuated in the in kit(W/W-v) mice. Adipocytes were present in bone marrow of tibia and lumbar vertebra in WT mice and bone marrow adiposity increased following ovx. In contrast, marrow adipocytes were not detected in either intact or ovx kit(W/W-v) mice. However, ovx in WT and kit(W/W-v) mice resulted in statistically indistinguishable changes in cortical and cancellous bone mass, cortical and cancellous bone formation rate, and cancellous osteoblast and osteoclast-lined bone perimeters. In conclusion, our findings do not support a causal role for increased bone marrow fat as a mediator of ovx-induced osteopenia in mice. Copyright © 2012 Elsevier Inc. All rights reserved.

Differences in the Importance of Mast Cells, Basophils, IgE, and IgG versus That of CD4+ T Cells and ILC2 Cells in Primary and Secondary Immunity to Strongyloides venezuelensis.

PubMed

Mukai, Kaori; Karasuyama, Hajime; Kabashima, Kenji; Kubo, Masato; Galli, Stephen J

2017-05-01

There is evidence that mast cells, basophils, and IgE can contribute to immune responses to parasites; however, the relative levels of importance of these effector elements in parasite immunity are not fully understood. Previous work in Il3 -deficient and c- kit mutant Kit W / W-v mice indicated that interleukin-3 and c-Kit contribute to expulsion of the intestinal nematode Strongyloides venezuelensis during primary infection. Our findings in mast cell-deficient Kit W-sh / W-sh mice and two types of mast cell-deficient mice that have normal c- kit ("Hello Kit ty" and MasTRECK mice) confirmed prior work in Kit W / W-v mice that suggested that mast cells play an important role in S. venezuelensis egg clearance in primary infections. We also assessed a possible contribution of basophils in immune responses to S. venezuelensis By immunohistochemistry, we found that numbers of basophils and mast cells were markedly increased in the jejunal mucosa during primary infections with S. venezuelensis Studies in basophil-deficient Mcpt8 DTR mice revealed a small but significant contribution of basophils to S. venezuelensis egg clearance in primary infections. Studies in mice deficient in various components of immune responses showed that CD4 + T cells and ILC2 cells, IgG, FcRγ, and, to a lesser extent, IgE and FcεRI contribute to effective immunity in primary S. venezuelensis infections. These findings support the conclusion that the hierarchy of importance of immune effector mechanisms in primary S. venezuelensis infection is as follows: CD4 + T cells/ILC2 cells, IgG, and FcRγ>mast cells>IgE and FcεRI>basophils. In contrast, in secondary S. venezuelensis infection, our evidence indicates that the presence of CD4 + T cells is of critical importance but mast cells, antibodies, and basophils have few or no nonredundant roles. Copyright © 2017 American Society for Microbiology.

c-kit mutation in a population of patients with mastocytosis.

PubMed

Nagata, H; Okada, T; Worobec, A S; Semere, T; Metcalfe, D D

1997-01-01

The c-kit Asp816Val activating mutation is found in all patients with mastocytosis with an associated hematologic disorder, and at least in a subset of patients with indolent mastocytosis. The case of an 11-month-old child is presented who was categorized as having indolent mastocytosis, and where the Asp816Val mutation was identified in lesional skin, but not in bone marrow or in peripheral blood mononuclear cell populations. The significance of these findings is discussed.

KIT mutations in Russian patients with mucosal melanoma.

PubMed

Abysheva, Svetlana N; Iyevleva, Aglaya G; Efimova, Nina V; Mokhina, Yulia B; Sabirova, Feruza A; Ivantsov, Alexandr O; Artemieva, Anna S; Togo, Alexandr V; Moiseyenko, Vladimir M; Matsko, Dmitry E; Imyanitov, Evgeny N

2011-12-01

A single institution series of 48 mucosal melanomas (MMs) has been analyzed for the presence of KIT mutations using high-resolution melting and sequencing of abnormally melted DNA fragments. The analysis of exons 9, 11, 13, and 17 has revealed eight of 48 (17%) nonsynonymous alterations, including zero of seven head and neck, six of 24 anorectal, one of 15 genitourinary, one of one gastric, and zero of one mediastinal MMs. Seven of these mutations were potentially associated with the tumor sensitivity to KIT tyrosine kinase inhibitors. One tumor harbored somatically acquired silent nucleotide substitution c.1383A>G (T461T). This study adds to the evidence that a substantial portion of MMs carry a therapeutically relevant mutation in the KIT oncogene.

Epithelial innervation of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy.

PubMed

Guthoff, Rudolf F; Wienss, Holger; Hahnel, Christian; Wree, Andreas

2005-07-01

Evaluation of a new method to visualize distribution and morphology of human corneal nerves (Adelta- and C-fibers) by means of fluorescence staining, confocal laser scanning microscopy, and 3-dimensional (3D) reconstruction. Trephinates of corneas with a diagnosis of Fuchs corneal dystrophy were sliced into layers of 200 microm thickness using a Draeger microkeratome (Storz, Germany). The anterior lamella was stained with the Life/Dead-Kit (Molecular Probes Inc.), examined by the confocal laser scanning microscope "Odyssey XL," step size between 0.5 and 1 microm, and optical sections were digitally 3D-reconstructed. Immediate staining of explanted corneas by the Life/Dead-Kit gave a complete picture of the nerves in the central human cornea. Thin nerves running parallel to the Bowman layer in the subepithelial plexus perforate the Bowman layer orthogonally through tube-like structures. Passing the Bowman layer, Adelta- and C-fibers can be clearly distinguished by fiber diameter, and, while running in the basal epithelial plexus, by their spatial arrangement. Adelta-fibers run straight and parallel to the Bowman layer underneath the basal cell layer. C-fibers, after a short run parallel to the Bowman layer, send off multiple branches penetrating epithelial cell layers orthogonally, ending blindly in invaginations of the superficial cells. In contrast to C-fibers, Adelta-fibers show characteristic bulbous formations when kinking into the basal epithelial plexus. Ex-vivo fluorescence staining of the cornea and 3D reconstructions of confocal scans provide a fast and easily reproducible tool to visualize nerves of the anterior living cornea at high resolution. This may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.

MicroRNA-132 targets HB-EGF upon IgE-mediated activation in murine and human mast cells.

PubMed

Molnár, Viktor; Érsek, Barbara; Wiener, Zoltán; Tömböl, Zsófia; Szabó, Péter M; Igaz, Péter; Falus, András

2012-03-01

MicroRNAs provide an additional layer in the regulation of gene expression acting as repressors with several targets at the posttranscriptional level. This study describes microRNA expression patterns during differentiation and activation of mast cells. The expression levels of 567 different mouse miRNAs were compared by microarray between c-Kit+ committed progenitors, mucosal mast cells, resting and IgE-crosslinked BMMCs in vitro. The strongest upregulation of miR-132 upon IgE-mediated activation was validated in human cord blood-derived mast cells as well. HB-EGF growth factor also upregulated upon activation and was ranked high by more prediction algorithms. Co-transfection of miR-132 mimicking precursor and the 3'UTR of human Hbegf-containing luciferase vector proves that the predicted binding site is functional. In line with this, neutralization of miR-132 by anti-miR inhibitor leads to sustained production of HB-EGF protein in activated mast cells. Our data provide a novel example for negative regulation of a growth factor by an upregulated miRNA. © Springer Basel AG 2011

Novel somatic KIT exon 8 mutation with dramatic response to imatinib in a patient with mucosal melanoma: a case report.

PubMed

Rapisuwon, Suthee; Parks, Kellie; Al-Refaie, Waddah; Atkins, Michael B

2014-10-01

Primary mucosal melanomas represent ∼1.3% of all cases of melanoma diagnosed in the USA. The sinonasal location is the most common primary site. Mutations in the KIT gene occur in 10-22% of mucosal melanomas. Tumor response to imatinib mesylate has been reported in about half of the patients with tumors harboring KIT mutations. Responses are almost exclusively restricted to tumors with mutations in KIT exon 9 or 11. We report a case of a patient with a sinonasal mucosal melanoma with a novel exon 8 mutation (C443S) who had marked initial response to imatinib. Somatic exon 8 KIT mutations have not been previously reported in mucosal melanoma or in other human solid tumors; however, such mutations have been reported in canine and feline mast cell tumors. Protein transcripts from exon 8 play an important role in the structural and functional integrity of the extracellular domain of KIT. In preclinical studies, a mutation in exon 8 led to autophosphorylation, independent of KIT ligand, and constitutive activation of the tyrosine kinase. This biology may explain the successful application of imatinib in animals with tumors harboring exon 8 KIT mutations and in our patient with mucosal melanoma. This report expands the population of patients with melanoma who might benefit from imatinib to those with somatic exon 8 KIT mutations. Such mutations should be looked for in patients with mucosal melanoma.

29 CFR 1910.421 - Pre-dive procedures.

Code of Federal Regulations, 2010 CFR

2010-07-01

... transportation; and (5) The nearest U.S. Coast Guard Rescue Coordination Center. (c) First aid supplies. (1) A first aid kit appropriate for the diving operation and approved by a physician shall be available at the dive location. (2) When used in a decompression chamber or bell, the first aid kit shall be suitable...

Comparison of the CT OligoGen kit with cobas 4800 assay for detection of Chlamydia trachomatis.

PubMed

Parra-Sánchez, Manuel; Marcuello-López, Ana; García-Rey, Silvia; Zakariya-Yousef, Ismail; Sivianes-Valdecantos, Nieves; Sierra-Atienza, Celestina; Bernal-Martínez, Samuel; Pueyo-Rodrígez, Isabel; Martín-Mazuelos, Estrella; Palomares-Folía, José Carlos

2015-12-01

A prospective study was designed to assess the performance of the new CT OligoGen kit and the cobas 4800 assay for detection of Chlamydia trachomatis. A set of samples that included urine samples (n=212), endocervical (n=167), rectal (n=53), pharyngeal (n=7) and urethral swabs (n=3). The samples were sent from a regional sexually transmitted diseases (STD) clinic in Seville, Spain, and were collected from 261 men and 181 women. Discordant results were re-analyzed and clinical data and other tests were reviewed in order to resolve them. Sensitivity, specificity, positive predicative value (PPV), negative predictive value (NPV) and kappa value for C. trachomatis detection using the CT OligoGen kit were 98.5%, 100%, 100%, 95.4% and 0.97, respectively. This new kit had a high sensitivity, specificity, PPV and NPV for C. trachomatis, therefore the performance profile confirms the usefulness and reliable results of this new assay. Copyright © 2015 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

A Contractile Network of Interstitial Cells of Cajal in the Supratarsal Mueller's Smooth Muscle Fibers With Sparse Sympathetic Innervation

PubMed Central

Yuzuriha, Shunsuke; Matsuo, Kiyoshi; Ban, Ryokuya; Yano, Shiharu; Moriizumi, Tetsuji

2012-01-01

Background: We previously reported that the supratarsal Mueller's muscle is innervated by both sympathetic efferent fibers and trigeminal proprioceptive afferent fibers, which function as mechanoreceptors-inducing reflexive contractions of both the levator and frontalis muscles. Controversy still persists regarding the role of the mechanoreceptors in Mueller's muscle; therefore, we clinically and histologically investigated Mueller's muscle. Methods: We evaluated the role of phenylephrine administration into the upper fornix in contraction of Mueller's smooth muscle fibers and how intraoperative stretching of Mueller's muscle alters the degree of eyelid retraction in 20 patients with aponeurotic blepharoptosis. In addition, we stained Mueller's muscle in 7 cadavers with antibodies against α-smooth muscle actin, S100, tyrosine hydroxylase, c-kit, and connexin 43. Results: Maximal eyelid retraction occurred approximately 3.8 minutes after administration of phenylephrine and prolonged eyelid retraction for at least 20 minutes after administration. Intraoperative stretching of Mueller's muscle increased eyelid retraction due to its reflexive contraction. The tyrosine hydroxylase antibody sparsely stained postganglionic sympathetic nerve fibers, whereas the S100 and c-kit antibodies densely stained the interstitial cells of Cajal (ICCs) among Mueller's smooth muscle fibers. A connexin 43 antibody failed to stain Mueller's muscle. Conclusions: A contractile network of ICCs may mediate neurotransmission within Mueller's multiunit smooth muscle fibers that are sparsely innervated by postganglionic sympathetic fibers. Interstitial cells of Cajal may also serve as mechanoreceptors that reflexively contract Mueller's smooth muscle fibers, forming intimate associations with intramuscular trigeminal proprioceptive fibers to induce reflexive contraction of the levator and frontalis muscles. PMID:22359687

Can job redesign interventions influence a broad range of employee outcomes by changing multiple job characteristics? A quasi-experimental study.

PubMed

Holman, David; Axtell, Carolyn

2016-07-01

Many job redesign interventions are based on a multiple mediator-multiple outcome model in which the job redesign intervention indirectly influences a broad range of employee outcomes by changing multiple job characteristics. As this model remains untested, the aim of this study is to test a multiple mediator-multiple outcome model of job redesign. Multilevel analysis of data from a quasi-experimental job redesign intervention in a call center confirmed the hypothesized model and showed that the job redesign intervention affected a broad range of employee outcomes (i.e., employee well-being, psychological contract fulfillment, and supervisor-rated job performance) through changes in 2 job characteristics (i.e., job control and feedback). The results provide further evidence for the efficacy and mechanisms of job redesign interventions. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Comparative analysis of Edwardsiella isolates from fish in the eastern United States identifies two distinct genetic taxa amongst organisms phenotypically classified as E. tarda

USGS Publications Warehouse

Griffin, Matt J.; Quiniou, Sylvie M.; Cody, Theresa; Tabuchi, Maki; Ware, Cynthia; Cipriano, Rocco C.; Mauel, Michael J.; Soto, Esteban

2013-01-01

Edwardsiella tarda, a Gram-negative member of the family Enterobacteriaceae, has been implicated in significant losses in aquaculture facilities worldwide. Here, we assessed the intra-specific variability of E. tarda isolates from 4 different fish species in the eastern United States. Repetitive sequence mediated PCR (rep-PCR) using 4 different primer sets (ERIC I & II, ERIC II, BOX, and GTG5) and multi-locus sequence analysis of 16S SSU rDNA, groEl, gyrA, gyrB, pho, pgi, pgm, and rpoA gene fragments identified two distinct genotypes of E. tarda (DNA group I; DNA group II). Isolates that fell into DNA group II demonstrated more similarity to E. ictaluri than DNA group I, which contained the reference E. tarda strain (ATCC #15947). Conventional PCR analysis using published E. tarda-specific primer sets yielded variable results, with several primer sets producing no observable amplification of target DNA from some isolates. Fluorometric determination of G + C content demonstrated 56.4% G + C content for DNA group I, 60.2% for DNA group II, and 58.4% for E. ictaluri. Surprisingly, these isolates were indistinguishable using conventional biochemical techniques, with all isolates demonstrating phenotypic characteristics consistent with E. tarda. Analysis using two commercial test kits identified multiple phenotypes, although no single metabolic characteristic could reliably discriminate between genetic groups. Additionally, anti-microbial susceptibility and fatty acid profiles did not demonstrate remarkable differences between groups. The significant genetic variation (<90% similarity at gyrA, gyrB, pho, phi and pgm; <40% similarity by rep-PCR) between these groups suggests organisms from DNA group II may represent an unrecognized, genetically distinct taxa of Edwardsiella that is phenotypically indistinguishable from E. tarda.

Resveratrol improves prostate fibrosis during progression of urinary dysfunction in chronic prostatitis.

PubMed

He, Yi; Zeng, Hui-Zhi; Yu, Yang; Zhang, Jia-Shu; Duan, Xingping; Zeng, Xiao-Na; Gong, Feng-Tao; Liu, Qi; Yang, Bo

2017-09-01

We investigated whether prostate fibrosis was associated with urinary dysfunction in chronic prostatitis (CP) and whether resveratrol improved urinary dysfunction and the underlying molecular mechanism. Rat model of CP was established via subcutaneous injections of DPT vaccine and subsequently treated with resveratrol. Bladder pressure and volume tests investigated the effect of resveratrol on urinary dysfunction in CP rats. Western blotting and immunohistochemical staining examined the expression level of C-kit/SCF and TGF-β/Wnt/β-catenin. Compared to the control group, the maximum capacity of the bladder, residual urine volume and maximum voiding pressure, the activity of C-kit/SCF and TGF-β/Wnt/β-catenin pathways were increased significantly in the CP group. Resveratrol treatment significantly improved these factors. CP induced significantly prostate fibrosis, which exhibits a close relationship with urinary dysfunction. Resveratrol improved fibrosis, which may be associated with the suppression of C-kit/SCF and TGF-β/Wnt/β-catenin pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Mast cells and matrix metalloproteinase 9 expression in actinic cheilitis and lip squamous cell carcinoma.

PubMed

Souza Freitas, Valéria; de Andrade Santos, Pedro Paulo; de Almeida Freitas, Roseana; Pereira Pinto, Leão; de Souza, Lélia Batista

2011-09-01

The aim of this study was to evaluate mast cell (MC) density and migration and their association with matrix metalloproteinase (MMP) 9 expression in squamous cell carcinoma (SCC) and actinic cheilitis (AC). Tryptase, c-Kit, and MMP-9 expression was evaluated in 20 cases of SCC, 20 cases of AC, and 7 cases of normal lip (control samples) by immunohistochemistry techniques. Tryptase(+) and c-Kit(+) MC densities were significantly higher in SCCs than in ACs and control samples (P < .001). However, no significant difference was found when comparing tryptase(+) and c-Kit(+) MC densities between ACs and control samples (P values .185 and .516, respectively). MMP-9 was strongly expressed in SCCs and moderately expressed in ACs and control samples. A highly significant association was found between tryptase(+) MC density and the expression of MMP-9 (P < .001). The increase in MC density associated with the strong expression of MMP-9 may favor SCC progression. Copyright © 2011 Mosby, Inc. All rights reserved.

Cognitive and affective mechanisms of pain and fatigue in multiple sclerosis.

PubMed

Arewasikporn, Anne; Turner, Aaron P; Alschuler, Kevin N; Hughes, Abbey J; Ehde, Dawn M

2018-06-01

To examine the extent to which pain catastrophizing, fatigue catastrophizing, positive affect, and negative affect simultaneously mediated the associations between common symptoms of multiple sclerosis (MS; i.e., pain, fatigue) and impact on daily life, depressive symptoms, and resilience. Participants were community-dwelling adults with MS (N = 163) reporting chronic pain, fatigue, and/or moderate depressive symptoms. Multiple mediation path analysis was used to model potential mediators of pain and fatigue separately, using baseline data from a randomized controlled trial comparing two symptom self-management interventions. In the pain model, pain catastrophizing was a mediator of pain intensity with pain interference and depression. Negative affect was a mediator of pain intensity with depression and resilience. In the fatigue model, fatigue catastrophizing was a mediator of fatigue intensity with fatigue impact and depression. Positive affect was a mediator of fatigue intensity with depression and resilience. These findings provide preliminary support for the presence of differential effects of cognitive-affective mediators and suggest potential targets for psychological interventions based on an individual's clinical presentation. The differential mediating effects also support the inclusion of both positive and negative aspects of psychological health in models of pain and fatigue, which would not be otherwise apparent if negative constructs were examined in isolation. To our knowledge, this is the first study to utilize a multivariate path analysis approach to examine cognitive-affective mediators of pain and fatigue in MS, while also examining positive and negative constructs concurrently. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Cytoskeleton in Mast Cell Signaling

PubMed Central

Dráber, Pavel; Sulimenko, Vadym; Dráberová, Eduarda

2012-01-01

Mast cell activation mediated by the high affinity receptor for IgE (FcεRI) is a key event in allergic response and inflammation. Other receptors on mast cells, as c-Kit for stem cell factor and G protein-coupled receptors (GPCRs) synergistically enhance the FcεRI-mediated release of inflammatory mediators. Activation of various signaling pathways in mast cells results in changes in cell morphology, adhesion to substrate, exocytosis, and migration. Reorganization of cytoskeleton is pivotal in all these processes. Cytoskeletal proteins also play an important role in initial stages of FcεRI and other surface receptors induced triggering. Highly dynamic microtubules formed by αβ-tubulin dimers as well as microfilaments build up from polymerized actin are affected in activated cells by kinases/phosphatases, Rho GTPases and changes in concentration of cytosolic Ca2+. Also important are nucleation proteins; the γ-tubulin complexes in case of microtubules or Arp 2/3 complex with its nucleation promoting factors and formins in case of microfilaments. The dynamic nature of microtubules and microfilaments in activated cells depends on many associated/regulatory proteins. Changes in rigidity of activated mast cells reflect changes in intermediate filaments build up from vimentin. This review offers a critical appraisal of current knowledge on the role of cytoskeleton in mast cells signaling. PMID:22654883

Fat-associated lymphoid cluster in Cyprinus carpio: Characterisation and its relation with peritoneal haemangiosarcoma.

PubMed

Madera-Sandoval, Ruth L; Reyes-Maldonado, Elba; Dzul-Caamal, Ricardo; Gallegos-Rangel, Esperanza; Domínguez-López, María Lilia; García-Latorre, Ethel; Vega-López, Armando

2015-06-01

FALC cells are natural helper cells producing Th2-type cytokines, which express c-kit, Sca-1, IL7R and CD45 in mouse and human. These cells are involved in allergic responses and contribute to the inflammatory reactions of adipose tissue; however, a lack of information prevails about the presence of these cells in other species. The aim of the study was to identify and characterise FALC cells in the common carp (Cyprinus carpio) using immunohistochemistry and molecular biology techniques as well as to explore their relationships with their microenvironment. Histological description of the FALC was performed using H&E and polyclonal antibodies were used against cell-surface markers such as c-kit, Sca-1 and CD45. Furthermore, gene expression of c-kit, Sca-1 and IL7R was assessed. C. carpio FALC cells express the same surface markers reported in FALC of the mouse at both the pre- and post-transcriptional level. By exposure to the soluble fraction of helminths, FALC cells produce abundant Th2 cytokines (IL-5, IL-6 and IL-13) but do not synthesise IL-1α. Additionally, FALC cells probably participate in vascular remodelling of the intestine vessels, inducing tumours because a malignant haemangiosarcoma in the peritoneal cavity was found. In this tumour, abundant FALC with their characteristic cell-surface markers were detected. The findings of this study suggest the involvement of some proto-oncogenes such as c-kit and Sca-1, and the deregulation of Src kinases modulated by CD45 present in C. carpio FALC with the ontogeny of peritoneal haemangiosarcoma in this fish species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of blanching treatments on antioxidant activity of frozen green capsicum (Capsicum annuum L. var bell pepper) using radical scavenging activity (DPPH) assay

NASA Astrophysics Data System (ADS)

Azizzuddin, Norafida; Abdullah, Aminah

2016-11-01

Blanching treatments are needed to deactivate enzymes in frozen vegetables. Antioxidant activity using DPPH radical scavenging activity assay were evaluated in steaming, boiling water, and microwave blanching at different temperature, time and microwave power level on frozen green capsicum. Green capsicum was chosen for frozen treatment compared to other capsicum with different maturity index because of the firm texture. The objective of this study was to compare the antioxidant activity of frozen green capsicum between conventional and Oxi Count Kit® assay for DPPH radical scavenging activity. Results showed frozen green capsicum blanched using microwave at high level/90 seconds (sample J) contained higher level of DPPH in both conventional method and Oxi Count Kit® compared to other treatments. However, there were no significant differences between sample J and fresh sample (sample A). Overall, the sequences from highest to lowest in blanching treatments for both DPPH conventional method, and DPPH Oxi Count Kit® were J (microwave high level/90 seconds) > A (Fresh) > H (Microwave Medium Level/120 seconds) > D (Boiling Water 80°C/150 seconds) > K (Microwave High Level/120 seconds) > I (Microwave Medium Level/150 seconds) > F (Microwave Low Level/150 seconds)> B (Steam 100°C/150 seconds) > E (Boiling Water 100°C /120 seconds) > G (Microwave Low Level /180 seconds)> C (Steam 100°C/180 seconds). Almost all frozen green capsicum samples showed no significant differences for comparison between test using DPPH conventional method and Oxi Count Kit®. Frozen storage for 0, and 3rd months showed no significant differences which indicate no changes on antioxidant activity during frozen storage at -18°C.

Clinical, Cytological, Histological and Immunohistochemical Features of Cutaneous Mast Cell Tumours in Ferrets (Mustela putorius furo).

PubMed

Vilalta, L; Meléndez-Lazo, A; Doria, G; Ramis, A; Solano-Gallego, L; Pastor, J; Martorell, J

2016-11-01

Cutaneous mast cell tumours (cMCTs) are one of the most common cutaneous tumours in ferrets (Mustela putorius furo). However, limited information is available regarding cytological and histological features of these tumours and studies evaluating KIT expression are lacking in this species. The aims of this prospective study were to describe the most common clinical, cytological and histological features of cMCTs in ferrets and to compare the usefulness of different staining techniques in the diagnosis of these tumours in ferrets as well as evaluating KIT expression in neoplastic mast cells (MCs) by immunohistochemistry. Macroscopically, the tumours were small, round to plaque-like and frequently associated with surface crusting. The most common locations were the extremities and the trunk. MC granules were stained in all cases using toluidine blue (TB) and Wright-Giemsa stains in cytological specimens, but none stained with modified Wright's stain. Haematoxylin and eosin and TB on histological sections failed to stain MC granules in all the cases. Cytological and histological examination revealed low to moderate anisocytosis and anisokaryosis. An infiltrative rather than a delineated or encapsulated growth pattern was noted histologically in all cases. Eosinophilic infiltration was not uncommon and 'collagenolysis' was detected on cytological and histological examination. KIT expression was detected in all cases evaluated. In approximately one third of the cases the MCs exhibited KIT labelling pattern I and in the remaining ferrets, KIT pattern III. No correlation was found between KIT expression pattern and biological behaviour. Copyright © 2016 Elsevier Ltd. All rights reserved.

Loss of Function of TET2 Cooperates with Constitutively Active KIT in Murine and Human Models of Mastocytosis

PubMed Central

De Vita, Serena; Schneider, Rebekka K.; Garcia, Michael; Wood, Jenna; Gavillet, Mathilde; Ebert, Benjamin L.; Gerbaulet, Alexander; Roers, Axel; Levine, Ross L.; Mullally, Ann; Williams, David A.

2014-01-01

Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM. PMID:24788138

Double Knockdown of Prolyyl Hydroxylase and Factor Inhibiting HIF with Non-Viral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction

PubMed Central

Huang, Mei; Nguyen, Patricia; Jia, Fangjun; Hu, Shijun; Gong, Yongquan; de Almeida, Patricia E.; Wang, Li; Nag, Divya; Kay, Mark A.; Giaccia, Amato J; Robbins, Robert C.; Wu, Joseph C.

2011-01-01

Background Under normoxic conditions, hypoxia inducible factor-1 alpha (HIF-1α) is rapidly degraded by two hydroxylases, prolyl hydroxylase (PHD) and factor inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its up-regulation may be an effective therapeutic option for ischemic heart failure. Methods and Results PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin sequences for inhibiting PHD isoenzyme 2 (shPHD2) and FIH (shFIH) were inserted into novel non-viral minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit+ cardiac progenitor cells (CPCs) demonstrated higher expression of angiogenesis factors in the double knockdown group compared to the single knockdown and shScramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially following LAD ligation in adult FVB mice (n=60). Functional studies using magnetic resonance imaging (MRI), echocardiography, and pressure-volume (PV) loops showed greater improvement in cardiac function in the double knockdown group. To assess mechanism(s) of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double knockdown group. Fluorescence activated cell sorting (FACS) showed significantly higher activation of endogenous c-kit+ cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser capture microdissection (LCM) analysis confirmed up-regulation of HIF-1α protein and angiogenesis genes, respectively. Conclusions We demonstrated that HIF-1α up-regulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function. PMID:21911818

Remission of Psoriasis in a Patient with Hepatocellular Carcinoma Treated with Sorafenib.

PubMed

Antoniou, Efstathios A; Koutsounas, Ioannis; Damaskos, Christos; Koutsounas, Sotiris

Psoriasis is a chronic, immune-mediated and angiogenesis-dependent disease. Activated keratinocytes in psoriatic lesions produce pro-angiogenic cytokines, including vascular endothelial growth factor (VEGF), which binds to vascular endothelial growth factor receptor (VEGFR) and promotes cell proliferation and angiogenesis. Sorafenib (BAY 43-9006) is a molecular multikinase inhibitor of RAF kinase, platelet-derived growth factor (PDGF), VEGFR-1, -2, -3, platelet-derived growth factor receptor (PDGFR)-β and c-Kit. This molecule inhibits tumor cell proliferation and angiogenesis and it is currently approved for the treatment of hepatocellular carcinoma (HCC). We present the complete remission of resistant psoriasis in a hepatitis C virus (HCV)-infected cirrhotic patient who was treated with sorafenib, for recurrent HCC. Several targeted therapies have demonstrated efficacy against psoriasis. More research and well-designed studies, both in novel drugs and those already marketed for other indications, are needed to determine their value as potential novel therapies for psoriasis. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

3D vision upgrade kit for TALON robot

NASA Astrophysics Data System (ADS)

Edmondson, Richard; Vaden, Justin; Hyatt, Brian; Morris, James; Pezzaniti, J. Larry; Chenault, David B.; Tchon, Joe; Barnidge, Tracy; Kaufman, Seth; Pettijohn, Brad

2010-04-01

In this paper, we report on the development of a 3D vision field upgrade kit for TALON robot consisting of a replacement flat panel stereoscopic display, and multiple stereo camera systems. An assessment of the system's use for robotic driving, manipulation, and surveillance operations was conducted. The 3D vision system was integrated onto a TALON IV Robot and Operator Control Unit (OCU) such that stock components could be electrically disconnected and removed, and upgrade components coupled directly to the mounting and electrical connections. A replacement display, replacement mast camera with zoom, auto-focus, and variable convergence, and a replacement gripper camera with fixed focus and zoom comprise the upgrade kit. The stereo mast camera allows for improved driving and situational awareness as well as scene survey. The stereo gripper camera allows for improved manipulation in typical TALON missions.

Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma.

PubMed

Patwardhan, Parag P; Ivy, Kathryn S; Musi, Elgilda; de Stanchina, Elisa; Schwartz, Gary K

2016-01-26

Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10-18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma.

Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma

PubMed Central

Musi, Elgilda; de Stanchina, Elisa; Schwartz, Gary K.

2016-01-01

Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10–18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma. PMID:26675259

TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells.

PubMed

Simeone, Ann-Marie; McMurtry, Vanity; Nieves-Alicea, René; Saavedra, Joseph E; Keefer, Larry K; Johnson, Marcella M; Tari, Ana M

2008-01-01

Tumor invasion and metastasis remain a major cause of mortality in breast cancer patients. High concentrations of nitric oxide (NO) suppress tumor invasion and metastasis in vivo. NO prodrugs generate large amounts of NO upon metabolism by appropriate intracellular enzymes, and therefore could have potential in the prevention and therapy of metastatic breast cancer. The present study was designed to determine the effects of the NO-releasing prodrug O2-(2,4-dinitrophenyl) 1- [(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) on breast cancer invasion and the mechanisms involved. MDA-MB-231, MDA-MB-231/F10, and MCF-7/COX-2 were the three breast cancer cell lines tested. NO levels were determined spectrophotometrically using a NO assay kit. Invasion and the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs were determined using Matrigel invasion assays, an MMP array kit and ELISAs. The activity and expression of extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase mitogen-activated protein kinases were determined using western blot analyses. Under conditions by which JS-K was not cytotoxic, JS-K significantly decreased (P < 0.05) the invasiveness of breast cancer cells across the Matrigel basement membrane, which was directly correlated with NO production. JS-43-126, a non-NO-releasing analog of JS-K, had no effect on NO levels or invasion. JS-K increased (P < 0.05) TIMP-2 production, and blocking TIMP-2 activity with a neutralizing antibody significantly increased (P < 0.05) the invasive activity of JS-K-treated cells across Matrigel. JS-K decreased p38 activity, whereas the activity and the expression of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase were unaffected. We report the novel findings that JS-K inhibits breast cancer invasion across the Matrigel basement membrane, and NO production is vital for this activity. Upregulation of TIMP-2 production is one mechanism by which JS-K mediates its anti-invasive effects. JS-K and other NO prodrugs may represent an innovative biological approach in the prevention and treatment of metastatic breast cancer.

Prognostic Importance of C-KIT Mutations in Core Binding Factor Acute Myeloid Leukemia: A Systematic Review.

PubMed

Ayatollahi, Hossein; Shajiei, Arezoo; Sadeghian, Mohammad Hadi; Sheikhi, Maryam; Yazdandoust, Ehsan; Ghazanfarpour, Masumeh; Shams, Seyyede Fatemeh; Shakeri, Sepideh

2017-03-01

Acute myeloid leukemia (AML) is defined as leukemic blast reproduction in bone marrow. Chromosomal abnormalities form different subgroups with joint clinical specifications and results. t(8;21)(q22;q22) and inv(16)(p13;q22) form core binding factor-AML (CBF-AML). c-kit mutation activation occurs in 12.8-46.1% of adults with CBF leukemia. These mutations occur in 20-25% of t(8;21) and 30% of inv(16) cases. In this systematic review, we searched different databases, including PubMed, Scopus, and Embase. Selected articles were measured based on the inclusion criteria of this study and initially compared in terms of titles or abstracts. Finally, articles relevant to the subject of this review were retrieved in full text. Twenty-two articles matched the inclusion criteria and were selected for this review. In this study, c-kit mutations were associated with poor prognosis in AML patients with t(8;21) and inv(16). In addition, these mutations had better prognostic effects on AML patients with inv(16) compared with those with t(8;21). According to the results of this study, c-kit mutations have intense, harmful effects on the relapse and white blood cell increase in CBF-AML adults. However, these mutations have no significant prognostic effects on patients. Copyright © 2016 King Faisal Specialist Hospital & Research Centre. Published by Elsevier Ltd. All rights reserved.

Tribbles 3 Mediates Endoplasmic Reticulum Stress-Induced Insulin Resistance in Skeletal Muscle

PubMed Central

Koh, Ho-Jin; Toyoda, Taro; Didesch, Michelle M.; Lee, Min-Young; Sleeman, Mark W.; Kulkarni, Rohit N.; Musi, Nicolas; Hirshman, Michael F.; Goodyear, Laurie J.

2013-01-01

Endoplasmic Reticulum (ER) stress has been linked to insulin resistance in multiple tissues but the role of ER stress in skeletal muscle has not been explored. ER stress has also been reported to increase tribbles 3 (TRB3) expression in multiple cell lines. Here, we report that high fat feeding in mice, and obesity and type 2 diabetes in humans significantly increases TRB3 and ER stress markers in skeletal muscle. Overexpression of TRB3 in C2C12 myotubes and mouse tibialis anterior muscles significantly impairs insulin signaling. Incubation of C2C12 cells and mouse skeletal muscle with ER stressors thapsigargin and tunicamycin increases TRB3 and impairs insulin signaling and glucose uptake, effects reversed in cells overexpressing RNAi for TRB3 and in muscles from TRB3 knockout mice. Furthermore, TRB3 knockout mice are protected from high fat diet-induced insulin resistance in skeletal muscle. These data demonstrate that TRB3 mediates ER stress-induced insulin resistance in skeletal muscle. PMID:23695665

Using Observational Data to Estimate the Effect of Hand Washing and Clean Delivery Kit Use by Birth Attendants on Maternal Deaths after Home Deliveries in Rural Bangladesh, India and Nepal

PubMed Central

Seward, Nadine; Prost, Audrey; Copas, Andrew; Corbin, Marine; Li, Leah; Colbourn, Tim; Osrin, David; Neuman, Melissa; Azad, Kishwar; Kuddus, Abdul; Nair, Nirmala; Tripathy, Prasanta; Manandhar, Dharma; Costello, Anthony; Cortina-Borja, Mario

2015-01-01

Background Globally, puerperal sepsis accounts for an estimated 8–12% of maternal deaths, but evidence is lacking on the extent to which clean delivery practices could improve maternal survival. We used data from the control arms of four cluster-randomised controlled trials conducted in rural India, Bangladesh and Nepal, to examine associations between clean delivery kit use and hand washing by the birth attendant with maternal mortality among home deliveries. Methods We tested associations between clean delivery practices and maternal deaths, using a pooled dataset for 40,602 home births across sites in the three countries. Cross-sectional data were analysed by fitting logistic regression models with and without multiple imputation, and confounders were selected a priori using causal directed acyclic graphs. The robustness of estimates was investigated through sensitivity analyses. Results Hand washing was associated with a 49% reduction in the odds of maternal mortality after adjusting for confounding factors (adjusted odds ratio (AOR) 0.51, 95% CI 0.28–0.93). The sensitivity analysis testing the missing at random assumption for the multiple imputation, as well as the sensitivity analysis accounting for possible misclassification bias in the use of clean delivery practices, indicated that the association between hand washing and maternal death had been over estimated. Clean delivery kit use was not associated with a maternal death (AOR 1.26, 95% CI 0.62–2.56). Conclusions Our evidence suggests that hand washing in delivery is critical for maternal survival among home deliveries in rural South Asia, although the exact magnitude of this effect is uncertain due to inherent biases associated with observational data from low resource settings. Our findings indicating kit use does not improve maternal survival, suggests that the soap is not being used in all instances that kit use is being reported. PMID:26295838

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

PubMed Central

Dubreuil, Patrice; Letard, Sébastien; Ciufolini, Marco; Gros, Laurent; Humbert, Martine; Castéran, Nathalie; Borge, Laurence; Hajem, Bérengère; Lermet, Anne; Sippl, Wolfgang; Voisset, Edwige; Arock, Michel; Auclair, Christian; Leventhal, Phillip S.; Mansfield, Colin D.; Moussy, Alain; Hermine, Olivier

2009-01-01

Background The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. Methodology/Principal Findings In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. Conclusions Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity. PMID:19789626

KIT amplification and gene mutations in acral/mucosal melanoma in Korea.

PubMed

Yun, Jina; Lee, Jeeyun; Jang, Jiryeon; Lee, Eui Jin; Jang, Kee Taek; Kim, Jung Han; Kim, Kyoung-Mee

2011-06-01

Mucosal and acral melanomas have demonstrated different genetic alterations and biological behavior compared with more common cutaneous melanomas. It was recently reported that gain-of-function KIT mutations and/or copy number increases are more common in mucosal and acral melanomas. Thus, we studied the frequency and pattern of KIT aberrations in mucosal and acral melanomas in Korea. We analyzed 97 patients who were pathologically confirmed with mucosal or acral melanoma between 1997 and 2010 at Samsung Medical Center. Of the 97 melanoma patients, 92 were screened for mutations in KIT exons 11, 13, 17, and 18, BRAF and NRAS genes. KIT copy number was assessed by quantitative, real-time PCR. Of the 97 patients, 55 (56.7%) were mucosal, 40 (41.2%) were acral melanoma, and two were of unknown primary origin. Among seven cases with KIT mutation, five (60.0%) occurred in exon 11, one (20.0%) in exon 17, and one (20.0%) in exon 13. Point mutations were the most common, resulting in substitutions in exon 11 (K558R, T574A, L576P, and V559A), exon 13 (N655K), and exon 17 (N822K). A novel Thr574Ala (c.1720A>G) KIT mutation, which has not been reported in melanoma or other tumor types, was identified in one genital melanoma case. Of the 97 mucosal or acral melanoma specimens, 49 were tested for KIT gene copy number changes using quantitative PCR. Increased KIT copy number was identified in 15 patients: seven (40%) of 20 acral melanomas and eight (31%) of 26 mucosal melanomas. Our study implicates that a significant proportion of acral and mucosal melanomas have KIT mutations in Asian population. © 2011 The Authors. APMIS © 2011 APMIS.

Adjusting dental ceramics: An in vitro evaluation of the ability of various ceramic polishing kits to mimic glazed dental ceramic surface.

PubMed

Steiner, René; Beier, Ulrike S; Heiss-Kisielewsky, Irene; Engelmeier, Robert; Dumfahrt, Herbert; Dhima, Matilda

2015-06-01

During the insertion appointment, the practitioner is often faced with the need to adjust ceramic surfaces to fit a restoration to the adjacent or opposing dentition and soft tissues. The purpose of this study was to assess the ceramic surface smoothness achieved with various commercially available ceramic polishing kits on different commonly used ceramic systems. The reliability of the cost of a polishing kit as an indicator of improved surface smoothness was assessed. A total of 350 ceramic surfaces representing 5 commonly available ceramic systems (IPS Empress Esthetic, IPS e.max Press, Cergo Kiss, Vita PM 9, Imagine PressX) were treated with 5 types of ceramic polishing systems (Cerapreshine, 94006C, Ceramiste, Optrafine, Zenostar) by following the manufacturers' guidelines. The surface roughness was measured with a profilometer (Taylor Hobson; Precision Taylor Hobson Ltd). The effects of ceramic systems and polishing kits of interest on surface roughness were analyzed by 2-way ANOVA, paired t test, and Bonferroni corrected significance level. The ceramic systems and polishing kits statistically affected surface roughness (P<.001).The polishing kit Zenostar on IPS e.max Press created the smoothest ceramic surface. No correlation could be established between the high cost of the polishing kit and low surface roughness. None of the commonly used ceramic polishing kits could create a surface smoother than that of glazed ceramic (P<.001). The inclusion of a diamond polishing paste step is recommended to improve surface smoothness (P<.001). The cost of ceramic polishing kits is not recommended as a reliable indicator of better performance of ceramic polishing kits (P>.30). Copyright © 2015 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

Mitomycin C induces apoptosis in rheumatoid arthritis fibroblast-like synoviocytes via a mitochondrial-mediated pathway.

PubMed

Yan, Chuqi; Kong, Dechao; Ge, Dong; Zhang, Yanming; Zhang, Xishan; Su, Changhui; Cao, Xiaojian

2015-01-01

Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterised by prominent synoviocyte hyperplasia and a potential imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Mitomycin C (MMC) has previously been demonstrated to inhibit fibroblast proliferation and to induce fibroblast apoptosis. However, the effects of MMC on the proliferation and apoptosis of human RA FLS and the potential mechanisms underlying its effects remain unknown. Cell viability was determined using the Cell Counting Kit-8 assay. Apoptotic cell death was analysed via Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. The production of intracellular reactive oxygen species (ROS) was assessed via flow cytometry, and the changes in mitochondrial membrane potential (ΔΨm) were visualized based on JC-1 staining via fluorescence microscopy. The expression of apoptosis-related proteins was determined via Western blot. Treatment with MMC significantly reduced cell viability and induced apoptosis in RA FLS. Furthermore, MMC exposure was found to stimulate the production of ROS and to disrupt the ΔΨm compared to the control treatment. Moreover, MMC increased the release of mitochondrial cytochrome c, the ratio of Bax/Bcl-2, the activation of caspase-9 and caspase-3, and the subsequent cleavage of poly(ADP-ribose) polymerase. Our findings suggest that MMC inhibits cell proliferation and induces apoptosis in RA FLS, and the mechanism underlying this MMC-induced apoptosis may involve a mitochondrial signalling pathway. © 2015 S. Karger AG, Basel.

UV Irradiation and Humic Acid Mediate Aggregation of Aqueous Fullerene (nC60) Nanoparticles

EPA Science Inventory

The transport and fate of engineered nanomaterials is affected by multiple environmental factors, including sunlight and natural organic matter. In this study, the initial aggregation kinetics of aqueous fullerene (nC60) nanoparticles before and after UVA irradiation was investig...

Acute carbon dioxide avoidance in Caenorhabditis elegans

PubMed Central

Hallem, Elissa A.; Sternberg, Paul W.

2008-01-01

Carbon dioxide is produced as a by-product of cellular respiration by all aerobic organisms and thus serves for many animals as an important indicator of food, mates, and predators. However, whether free-living terrestrial nematodes such as Caenorhabditis elegans respond to CO2 was unclear. We have demonstrated that adult C. elegans display an acute avoidance response upon exposure to CO2 that is characterized by the cessation of forward movement and the rapid initiation of backward movement. This response is mediated by a cGMP signaling pathway that includes the cGMP-gated heteromeric channel TAX-2/TAX-4. CO2 avoidance is modulated by multiple signaling molecules, including the neuropeptide Y receptor NPR-1 and the calcineurin subunits TAX-6 and CNB-1. Nutritional status also modulates CO2 responsiveness via the insulin and TGFβ signaling pathways. CO2 response is mediated by a neural circuit that includes the BAG neurons, a pair of sensory neurons of previously unknown function. TAX-2/TAX-4 function in the BAG neurons to mediate acute CO2 avoidance. Our results demonstrate that C. elegans senses and responds to CO2 using multiple signaling pathways and a neural network that includes the BAG neurons and that this response is modulated by the physiological state of the worm. PMID:18524955

Acute carbon dioxide avoidance in Caenorhabditis elegans.

PubMed

Hallem, Elissa A; Sternberg, Paul W

2008-06-10

Carbon dioxide is produced as a by-product of cellular respiration by all aerobic organisms and thus serves for many animals as an important indicator of food, mates, and predators. However, whether free-living terrestrial nematodes such as Caenorhabditis elegans respond to CO2 was unclear. We have demonstrated that adult C. elegans display an acute avoidance response upon exposure to CO2 that is characterized by the cessation of forward movement and the rapid initiation of backward movement. This response is mediated by a cGMP signaling pathway that includes the cGMP-gated heteromeric channel TAX-2/TAX-4. CO2 avoidance is modulated by multiple signaling molecules, including the neuropeptide Y receptor NPR-1 and the calcineurin subunits TAX-6 and CNB-1. Nutritional status also modulates CO2 responsiveness via the insulin and TGFbeta signaling pathways. CO2 response is mediated by a neural circuit that includes the BAG neurons, a pair of sensory neurons of previously unknown function. TAX-2/TAX-4 function in the BAG neurons to mediate acute CO2 avoidance. Our results demonstrate that C. elegans senses and responds to CO2 using multiple signaling pathways and a neural network that includes the BAG neurons and that this response is modulated by the physiological state of the worm.

Two variants in the KIT gene as candidate causative mutations for a dominant white and a white spotting phenotype in the donkey.

PubMed

Haase, B; Rieder, S; Leeb, T

2015-06-01

White spotting phenotypes have been intensively studied in horses, and although similar phenotypes occur in the donkey, little is known about the molecular genetics underlying these patterns in donkeys. White spotting in donkeys can range from only a few white areas to almost complete depigmentation and is characterised by a loss of pigmentation usually progressing from a white spot in the hip area. Completely white-born donkeys are rare, and the phenotype is characterised by the complete absence of pigment resulting in pink skin and a white coat. A dominant mode of inheritance has been demonstrated for spotting in donkeys. Although the mode of inheritance for the completely white phenotype in donkeys is not clear, the phenotype shows similarities to dominant white in horses. As variants in the KIT gene are known to cause a range of white phenotypes in the horse, we investigated the KIT gene as a potential candidate gene for two phenotypes in the donkey, white spotting and white. A mutation analysis of all 21 KIT exons identified a missense variant in exon 4 (c.662A>C; p.Tyr221Ser) present only in a white-born donkey. A second variant affecting a splice donor site (c.1978+2T>A) was found exclusively in donkeys with white spotting. Both variants were absent in 24 solid-coloured controls. To the authors' knowledge, this is the first study investigating genetic mechanisms underlying white phenotypes in donkeys. Our results suggest that two independent KIT alleles are probably responsible for white spotting and white in donkeys. © 2015 Stichting International Foundation for Animal Genetics.

Immune-Mediated Inflammation Promotes Subclinical Atherosclerosis in Recent-Onset Psoriatic Arthritis Patients without Conventional Cardiovascular Risk Factors

PubMed Central

Kolliker Frers, Rodolfo A.; Cosentino, Vanesa; Tau, Julia; Kerzberg, Eduardo M.; Urdapilleta, Adriana; Chiocconi, Monica; Kogan, Nora; Otero-Losada, Matilde; Capani, Francisco

2018-01-01

Studies on the inflammatory burden in recent-onset psoriatic arthritis (PsA) patients without conventional cardiovascular risk factors (CVRFs) are not available. This preliminary study focuses on cardiovascular risk in cutaneous psoriasis (CPs) and recent-onset PsA patients. Blood biochemistry (glucose, cholesterol, uric acid, lipid profile and apolipoprotein B) was analyzed using standard kits. Proatherogenic inflammation markers, C-reactive protein (CRP) and interleukin-6 (IL-6), and endothelial activators monocyte chemoattractant protein-1 (MCP-1) and soluble intercellular adhesion molecule-1 (sICAM-1), were determined by enzyme-linked immunosorbent assay. Ultrasound images allowed measuring carotid intima–media thickness (cIMT). Our study first shows an increase in cIMT, and in serum levels of sICAM-1 and CRP in recent-onset PsA patients not presenting conventional CVRFs over the non-medicated time-period, from disease diagnosis to the beginning of pharmacological treatment, compared with healthy subjects. The outcome highlights the importance of monitoring serum level of sICAM1, CRP, and cIMT, and the value of primary prevention in psoriatic patients even with no history of cardiovascular events. PMID:29535705

Immune-Mediated Inflammation Promotes Subclinical Atherosclerosis in Recent-Onset Psoriatic Arthritis Patients without Conventional Cardiovascular Risk Factors.

PubMed

Kolliker Frers, Rodolfo A; Cosentino, Vanesa; Tau, Julia; Kerzberg, Eduardo M; Urdapilleta, Adriana; Chiocconi, Monica; Kogan, Nora; Otero-Losada, Matilde; Capani, Francisco

2018-01-01

Studies on the inflammatory burden in recent-onset psoriatic arthritis (PsA) patients without conventional cardiovascular risk factors (CVRFs) are not available. This preliminary study focuses on cardiovascular risk in cutaneous psoriasis (CPs) and recent-onset PsA patients. Blood biochemistry (glucose, cholesterol, uric acid, lipid profile and apolipoprotein B) was analyzed using standard kits. Proatherogenic inflammation markers, C-reactive protein (CRP) and interleukin-6 (IL-6), and endothelial activators monocyte chemoattractant protein-1 (MCP-1) and soluble intercellular adhesion molecule-1 (sICAM-1), were determined by enzyme-linked immunosorbent assay. Ultrasound images allowed measuring carotid intima-media thickness (cIMT). Our study first shows an increase in cIMT, and in serum levels of sICAM-1 and CRP in recent-onset PsA patients not presenting conventional CVRFs over the non-medicated time-period, from disease diagnosis to the beginning of pharmacological treatment, compared with healthy subjects. The outcome highlights the importance of monitoring serum level of sICAM1, CRP, and cIMT, and the value of primary prevention in psoriatic patients even with no history of cardiovascular events.

COMPARISON OF IMMUNOASSAY AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY METHODS FOR MEASURING 3,5,6-TRICHLORO-2PYRIDINOL IN MULTIPLE SAMPLE MEDIA

EPA Science Inventory

Two enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the determination of 3,5,6-trichloro-2-pyridinol (3,5,6-TCP) in multiple sample media (dust, soil, food, and urine). The dust and soil samples were analyzed by a commercial RaPID immunoassay testing kit. ...

Mutant KIT as imatinib-sensitive target in metastatic sinonasal carcinoma.

PubMed

Dieter, S M; Heining, C; Agaimy, A; Huebschmann, D; Bonekamp, D; Hutter, B; Ehrenberg, K R; Fröhlich, M; Schlesner, M; Scholl, C; Schlemmer, H-P; Wolf, S; Mavratzas, A; Jung, C S; Gröschel, S; von Kalle, C; Eils, R; Brors, B; Penzel, R; Kriegsmann, M; Reuss, D E; Schirmacher, P; Stenzinger, A; Federspil, P A; Weichert, W; Glimm, H; Fröhling, S

2017-01-01

Sinonasal carcinomas (SNCs) comprise various rare tumor types that are characterized by marked histologic diversity and largely unknown molecular profiles, yet share an overall poor prognosis owing to an aggressive clinical course and frequent late-stage diagnosis. The lack of effective systemic therapies for locally advanced or metastatic SNC poses a major challenge to therapeutic decision making for individual patients. We here aimed to identify actionable genetic alterations in a patient with metastatic SNC whose tumor, despite all diagnostic efforts, could not be assigned to any known SNC category and was refractory to multimodal therapy. We used whole-exome and transcriptome sequencing to identify a KIT exon 11 mutation (c.1733_1735del, p.D579del) as potentially druggable target in this patient and carried out cancer hotspot panel sequencing to detect secondary resistance-conferring mutations in KIT. Furthermore, as a step towards clinical exploitation of the recently described signatures of mutational processes in cancer genomes, we established and applied a novel bioinformatics algorithm that enables supervised analysis of the mutational catalogs of individual tumors. Molecularly guided treatment with imatinib in analogy to the management of gastrointestinal stromal tumor (GIST) resulted in a dramatic and durable response with remission of nearly all tumor manifestations, indicating a dominant driver function of mutant KIT in this tumor. KIT dependency was further validated by a secondary KIT exon 17 mutation (c.2459_2462delATTCinsG, p.D820_S821delinsG) that was detected upon tumor progression after 10 months of imatinib treatment and provided a rationale for salvage therapy with regorafenib, which has activity against KIT exon 11/17 mutant GIST. These observations highlight the potential of unbiased genomic profiling for uncovering the vulnerabilities of individual malignancies, particularly in rare and unclassifiable tumors, and underscore that KIT exon 11 mutations represent tractable therapeutic targets across different histologies. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

SCF-KIT signaling induces endothelin-3 synthesis and secretion: Thereby activates and regulates endothelin-B-receptor for generating temporally- and spatially-precise nitric oxide to modulate SCF- and or KIT-expressing cell functions.

PubMed

Chen, Lei L; Zhu, Jing; Schumacher, Jonathan; Wei, Chongjuan; Ramdas, Latha; Prieto, Victor G; Jimenez, Arnie; Velasco, Marco A; Tripp, Sheryl R; Andtbacka, Robert H I; Gouw, Launce; Rodgers, George M; Zhang, Liansheng; Chan, Benjamin K; Cassidy, Pamela B; Benjamin, Robert S; Leachman, Sancy A; Frazier, Marsha L

2017-01-01

We demonstrate that SCF-KIT signaling induces synthesis and secretion of endothelin-3 (ET3) in human umbilical vein endothelial cells and melanoma cells in vitro, gastrointestinal stromal tumors, human sun-exposed skin, and myenteric plexus of human colon post-fasting in vivo. This is the first report of a physiological mechanism of ET3 induction. Integrating our finding with supporting data from literature leads us to discover a previously unreported pathway of nitric oxide (NO) generation derived from physiological endothelial NO synthase (eNOS) or neuronal NOS (nNOS) activation (referred to as the KIT-ET3-NO pathway). It involves: (1) SCF-expressing cells communicate with neighboring KIT-expressing cells directly or indirectly (cleaved soluble SCF). (2) SCF-KIT signaling induces timely local ET3 synthesis and secretion. (3) ET3 binds to ETBR on both sides of intercellular space. (4) ET3-binding-initiated-ETBR activation increases cytosolic Ca2+, activates cell-specific eNOS or nNOS. (5) Temporally- and spatially-precise NO generation. NO diffuses into neighboring cells, thus acts in both SCF- and KIT-expressing cells. (6) NO modulates diverse cell-specific functions by NO/cGMP pathway, controlling transcriptional factors, or other mechanisms. We demonstrate the critical physiological role of the KIT-ET3-NO pathway in fulfilling high demand (exceeding basal level) of endothelium-dependent NO generation for coping with atherosclerosis, pregnancy, and aging. The KIT-ET3-NO pathway most likely also play critical roles in other cell functions that involve dual requirement of SCF-KIT signaling and NO. New strategies (e.g. enhancing the KIT-ET3-NO pathway) to harness the benefit of endogenous eNOS and nNOS activation and precise NO generation for correcting pathophysiology and restoring functions warrant investigation.

SCF-KIT signaling induces endothelin-3 synthesis and secretion: Thereby activates and regulates endothelin-B-receptor for generating temporally- and spatially-precise nitric oxide to modulate SCF- and or KIT-expressing cell functions

PubMed Central

Zhu, Jing; Schumacher, Jonathan; Wei, Chongjuan; Ramdas, Latha; Prieto, Victor G.; Jimenez, Arnie; Velasco, Marco A.; Tripp, Sheryl R.; Andtbacka, Robert H. I.; Gouw, Launce; Rodgers, George M.; Zhang, Liansheng; Chan, Benjamin K.; Cassidy, Pamela B.; Benjamin, Robert S.; Leachman, Sancy A.; Frazier, Marsha L.

2017-01-01

We demonstrate that SCF-KIT signaling induces synthesis and secretion of endothelin-3 (ET3) in human umbilical vein endothelial cells and melanoma cells in vitro, gastrointestinal stromal tumors, human sun-exposed skin, and myenteric plexus of human colon post-fasting in vivo. This is the first report of a physiological mechanism of ET3 induction. Integrating our finding with supporting data from literature leads us to discover a previously unreported pathway of nitric oxide (NO) generation derived from physiological endothelial NO synthase (eNOS) or neuronal NOS (nNOS) activation (referred to as the KIT-ET3-NO pathway). It involves: (1) SCF-expressing cells communicate with neighboring KIT-expressing cells directly or indirectly (cleaved soluble SCF). (2) SCF-KIT signaling induces timely local ET3 synthesis and secretion. (3) ET3 binds to ETBR on both sides of intercellular space. (4) ET3-binding-initiated-ETBR activation increases cytosolic Ca2+, activates cell-specific eNOS or nNOS. (5) Temporally- and spatially-precise NO generation. NO diffuses into neighboring cells, thus acts in both SCF- and KIT-expressing cells. (6) NO modulates diverse cell-specific functions by NO/cGMP pathway, controlling transcriptional factors, or other mechanisms. We demonstrate the critical physiological role of the KIT-ET3-NO pathway in fulfilling high demand (exceeding basal level) of endothelium-dependent NO generation for coping with atherosclerosis, pregnancy, and aging. The KIT-ET3-NO pathway most likely also play critical roles in other cell functions that involve dual requirement of SCF-KIT signaling and NO. New strategies (e.g. enhancing the KIT-ET3-NO pathway) to harness the benefit of endogenous eNOS and nNOS activation and precise NO generation for correcting pathophysiology and restoring functions warrant investigation. PMID:28880927

[Development of ELISA-kit of quantitative analysis for Zearalenone].

PubMed

Wang, Yu-ping; Ji, Rong; Jiang, Tao; Kang, Wei-jun

2006-03-01

To develope a rapid, sensitive, quantitative ELISA-kit for Zearalenone and determine zearalenone in cereals. On the base of monoclonal antibodies against ZEN, apply indirect ELISA to study the performance parameter of the kit. The limited concentration of detection of the ELISA-kit was 1ng/ml, linear range was 1-200 ng/ml, the linear equation was Y = 0.99 - 0.40 x (R2 = 0.99). The inhibition concentration of 50% against ZEN was 16.3 ng/ml. The average recovery rate of spiked corn and wheat was 96.5% and 95.5%, respectively, the coefficient of variant was 13.2% and 10.9%, respectively. The kit can be stored at 4 degrees C over 6 months. The cross reaction rate with the other mycotoxins was less than 1%, and coefficient of variant within-laboratory and between-laboratory was less than 15% and less than 20%, respectively. Detecting the VICAM sample with ELISA method and HPLC method, the results were within the range of the sample, and there was no statistic difference between the two methods. This ELISA-kit was quick, sensitive, stable and specific and can be used to determine ZEN in cereals.

Medical issues in flight and updating the emergency medical kit.

PubMed

Verjee, Mohamud A; Crone, Robert; Ostrovskiy, Grigory

2018-01-01

Airline travel is more affordable than ever and likely safer than ever too. Within half a day, a passenger can be on the other side of the world. However, medical care in-flight has been an issue for those with medical conditions and for those who fall sick during a journey. While airlines have the advice of multiple recognized organizations on needs and standards of care, in-flight emergencies occur at various levels. An emergency medical kit (EMK) together with trained cabin crew can be very effective at resolving the minor problems that arise and reducing the risk of escalation. On occasion, an overhead plea may be announced for additional medical expertise. Having the right content in a medical kit is more important in modern day travel, coupled with advances in equipment and passenger expectations. The authors address current issues of illness and other relevant conditions and suggest a content enhancement for an onboard EMK.

Medical issues in flight and updating the emergency medical kit

PubMed Central

Verjee, Mohamud A; Crone, Robert; Ostrovskiy, Grigory

2018-01-01

Airline travel is more affordable than ever and likely safer than ever too. Within half a day, a passenger can be on the other side of the world. However, medical care in-flight has been an issue for those with medical conditions and for those who fall sick during a journey. While airlines have the advice of multiple recognized organizations on needs and standards of care, in-flight emergencies occur at various levels. An emergency medical kit (EMK) together with trained cabin crew can be very effective at resolving the minor problems that arise and reducing the risk of escalation. On occasion, an overhead plea may be announced for additional medical expertise. Having the right content in a medical kit is more important in modern day travel, coupled with advances in equipment and passenger expectations. The authors address current issues of illness and other relevant conditions and suggest a content enhancement for an onboard EMK. PMID:29750057

33 CFR 149.323 - What are the requirements for first aid kits?

Code of Federal Regulations, 2014 CFR

2014-07-01

... organization, such as the American Red Cross, for the maximum number of persons on the deepwater port. (b) The first aid kit must be maintained in a space designated as a medical treatment room or, if there is no medical treatment room, under the custody of the person in charge. (c) The operator must ensure that each...

33 CFR 149.323 - What are the requirements for first aid kits?

Code of Federal Regulations, 2012 CFR

2012-07-01

... organization, such as the American Red Cross, for the maximum number of persons on the deepwater port. (b) The first aid kit must be maintained in a space designated as a medical treatment room or, if there is no medical treatment room, under the custody of the person in charge. (c) The operator must ensure that each...

33 CFR 149.323 - What are the requirements for first aid kits?

Code of Federal Regulations, 2011 CFR

2011-07-01

... organization, such as the American Red Cross, for the maximum number of persons on the deepwater port. (b) The first aid kit must be maintained in a space designated as a medical treatment room or, if there is no medical treatment room, under the custody of the person in charge. (c) The operator must ensure that each...

33 CFR 149.323 - What are the requirements for first aid kits?

Code of Federal Regulations, 2013 CFR

2013-07-01

... organization, such as the American Red Cross, for the maximum number of persons on the deepwater port. (b) The first aid kit must be maintained in a space designated as a medical treatment room or, if there is no medical treatment room, under the custody of the person in charge. (c) The operator must ensure that each...

Pilot Fullerton uses hairbrush on middeck

NASA Image and Video Library

1982-03-30

STS003-22-119 (30 March 1982) --- Astronaut Gordon Fullerton, STS-3 pilot, wearing communications kit assembly (ASSY) mini-headset (HDST), uses hygiene kit hair brush on aft middeck. He makes light of his lack of hair during a freshening up session. He makes a token effort with a hair brush. Side hatch and panel ML31C appear behind him. Photo credit: NASA

Molecular screening strategies for NF1-like syndromes with café-au-lait macules

PubMed Central

Zhang, Jia; Li, Ming; Yao, Zhirong

2016-01-01

Multiple café-au-lait macules (CALM) are usually associated with neurofibromatosis type 1 (NF1), one of the most common hereditary disorders. However, a group of genetic disorders presenting with CALM have mutations that are involved in human skin pigmentation regulation signaling pathways, including KIT ligand/KIT proto-oncogene receptor tyrosine kinase and Ras/mitogen-activated protein kinase. These disorders, which include Legius syndrome, Noonan syndrome with multiple lentigines or LEOPARD syndrome, and familial progressive hyperpigmentation) are difficult to distinguish from NF1 at early stages, using skin appearance alone. Furthermore, certain syndromes are clinically overlapping and molecular testing is a vital diagnostic method. The present review aims to provide an overview of these ‘NF1-like’ inherited diseases and recommend a cost-effective strategy for making a clear diagnosis among these diseases with an ambiguous borderline. PMID:27666661

Molecular screening strategies for NF1-like syndromes with café-au-lait macules (Review).

PubMed

Zhang, Jia; Li, Ming; Yao, Zhirong

2016-11-01

Multiple café-au-lait macules (CALM) are usually associated with neurofibromatosis type 1 (NF1), one of the most common hereditary disorders. However, a group of genetic disorders presenting with CALM have mutations that are involved in human skin pigmentation regulation signaling pathways, including KIT ligand/KIT proto‑oncogene receptor tyrosine kinase and Ras/mitogen‑activated protein kinase. These disorders, which include Legius syndrome, Noonan syndrome with multiple lentigines or LEOPARD syndrome, and familial progressive hyperpigmentation) are difficult to distinguish from NF1 at early stages, using skin appearance alone. Furthermore, certain syndromes are clinically overlapping and molecular testing is a vital diagnostic method. The present review aims to provide an overview of these 'NF1‑like' inherited diseases and recommend a cost‑effective strategy for making a clear diagnosis among these diseases with an ambiguous borderline.

Elevated Apoptosis in the Liver of Dairy Cows with Ketosis.

PubMed

Du, Xiliang; Chen, Liang; Huang, Dan; Peng, Zhicheng; Zhao, Chenxu; Zhang, Yuming; Zhu, Yiwei; Wang, Zhe; Li, Xinwei; Liu, Guowen

2017-01-01

Dairy cows with ketosis are characterized by oxidative stress and hepatic damage. The aim of this study was to investigate hepatic oxidative stress and the apoptotic status of ketotic cows, as well as the underlying apoptosis pathway. The blood aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (GGT) activities and the haptoglobin (HP), serum amyloid A (SAA) and serum apoptotic cytokeratin 18 neo-epitope M30 (CK18 M30) concentrations were determined by commercially available kits and ELISA kits, respectively. Liver histology, TUNEL and Oil red O staining were performed in liver tissue samples. TG contents were measured using an enzymatic kit; Caspase 3 assays were carried out using the Caspase 3 activity assay kit; oxidation and antioxidant markers were measured using biochemical kits; apoptosis pathway were determined by qRT-PCR and western blot. Ketotic cows displayed hepatic fat accumulation. The hepatic malondialdehyde (MDA) content was significantly increased, but the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were markedly decreased in ketotic cows compared with control cows, indicating that ketotic cows displayed severe oxidative stress. Significantly higher serum levels of the hepatic damage markers AST, ALT, GGT and GLDH were observed in ketotic cows than in control cows. The blood concentration of the apoptotic marker CK18 M30 and the number of TUNEL-positive cells in the liver of ketotic cows were 1.19- and 2.61-fold, respectively, higher than the values observed in control cows. Besides, Caspase 3 activity was significantly increased in the liver of ketosis cows. Importantly, the levels of phosphorylated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) were significantly increased but the level of phosphorylated extracellular signal-regulated kinase1/2 (ERK1/2) was markedly decreased, which further promoted tumor protein 53 (p53) expression and inhibited nuclear factor E2-related factor 2 (Nrf2) expression. The apoptosis-related molecules p21, MDM2, Caspase 3, Caspase 9 and Bax were expressed at significantly higher levels in ketotic cows than in healthy cows, whereas the anti-apoptosis molecule Bcl-2 was expressed at significantly lower levels. Based on these results, ketotic cows display severe hepatic oxidative stress. The hepatic MAPK-p53-Nrf2 apoptotic pathway is over induced and partially mediated apoptotic damage in the liver. © 2017 The Author(s). Published by S. Karger AG, Basel.

Platelet-Rich Plasma Preparation Types Show Impact on Chondrogenic Differentiation, Migration, and Proliferation of Human Subchondral Mesenchymal Progenitor Cells.

PubMed

Kreuz, Peter Cornelius; Krüger, Jan Philipp; Metzlaff, Sebastian; Freymann, Undine; Endres, Michaela; Pruss, Axel; Petersen, Wolf; Kaps, Christian

2015-10-01

To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen in vitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P = .00041; Regen, P = .00029; Dr.PRP, P = .00051; PRP-A, P < .0001; and PRP-C, P < .0001) stimulated migration of MPCs. All platelet concentrates but one (Dr.PRP, P = .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P = .027; Regen, P = .0029; PRP-A, P = .00021; and PRP-C, P = .00069) in DNA content. Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP preparations did induce such differentiation. These findings suggest differing outcomes with PRP treatment in stem cell-based cartilage repair. Our findings may help to explain the variability of results in studies examining the use of PRP clinically. Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Mast cells mediate neutrophil recruitment during atherosclerotic plaque progression.

PubMed

Wezel, Anouk; Lagraauw, H Maxime; van der Velden, Daniël; de Jager, Saskia C A; Quax, Paul H A; Kuiper, Johan; Bot, Ilze

2015-08-01

Activated mast cells have been identified in the intima and perivascular tissue of human atherosclerotic plaques. As mast cells have been described to release a number of chemokines that mediate leukocyte fluxes, we propose that activated mast cells may play a pivotal role in leukocyte recruitment during atherosclerotic plaque progression. Systemic IgE-mediated mast cell activation in apoE(-/-)μMT mice resulted in an increase in atherosclerotic lesion size as compared to control mice, and interestingly, the number of neutrophils was highly increased in these lesions. In addition, peritoneal mast cell activation led to a massive neutrophil influx into the peritoneal cavity in C57Bl6 mice, whereas neutrophil numbers in mast cell deficient Kit(W(-sh)/W(-sh)) mice were not affected. Within the newly recruited neutrophil population, increased levels of CXCR2(+) and CXCR4(+) neutrophils were observed after mast cell activation. Indeed, mast cells were seen to contain and release CXCL1 and CXCL12, the ligands for CXCR2 and CXCR4. Intriguingly, peritoneal mast cell activation in combination with anti-CXCR2 receptor antagonist resulted in decreased neutrophil recruitment, thus establishing a prominent role for the CXCL1/CXCR2 axis in mast cell-mediated neutrophil recruitment. Our data suggest that chemokines, and in particular CXCL1, released from activated mast cells induce neutrophil recruitment to the site of inflammation, thereby aggravating the ongoing inflammatory response and thus affecting plaque progression and destabilization. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

miR-148b-3p functions as a tumor suppressor in GISTs by directly targeting KIT.

PubMed

Wang, Yu; Li, Jun; Kuang, Dong; Wang, Xiaoyan; Zhu, Yuanli; Xu, Sanpeng; Chen, Yaobing; Cheng, Henghui; Zhao, Qiu; Duan, Yaqi; Wang, Guoping

2018-04-16

Gain-of-function mutations and overexpression of KIT are characteristic features of gastrointestinal stromal tumor (GIST). Dysregulation in miRNA expression may lead to KIT overexpression and tumorigenesis. miRNA microarray analysis and real-time PCR were used to determine the miRNA expression profiles in a cohort of 69 clinical samples including 50 CD117 IHC+ /KIT mutation GISTs and 19 CD117 IHC- /wild-type GISTs. GO enrichment and KEGG pathway analyses were performed to reveal the predicted targets of the dysregulated miRNAs. Of the dysregulated miRNAs whose expression was inversely correlated with that of KIT miRNAs were predicted by bioinformatics analysis and confirmed by luciferase reporter assay. Cell counting kit-8 (CCK-8) and flow cytometry were used to measure the cell proliferation, cycle arrest and apoptosis. Wound healing and transwell assays were used to evaluate migration and invasion. A xenograft BALB/c nude mouse model was applied to investigate the tumorigenesis in vivo. Western blot and qRT-PCR were used to investigate the protein and mRNA levels of KIT and its downstream effectors including ERK, AKT and STAT3. Of the six miRNAs whose expression was inversely correlated with that of KIT, we found that miR-148b-3p was significantly downregulated in the CD117 IHC+ /KIT mutation GIST cohort. This miRNA was subsequently found to inhibit proliferation, migration and invasion of GIST882 cells. Mechanistically, miR-148b-3p was shown to regulate KIT expression through directly binding to the 3'-UTR of the KIT mRNA. Restoration of miR-148b-3p expression in GIST882 cells led to reduced expression of KIT and the downstream effectors proteins ERK, AKT and STAT3. However, overexpression of KIT reversed the inhibitory effect of miR-148b-3p on cell proliferation, migration and invasion. Furthermore, we found that reduced miR-148b-3p expression correlated with poor overall survival (OS) and disease-free survival (DFS) in GIST patients. miR-148b-3p functions as an important regulator of KIT expression and a potential prognostic biomarker for GISTs.

Effects of gestational maternal undernutrition on growth, carcass composition and meat quality of rabbit offspring.

PubMed

Symeon, George K; Goliomytis, Michael; Bizelis, Iosif; Papadomichelakis, George; Pagonopoulou, Olga; Abas, Zafeiris; Deligeorgis, Stelios G; Chadio, Stella E

2015-01-01

An experiment was conducted in order to evaluate the effects of gestational undernutrition of rabbit does on growth, carcass composition and meat quality of the offsprings. Thirty primiparous non lactating rabbit does were artificially inseminated and randomly divided in three treatment groups: Control (C; fed to 100% of maintenance requirements throughout gestation, n = 10), early undernourished (EU; fed to 50% of maintenance requirements during days 7-19 of gestation, n = 10) and late undernourished (LU; fed to 50% of maintenance requirements during days 20-27 of gestation, n = 10). During the 4th week of the gestation period, LU does significantly lost weight compared to C and EU groups (P<0.05). At kindling, C does produced litters with higher proportions of stillborn kits (P<0.05) while the total litter size (alive and stillborn kits) was not different among groups (10.7, 12.8 and 12.7 kits in C, EU and LU groups, respectively). Kit birth weight tended to be lower in the LU group. During fattening, body weight and feed intake were not different among offsprings of the three experimental groups. Moreover, the maternal undernutrition did not have any impact on carcass composition of the offsprings in terms of carcass parts and internal organs weights as well as meat quality of L. lumborum muscle (pH24, colour, water holding capacity and shear values) at slaughter (70 days of age). Therefore, it can be concluded that the gestational undernutrition of the mother does not have detrimental effects on the productive and quality traits of the offsprings.

Prevalence of exon 11 internal tandem duplications in the C-KIT proto-oncogene in Australian canine mast cell tumours.

PubMed

Tamlin, V S; Kessell, A E; Mccoy, R J; Dobson, E C; Smith, T S; Hebart, M; Brown, L; Mitrovic, D; Peaston, A E

2017-10-01

To measure the prevalence of internal tandem duplications (ITDs) in exon 11 of the proto-oncogene C-KIT in a sample of Australian cutaneous canine mast cell tumours (MCTs) drawn from general practice and to evaluate relationships between tumour mutation status and prognostic factors including signalment, tumour histological grade, tumour anatomical location and tumour size. C-KIT exon 11 ITDs were detected by PCR in DNA extracted from formalin-fixed, paraffin-embedded canine MCTs sourced from three veterinary diagnostic laboratories in Adelaide and Melbourne. Tumours were graded according to two different systems (Patnaik and Kiupel systems) by board-certified anatomical pathologists blinded to the PCR results. Relationships between tumour mutation status and prognostic factors were evaluated using a generalised binary logistic regression analysis. ITDs were identified in 13 of 74 cutaneous canine MCT samples, giving an overall prevalence of 17.6% (95% confidence interval: 8.9-26.2%). ITDs were detected in 10 of 18 Patnaik grade III MCTs (55.6%) and 11 of 22 Kiupel high-grade MCTs (50%). Wald chi-square analysis revealed that detection of tumour ITDs was significantly associated with both Patnaik's and Kiupel's histologic grading systems (each: P < 0.001). The presence of the ITDs in MCTs was not associated with signalment, tumour anatomical location or tumour size. The prevalence of C-KIT exon 11 ITDs in Australian canine MCTs is similar to the prevalence in overseas canine populations (overall prevalence in Australia approximately 18%). ITDs were more frequently identified in higher grade MCTs. © 2017 Australian Veterinary Association.

Validation of an Anaplasma marginale cELISA for use in the diagnosis of A. ovis infections in domestic sheep and Anaplasma spp. in wild ungulates.

PubMed

Scoles, Glen A; Goff, Will L; Lysyk, Timothy J; Lewis, Gregory S; Knowles, Donald P

2008-07-27

A commercially available (cELISA) kit for diagnosing Anaplasma marginale infection in cattle was validated for diagnosing A ovis infection in sheep using the bovine serum controls as supplied by the manufacturer (BcELISA) and sheep serum controls from pathogen-free sheep (OcELISA). True positives were identified using two previously established assays, a nested PCR (nPCR) test and an indirect immunofluorescent assay (IFA). The BcELISA was also applied to sera from various species of wild ruminants, comparing the results with the IFA. Receiver operating characteristic (ROC) analysis indicated that the predicted threshold inhibition for the BcELISA was 19.2. The sensitivity for the BcELISA was 98.2% and the specificity was 96.3%. The predicted threshold inhibition decreased to 14.3 for the OcELISA; the sensitivity was 96.5% and the specificity was 98.1%. There was >/=90% concordance between IFA and nPCR, as well as between the BcELISA at 19% inhibition cutoff and either IFA or PCR. Concordance between the cELISA and IFA using sera from elk, mule deer, bighorn sheep, pronghorn antelope, and black-tailed deer ranged from 64% to 100%. This commercially available cELISA test kit can be used very effectively to test domestic sheep for infection with A. ovis using the kit-supplied controls (i.e. the BcELISA) and a 19% inhibition cutoff; the kit may also be useful for detecting intra-erythrocytic Anaplasma infections in wild ruminants.

The identification of specialized pacemaking cells in the anal sphincters.

PubMed

Shafik, Ahmed; El Sibai, Olfat; Ahmed, Ismail

2006-07-01

Interstitial cells of Cajal (ICC) are claimed to generate the electrical activity in the colon and stomach. As the external (EAS) and internal (IAS) anal sphincters exhibit resting electrical activity, we hypothesized the presence of ICC in these sphincters. This hypothesis was investigated in the current study. Specimens from the EAS and IAS were taken from normal areas of the anorectum which had been surgically excised by abdominoperineal operation for rectal cancer of 28 patients (16 men, 12 women, mean age 42.2+/-4.8 years). The specimens were subjected to c-kit immunohistochemistry. Controls for the specificity of the antisera consisted of tissue incubation with normal rabbit serum substituted for the primary antiserum. Fusiform, c-kit positive, ICC-like cells were detected in the anal sphincters; they had dendritic processes. They were clearly distinguishable from the non-branching, c-kit negative smooth and striated muscle cells of the anal sphincters. The specimens contained also c-kit positive mast cells, but they had a rounded body with no dendritic processes. Immunoreactivity was absent in negative controls in which the primary antibody was omitted. We have identified, for the first time, cells in EAS and IAS with morphological and immunological phenotypes similar to ICCs of the gut. These cells appear to be responsible for initiating the slow waves recorded from the anal sphincters and for controlling their activity. A deficiency or absence of these cells may affect the anal motile activity. Studies are needed to explore the role of these cells in anal motility disorders.

Effects of Gestational Maternal Undernutrition on Growth, Carcass Composition and Meat Quality of Rabbit Offspring

PubMed Central

Symeon, George K.; Goliomytis, Michael; Bizelis, Iosif; Papadomichelakis, George; Pagonopoulou, Olga; Abas, Zafeiris; Deligeorgis, Stelios G.; Chadio, Stella E.

2015-01-01

An experiment was conducted in order to evaluate the effects of gestational undernutrition of rabbit does on growth, carcass composition and meat quality of the offsprings. Thirty primiparous non lactating rabbit does were artificially inseminated and randomly divided in three treatment groups: Control (C; fed to 100% of maintenance requirements throughout gestation, n = 10), early undernourished (EU; fed to 50% of maintenance requirements during days 7–19 of gestation, n = 10) and late undernourished (LU; fed to 50% of maintenance requirements during days 20-27 of gestation, n = 10). During the 4th week of the gestation period, LU does significantly lost weight compared to C and EU groups (P<0.05). At kindling, C does produced litters with higher proportions of stillborn kits (P<0.05) while the total litter size (alive and stillborn kits) was not different among groups (10.7, 12.8 and 12.7 kits in C, EU and LU groups, respectively). Kit birth weight tended to be lower in the LU group. During fattening, body weight and feed intake were not different among offsprings of the three experimental groups. Moreover, the maternal undernutrition did not have any impact on carcass composition of the offsprings in terms of carcass parts and internal organs weights as well as meat quality of L. lumborum muscle (pH24, colour, water holding capacity and shear values) at slaughter (70 days of age). Therefore, it can be concluded that the gestational undernutrition of the mother does not have detrimental effects on the productive and quality traits of the offsprings. PMID:25671602

Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.

PubMed

Bidmon, Nicole; Kind, Sonja; Welters, Marij J P; Joseph-Pietras, Deborah; Laske, Karoline; Maurer, Dominik; Hadrup, Sine Reker; Schreibelt, Gerty; Rae, Richard; Sahin, Ugur; Gouttefangeas, Cécile; Britten, Cedrik M; van der Burg, Sjoerd H

2018-07-01

Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users to generate large-scale TERS, on-site using the most commonly used electroporation (EP) devices, an RNA-based kit approach, providing stable TCR mRNA and an optimized manufacturing protocol were established. In preparation for the release of this immuno-control kit, we established optimal EP conditions on six devices and initiated an extended RNA stability study. Furthermore, we coordinated on-site production of TERS with 4 participants. Finally, a proficiency panel was organized to test the unsupervised production of TERS at different laboratories using the kit approach. The results obtained show the feasibility and robustness of the kit approach for versatile in-house production of cellular control samples. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Large-Pore 3D Cubic Mesoporous (KIT-6) Hybrid Bearing a Hard-Soft Donor Combined Ligand for Enhancing U(VI) Capture: An Experimental and Theoretical Investigation.

PubMed

Yuan, Li-Yong; Zhu, Lin; Xiao, Cheng-Liang; Wu, Qun-Yan; Zhang, Nan; Yu, Ji-Pan; Chai, Zhi-Fang; Shi, Wei-Qun

2017-02-01

A preorganized tetradentate phenanthrolineamide (DAPhen) ligand with hard and soft donors combined in the same molecule has been found to possess high extraction ability toward actinides over lanthanides from acidic aqueous solution in our previous work. Herein we grafted phenanthrolineamide groups onto a large-pore three-dimensional cubic silica support by the reaction of DAPhen siloxane with KIT-6 substrate to prepare a novel uranium-selective sorbent, KIT-6-DAPhen. The as-synthesized sorbent was well-characterized by scanning electron microscopy, high-resolution transmission electron microscopy, N 2 adsorption/desorption, X-ray diffraction, FT-IR, 13 C cross-polarization magic-angle spinning NMR, and TGA techniques, which confirmed the consummation of the functionalization. Subsequently, the effects of contact time, solution pH, initial U(VI) concentration, and the presence of competing metal ions on the U(VI) sorption onto KIT-6-DAPhen sorbent were investigated in detail. It was found that KIT-6-DAPhen showed largely enhanced sorption capacity and excellent selectivity toward U(VI). The maximum sorption capacity of KIT-6-DAPhen at pH 5.0 reaches 328 mg of U/g of sorbent, which is superior to most of functionalized mesoporous silica materials. Density functional theory coupled with quasi-relativistic small-core pseudopotentials was used to explore the sorption interaction between U(VI) and KIT-6-DAPhen, which gives a sorption reaction of KIT-6-DAPhen + [UO 2 (H 2 O) 5 ] 2+ + NO 3 - ⇄ [UO 2 (KIT-6-DAPhen)(NO 3 )] + + 5H 2 O. The findings of the present work provide new clues for developing new actinide sorbents by combining new ligands with various mesoporous matrixes.

A Frameshift Mutation in KIT is Associated with White Spotting in the Arabian Camel

PubMed Central

Holl, Heather; Isaza, Ramiro; Mohamoud, Yasmin; Ahmed, Ayeda; Almathen, Faisal; Youcef, Cherifi; Gaouar, Semir; Antczak, Douglas F.; Brooks, Samantha

2017-01-01

While the typical Arabian camel is characterized by a single colored coat, there are rare populations with white spotting patterns. White spotting coat patterns are found in virtually all domesticated species, but are rare in wild species. Theories suggest that white spotting is linked to the domestication process, and is occasionally associated with health disorders. Though mutations have been found in a diverse array of species, fewer than 30 genes have been associated with spotting patterns, thus providing a key set of candidate genes for the Arabian camel. We obtained 26 spotted camels and 24 solid controls for candidate gene analysis. One spotted and eight solid camels were whole genome sequenced as part of a separate project. The spotted camel was heterozygous for a frameshift deletion in KIT (c.1842delG, named KITW1 for White spotting 1), whereas all other camels were wild-type (KIT+/KIT+). No additional mutations unique to the spotted camel were detected in the EDNRB, EDN3, SOX10, KITLG, PDGFRA, MITF, and PAX3 candidate white spotting genes. Sanger sequencing of the study population identified an additional five KITW1/KIT+ spotted camels. The frameshift results in a premature stop codon five amino acids downstream, thus terminating KIT at the tyrosine kinase domain. An additional 13 spotted camels tested KIT+/KIT+, but due to phenotypic differences when compared to the KITW1/KIT+ camels, they likely represent an independent mutation. Our study suggests that there are at least two causes of white spotting in the Arabian camel, the newly described KITW1 allele and an uncharacterized mutation. PMID:28282952

Unifying mechanism for different fibrotic diseases

PubMed Central

Wernig, Gerlinde; Chen, Shih-Yu; Cui, Lu; Van Neste, Camille; Tsai, Jonathan M.; Kambham, Neeraja; Vogel, Hannes; Natkunam, Yaso; Gilliland, D. Gary; Nolan, Garry; Weissman, Irving L.

2017-01-01

Fibrotic diseases are not well-understood. They represent a number of different diseases that are characterized by the development of severe organ fibrosis without any obvious cause, such as the devastating diseases idiopathic pulmonary fibrosis (IPF) and scleroderma. These diseases have a poor prognosis comparable with endstage cancer and are uncurable. Given the phenotypic differences, it was assumed that the different fibrotic diseases also have different pathomechanisms. Here, we demonstrate that many endstage fibrotic diseases, including IPF; scleroderma; myelofibrosis; kidney-, pancreas-, and heart-fibrosis; and nonalcoholic steatohepatosis converge in the activation of the AP1 transcription factor c-JUN in the pathologic fibroblasts. Expression of the related AP1 transcription factor FRA2 was restricted to pulmonary artery hypertension. Induction of c-Jun in mice was sufficient to induce severe fibrosis in multiple organs and steatohepatosis, which was dependent on sustained c-Jun expression. Single cell mass cytometry revealed that c-Jun activates multiple signaling pathways in mice, including pAkt and CD47, which were also induced in human disease. αCD47 antibody treatment and VEGF or PI3K inhibition reversed various organ c-Jun–mediated fibroses in vivo. These data suggest that c-JUN is a central molecular mediator of most fibrotic conditions. PMID:28424250

Transcriptional profiling reveals regulated genes in the hippocampus during memory formation

NASA Technical Reports Server (NTRS)

Donahue, Christine P.; Jensen, Roderick V.; Ochiishi, Tomoyo; Eisenstein, Ingrid; Zhao, Mingrui; Shors, Tracey; Kosik, Kenneth S.

2002-01-01

Transcriptional profiling (TP) offers a powerful approach to identify genes activated during memory formation and, by inference, the molecular pathways involved. Trace eyeblink conditioning is well suited for the study of regional gene expression because it requires the hippocampus, whereas the highly parallel task, delay conditioning, does not. First, we determined when gene expression was most regulated during trace conditioning. Rats were exposed to 200 trials per day of paired and unpaired stimuli each day for 4 days. Changes in gene expression were most apparent 24 h after exposure to 200 trials. Therefore, we profiled gene expression in the hippocampus 24 h after 200 trials of trace eyeblink conditioning, on multiple arrays using additional animals. Of 1,186 genes on the filter array, seven genes met the statistical criteria and were also validated by real-time polymerase chain reaction. These genes were growth hormone (GH), c-kit receptor tyrosine kinase (c-kit), glutamate receptor, metabotropic 5 (mGluR5), nerve growth factor-beta (NGF-beta), Jun oncogene (c-Jun), transmembrane receptor Unc5H1 (UNC5H1), and transmembrane receptor Unc5H2 (UNC5H2). All these genes, except for GH, were downregulated in response to trace conditioning. GH was upregulated; therefore, we also validated the downregulation of the GH inhibitor, somatostatin (SST), even though it just failed to meet criteria on the arrays. By during situ hybridization, GH was expressed throughout the cell layers of the hippocampus in response to trace conditioning. None of the genes regulated in trace eyeblink conditioning were similarly affected by delay conditioning, a task that does not require the hippocampus. These findings demonstrate that transcriptional profiling can exhibit a repertoire of genes sensitive to the formation of hippocampal-dependent associative memories.

Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function

PubMed Central

Dudek, Steven M.; Chiang, Eddie T.; Camp, Sara M.; Guo, Yurong; Zhao, Jing; Brown, Mary E.; Singleton, Patrick A.; Wang, Lichun; Desai, Anjali; Arce, Fernando T.; Lal, Ratnesh; Van Eyk, Jennifer E.; Imam, Syed Z.

2010-01-01

Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement. PMID:20861316

Differential Effects of CSF-1R D802V and KIT D816V Homologous Mutations on Receptor Tertiary Structure and Allosteric Communication

PubMed Central

Da Silva Figueiredo Celestino Gomes, Priscila; Panel, Nicolas; Laine, Elodie; Pascutti, Pedro Geraldo; Solary, Eric; Tchertanov, Luba

2014-01-01

The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind. PMID:24828813

Advanced Oxidation Protein Products and Carbonylated Proteins as Biomarkers of Oxidative Stress in Selected Atherosclerosis-Mediated Diseases.

PubMed

Gryszczyńska, Bogna; Formanowicz, Dorota; Budzyń, Magdalena; Wanic-Kossowska, Maria; Pawliczak, Elżbieta; Formanowicz, Piotr; Majewski, Wacław; Strzyżewski, Krzysztof Wojciech; Kasprzak, Magdalena P; Iskra, Maria

2017-01-01

The main question of this study was to evaluate the intensity of oxidative protein modification shown as advanced oxidation protein products (AOPP) and carbonylated proteins, expressed as protein carbonyl content (C=O) in abdominal aortic aneurysms (AAA), aortoiliac occlusive disease (AIOD), and chronic kidney disease (CKD). The study was carried out in a group of 35 AAA patients and 13 AIOD patients. However, CKD patients were divided into two groups: predialysis (PRE) included 50 patients or hemodialysis (HD) consisted of 34 patients. AOPP and C=O were measured using colorimetric assay kit, while C-reactive protein concentration was measured by high-sensitivity assay (hsCRP). The concentration of AOPP in both AAA and AIOD groups was higher than in PRE and HD groups according to descending order: AAA~AIOD > HD > PRE. The content of C=O was higher in the PRE group in comparison to AIOD and AAA according to the descending order: PRE~HD > AAA~AIOD. AAA, AIOD, and CKD-related atherosclerosis (PRE and HD) contribute to the changes in the formation of AOPP and C=O. They may promote modification of proteins in a different way, probably due to the various factors that influence oxidative stress here.

The KIT gene is associated with the english spotting coat color locus and congenital megacolon in Checkered Giant rabbits (Oryctolagus cuniculus).

PubMed

Fontanesi, Luca; Vargiolu, Manuela; Scotti, Emilio; Latorre, Rocco; Faussone Pellegrini, Maria Simonetta; Mazzoni, Maurizio; Asti, Martina; Chiocchetti, Roberto; Romeo, Giovanni; Clavenzani, Paolo; De Giorgio, Roberto

2014-01-01

The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated ("mega") cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ=0.00 LOD  =75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5-10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon.

The KIT Gene Is Associated with the English Spotting Coat Color Locus and Congenital Megacolon in Checkered Giant Rabbits (Oryctolagus cuniculus)

PubMed Central

Fontanesi, Luca; Vargiolu, Manuela; Scotti, Emilio; Latorre, Rocco; Faussone Pellegrini, Maria Simonetta; Mazzoni, Maurizio; Asti, Martina; Chiocchetti, Roberto; Romeo, Giovanni; Clavenzani, Paolo; De Giorgio, Roberto

2014-01-01

The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated (“mega”) cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ = 0.00 LOD  = 75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5–10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon. PMID:24736498

Mast cell deficiency attenuates acupuncture analgesia for mechanical pain using c-kit gene mutant rats.

PubMed

Cui, Xiang; Liu, Kun; Xu, Dandan; Zhang, Youyou; He, Xun; Liu, Hao; Gao, Xinyan; Zhu, Bing

2018-01-01

Acupuncture therapy plays a pivotal role in pain relief, and increasing evidence demonstrates that mast cells (MCs) may mediate acupuncture analgesia. The present study aims to investigate the role of MCs in acupuncture analgesia using c-kit gene mutant-induced MC-deficient rats. WsRC-Ws/Ws rats and their wild-type (WT) littermates (WsRC-+/+) were used. The number of MCs in skin of ST36 area was compared in two rats after immunofluorescence labeling. Mechanical withdrawal latency (MWL), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL) were measured on bilateral plantar for pain threshold evaluation before and after each stimulus. Acupuncture- and moxibustion-like stimuli (43°C, 46°C heat, 1 mA electroacupuncture [EA], 3 mA EA, and manual acupuncture [MA]) were applied randomly on different days. Fewer MCs were observed in the skin of ST36 in mutant rats compared to WT rats ( P <0.001). For pain thresholds, MWL and MWT were higher in WsRC-Ws/Ws compared to WsRC-+/+ on bilateral paws ( P <0.05), but TWL was not different between the two rats ( P >0.05). Bilateral MWL and MWT in WsRC-+/+ rats increased significantly after each stimulus compared to baseline ( P <0.01, P <0.001). In WsRC-Ws/Ws rats, only noxious stimuli could produce anti-nociceptive effects for mechanical pain (46°C, 3 mA EA, MA) ( P <0.01, P <0.001). Additionally, the net increases in MWL and MWT induced by most stimuli were greater in WT than in mutant rats ( P <0.05). For thermal nociception, either high- or low-intensity stimuli could significantly augment TWL in two rats ( P <0.001), and the net increases of TWL evoked by most stimuli were to the same extent in two genetic variants. MCs influence the basic mechanical but not thermal pain threshold. MCs participate in acupuncture analgesia in mechanical but not in thermal nociception, in that MC deficiency may attenuate the mechanical analgesia evoked by high-intensity stimuli and eliminate analgesia provoked by low-intensity stimuli.

Postabsorptive hyperglucagonemia in patients with type 2 diabetes mellitus analyzed with a novel enzyme-linked immunosorbent assay.

PubMed

Matsuo, Toshihiro; Miyagawa, Jun-Ichiro; Kusunoki, Yoshiki; Miuchi, Masayuki; Ikawa, Takashi; Akagami, Takafumi; Tokuda, Masaru; Katsuno, Tomoyuki; Kushida, Akira; Inagaki, Takashi; Namba, Mitsuyoshi

2016-05-01

The aims of the present study were to investigate the performance of a novel sandwich enzyme-linked immunosorbent assay (ELISA) for measuring glucagon (1-29) with monoclonal antibodies against both the C- and N-terminal regions of glucagon (1-29), and to analyze the differences in plasma levels and responses of glucagon (1-29) to oral glucose loading in normal glucose tolerance (NGT) subjects and patients with type 2 diabetes mellitus. The cross-reactivity against proglucagon fragments using the ELISA kit and two types of conventional radioimmunoassay (RIA) kits was evaluated. A 75-g oral glucose tolerance test was carried out with NGT subjects and patients with type 2 diabetes mellitus, and the glucagon (1-29) concentration was measured using three types of kit. The ELISA kit clearly had the lowest cross-reactivity against miniglucagon (19-29) and glicentin (1-61). The oral glucose tolerance test was carried out with 30 NGT and 17 patients with type 2 diabetes mellitus. The glucagon (1-29) levels measured by the ELISA kit after glucose loading were significantly higher at all time-points in the type 2 diabetes mellitus group than in the NGT group. However, the glucagon (1-29) levels measured by one RIA kit were significantly higher in the NGT group, and those measured with the other RIA kit were approximately the same among the groups. The novel sandwich ELISA accurately determines plasma glucagon (1-29) concentrations with much less cross-reactivity against other proglucagon fragments than conventional RIA kits.

Whole-exome sequencing identifies recurrent SF3B1 R625 mutation and comutation of NF1 and KIT in mucosal melanoma.

PubMed

Hintzsche, Jennifer D; Gorden, Nicholas T; Amato, Carol M; Kim, Jihye; Wuensch, Kelsey E; Robinson, Steven E; Applegate, Allison J; Couts, Kasey L; Medina, Theresa M; Wells, Keith R; Wisell, Joshua A; McCarter, Martin D; Box, Neil F; Shellman, Yiqun G; Gonzalez, Rene C; Lewis, Karl D; Tentler, John J; Tan, Aik Choon; Robinson, William A

2017-06-01

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease.

Whole-exome sequencing identifies recurrent SF3B1 R625 mutation and comutation of NF1 and KIT in mucosal melanoma

PubMed Central

Hintzsche, Jennifer D.; Gorden, Nicholas T.; Amato, Carol M.; Kim, Jihye; Wuensch, Kelsey E.; Robinson, Steven E.; Applegate, Allison J.; Couts, Kasey L.; Medina, Theresa M.; Wells, Keith R.; Wisell, Joshua A.; McCarter, Martin D.; Box, Neil F.; Shellman, Yiqun G.; Gonzalez, Rene C.; Lewis, Karl D.; Tentler, John J.

2017-01-01

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease. PMID:28296713

Foreign Language Analysis and Recognition (FLARe) Initial Progress

DTIC Science & Technology

2012-11-29

University Language Modeling ToolKit CoMMA Count Mediated Morphological Analysis CRUD Create, Read , Update & Delete CPAN Comprehensive Perl Archive...DATES COVERED (From - To) 1 October 2010 – 30 September 2012 4. TITLE AND SUBTITLE Foreign Language Analysis and Recognition (FLARe) Initial Progress...AFRL-RH-WP-TR-2012-0165 FOREIGN LANGUAGE ANALYSIS AND RECOGNITION (FLARE) INITIAL PROGRESS Brian M. Ore

The antimicrobial peptide derived from insulin-like growth factor-binding protein 5, AMP-IBP5, regulates keratinocyte functions through Mas-related gene X receptors.

PubMed

Chieosilapatham, Panjit; Niyonsaba, François; Kiatsurayanon, Chanisa; Okumura, Ko; Ikeda, Shigaku; Ogawa, Hideoki

2017-10-01

In addition to their microbicidal properties, host defense peptides (HDPs) display various immunomodulatory functions, including keratinocyte production of cytokines/chemokines, proliferation, migration and wound healing. Recently, a novel HDP named AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5) was shown to exhibit antimicrobial activity against numerous pathogens, even at concentrations comparable to those of human β-defensins and LL-37. However, the immunomodulatory role of AMP-IBP5 in cutaneous tissue remains unknown. To investigate whether AMP-IBP5 triggers keratinocyte activation and to clarify its mechanism. Production of cytokines/chemokines and growth factors was determined by appropriate ELISA kits. Cell migration was assessed by in vitro wound closure assay, whereas cell proliferation was analyzed using BrdU incorporation assay complimented with XTT assay. MAPK and NF-κB activation was determined by Western blotting. Intracellular cAMP levels were assessed using cAMP enzyme immunoassay kit. Among various cytokines/chemokines and growth factors tested, AMP-IBP5 selectively increased the production of IL-8 and VEGF. Moreover, AMP-IBP5 markedly enhanced keratinocyte migration and proliferation. AMP-IBP5-induced keratinocyte activation was mediated by Mrg X1-X4 receptors with MAPK and NF-κB pathways working downstream, as evidenced by the inhibitory effects of MrgX1-X4 siRNAs and ERK-, JNK-, p38- and NF-κB-specific inhibitors. We confirmed that AMP-IBP5 indeed induced MAPK and NF-κB activation. Furthermore, AMP-IBP5-induced VEGF but not IL-8 production correlated with an increase in intracellular cAMP. Our findings suggest that in addition to its antimicrobial function, AMP-IBP5 might contribute to wound healing process through activation of keratinocytes. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Soluble stem cell factor receptor (CD117) and IL-2 receptor alpha chain (CD25) levels in the plasma of patients with mastocytosis: relationships to disease severity and bone marrow pathology.

PubMed

Akin, C; Schwartz, L B; Kitoh, T; Obayashi, H; Worobec, A S; Scott, L M; Metcalfe, D D

2000-08-15

Systemic mastocytosis is a disease of mast cell proliferation that may be associated with hematologic disorders. There are no features on examination that allow the diagnosis of systemic disease, and mast cell-derived mediators, which may be elevated in urine or blood, may also be elevated in individuals with severe allergic disorders. Thus, the diagnosis usually depends on results of bone marrow biopsy. To facilitate evaluation, surrogate markers of the extent and severity of the disease are needed. Because of the association of mastocytosis with hematologic disease, plasma levels were measured for soluble KIT (sKIT) and soluble interleukin-2 receptor alpha chain (sCD25), which are known to be cleaved in part from the mast cell surface and are elevated in some hematologic malignancies. Results revealed that levels of both soluble receptors are increased in systemic mastocytosis. Median plasma sKIT concentrations as expressed by AU/mL (1 AU = 1.4 ng/mL) were as follows: controls, 176 (n = 60); urticaria pigmentosa without systemic involvement, 194 (n = 8); systemic indolent mastocytosis, 511 (n = 30); systemic mastocytosis with an associated hematologic disorder, 1320 (n = 7); aggressive mastocytosis, 3390 (n = 3). Plasma sCD25 levels were elevated in systemic mastocytosis; the highest levels were associated with extensive bone marrow involvement. Levels of sKIT correlated with total tryptase levels, sCD25 levels, and bone marrow pathology. These results demonstrate that sKIT and sCD25 are useful surrogate markers of disease severity in patients with mastocytosis and should aid in diagnosis, in the selection of those needing a bone marrow biopsy, and in the documentation of disease progression. (Blood. 2000;96:1267-1273)

DeePMD-kit: A deep learning package for many-body potential energy representation and molecular dynamics

NASA Astrophysics Data System (ADS)

Wang, Han; Zhang, Linfeng; Han, Jiequn; E, Weinan

2018-07-01

Recent developments in many-body potential energy representation via deep learning have brought new hopes to addressing the accuracy-versus-efficiency dilemma in molecular simulations. Here we describe DeePMD-kit, a package written in Python/C++ that has been designed to minimize the effort required to build deep learning based representation of potential energy and force field and to perform molecular dynamics. Potential applications of DeePMD-kit span from finite molecules to extended systems and from metallic systems to chemically bonded systems. DeePMD-kit is interfaced with TensorFlow, one of the most popular deep learning frameworks, making the training process highly automatic and efficient. On the other end, DeePMD-kit is interfaced with high-performance classical molecular dynamics and quantum (path-integral) molecular dynamics packages, i.e., LAMMPS and the i-PI, respectively. Thus, upon training, the potential energy and force field models can be used to perform efficient molecular simulations for different purposes. As an example of the many potential applications of the package, we use DeePMD-kit to learn the interatomic potential energy and forces of a water model using data obtained from density functional theory. We demonstrate that the resulted molecular dynamics model reproduces accurately the structural information contained in the original model.

Multiplex method for initial complex testing of antibodies to blood transmitted diseases agents.

PubMed

Poltavchenko, Alexander G; Nechitaylo, Oleg V; Filatov, Pavel V; Ersh, Anna V; Gureyev, Vadim N

2016-10-01

Initial screening of donors and population at high risk of infection with blood transmitted diseases involves a number of analyses using monospesific diagnostic systems, and therefore is expensive labor- and time-consuming process. The goal of this work is to construct a multiplex test enabling to carry out rapid initial complex testing at a low price. The paper describes a kit making it possible to detect simultaneously antibodies to six agents of the most significant blood transmitted diseases: HIV virus, hepatitis B and C viruses, cytomegalovirus, T. pallidum and T. gondii in blood products. The kit comprises multiplex dot-immunoassay based on plane protein arrays (immune chips) using colloidal gold conjugates and silver development. It provides an opportunity to carry out complex analysis within 70min at room temperature, and there is no need of well-qualified personnel. We compared laboratory findings of the kit with monospecific kits for ELISA produced by two Russian commercial companies. Dot-assay results correlate well with data obtained using commercial kits for ELISA. Furthermore, multiplex analysis is quicker and cheaper in comparison with ELISA and can be carried out in non-laboratory conditions. The kit for multiplex dot-immunoassay of antibodies to blood transmitted agents can significantly simplify initial complex testing. Copyright © 2016 Elsevier B.V. All rights reserved.

Stem cell factor and interleukin-2/15 combine to enhance MAPK-mediated proliferation of human natural killer cells

PubMed Central

Benson, Don M.; Yu, Jianhua; Becknell, Brian; Wei, Min; Freud, Aharon G.; Ferketich, Amy K.; Trotta, Rossana; Perrotti, Danilo; Briesewitz, Roger

2009-01-01

Stem cell factor (SCF) promotes synergistic cellular proliferation in combination with several growth factors, and appears important for normal natural killer (NK)–cell development. CD34+ hematopoietic precursor cells (HPCs) require interleukin-15 (IL-15) for differentiation into human NK cells, and this effect can be mimicked by IL-2. Culture of CD34+ HPCs or some primary human NK cells in IL-2/15 and SCF results in enhanced growth compared with either cytokine alone. The molecular mechanisms responsible for this are unknown and were investigated in the present work. Activation of NK cells by IL-2/15 increases expression of c-kit whose kinase activity is required for synergy with IL-2/15 signaling. Mitogen-activated protein kinase (MAPK) signaling intermediaries that are activated both by SCF and IL-2/15 are enhanced in combination to facilitate earlier cell-cycle entry. The effect results at least in part via enhanced MAPK-mediated modulation of p27 and CDK4. Collectively the data reveal a novel mechanism by which SCF enhances cellular proliferation in combination with IL-2/15 in primary human NK cells. PMID:19060242

The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

PubMed

Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

2001-05-01

cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development. Copyright 2001 Academic Press.

The ERK/CREB pathway is involved in the c-Ski expression induced by low TGF-β1 concentrations during primary fibroblast proliferation.

PubMed

Li, Ping; Liu, Ping; Peng, Yan; Zhang, Zhuo-Hang; Li, Xiao-Ming; Xiong, Ren-Ping; Chen, Xing; Zhao, Yan; Ning, Ya-Lei; Yang, Nan; Zhang, Bo; Zhou, Yuan-Guo

2018-06-27

Increasing evidence has suggested that bidirectional regulation of cell proliferation is one important effect of TGF-β1 in wound healing. Increased c-Ski expression plays a role in promoting fibroblast proliferation at low TGF-β1 concentrations, but the mechanism by which low TGF-β1 concentrations regulate c-Ski levels remains unclear. In this study, the proliferation of rat primary fibroblasts was assessed with an ELISA BrdU kit. The mRNA and protein expression and phosphorylation levels of corresponding factors were measured by RT-qPCR, immunohistochemistry or Western blotting. We first found that low TGF-β1 concentrations not only promoted c-Ski mRNA and protein expression in rat primary fibroblasts but also increased the phosphorylation levels of Extracellular Signal-Regulated Kinases (ERK) and cAMP response element binding (CREB) protein. An ERK kinase (mitogen-activated protein kinase kinase, MEK) inhibitor significantly inhibited ERK1/2 phosphorylation levels, markedly reducing c-Ski expression and CREB phosphorylation levels and abrogating the growth-promoting effect of low TGF-β1 concentrations. At the same time, Smad2/3 phosphorylation levels were not significantly changed. Taken together, these results suggest that the increased cell proliferation induced by low TGF-β1 concentrations mediates c-Ski expression potentially through the ERK/CREB pathway rather than through the classic TGF-β1/Smad pathway.

3D display for enhanced tele-operation and other applications

NASA Astrophysics Data System (ADS)

Edmondson, Richard; Pezzaniti, J. Larry; Vaden, Justin; Hyatt, Brian; Morris, James; Chenault, David; Bodenhamer, Andrew; Pettijohn, Bradley; Tchon, Joe; Barnidge, Tracy; Kaufman, Seth; Kingston, David; Newell, Scott

2010-04-01

In this paper, we report on the use of a 3D vision field upgrade kit for TALON robot consisting of a replacement flat panel stereoscopic display, and multiple stereo camera systems. An assessment of the system's use for robotic driving, manipulation, and surveillance operations was conducted. A replacement display, replacement mast camera with zoom, auto-focus, and variable convergence, and a replacement gripper camera with fixed focus and zoom comprise the upgrade kit. The stereo mast camera allows for improved driving and situational awareness as well as scene survey. The stereo gripper camera allows for improved manipulation in typical TALON missions.

Choline Alleviates Parenteral Nutrition-Associated Duodenal Motility Disorder in Infant Rats.

PubMed

Zhu, Jie; Wu, Yang; Guo, Yonggao; Tang, Qingya; Lu, Ting; Cai, Wei; Huang, Haiyan

2016-09-01

Parenteral nutrition (PN) has been found to influence duodenal motility in animals. Choline is an essential nutrient, and its deficiency is related to PN-associated organ diseases. Therefore, this study was aimed to investigate the role of choline supplementation in an infant rat model of PN-associated duodenal motility disorder. Three-week-old Sprague-Dawley male rats were fed chow and water (controls), PN solution (PN), or PN plus intravenous choline (600 mg/kg) (PN + choline). Rats underwent jugular vein cannulation for infusion of PN solution or 0.9% saline (controls) for 7 days. Duodenal oxidative stress status, concentrations of plasma choline, phosphocholine, and betaine and serum tumor necrosis factor (TNF)-α were assayed. The messenger RNA (mRNA) and protein expression of c-Kit proto-oncogene protein (c-Kit) and membrane-bound stem cell factor (mSCF) together with the electrophysiological features of slow waves in the duodenum were also evaluated. Rats on PN showed increased reactive oxygen species; decreased total antioxidant capacity in the duodenum; reduced plasma choline, phosphocholine, and betaine; and enhanced serum TNF-α concentrations, which were reversed by choline intervention. In addition, PN reduced mRNA and protein expression of mSCF and c-Kit, which were inversed under choline administration. Moreover, choline attenuated depolarized resting membrane potential and declined the frequency and amplitude of slow waves in duodenal smooth muscles of infant rats induced by PN, respectively. The addition of choline to PN may alleviate the progression of duodenal motor disorder through protecting smooth muscle cells from injury, promoting mSCF/c-Kit signaling, and attenuating impairment of interstitial cells of Cajal in the duodenum during PN feeding. © 2015 American Society for Parenteral and Enteral Nutrition.

Ginsenoside Rg1 improves bone marrow haematopoietic activity via extramedullary haematopoiesis of the spleen

PubMed Central

Liu, Hua-Hsing; Chen, Fei-Peng; Liu, Rong-Kai; Lin, Chun-Lin; Chang, Ko-Tung

2015-01-01

Cyclophosphamide (CY) is a chemotherapeutic agent used for cancer and immunological diseases. It induces cytotoxicity of bone marrow and causes myelosuppression and extramedullary haematopoiesis (EMH) in treated patients. EMH is characterized with the emergence of multipotent haematopoietic progenitors most likely in the spleen and liver. Previous studies indicated that a Chinese medicine, ginsenoside Rg1, confers a significant effect to elevate the number of lineage (Lin−) Sca-1+ c-Kit+ haematopoietic stem and progenitor cells (HSPCs) and restore the function of bone marrow in CY-treated myelosuppressed mice. However, whether the amelioration of bone marrow by Rg1 accompanies an alleviation of EMH in the spleen was still unknown. In our study, the cellularity and weight of the spleen were significantly reduced after Rg1 treatment in CY-treated mice. Moreover, the number of c-Kit+ HSPCs was significantly decreased but not as a result of apoptosis, indicating that Rg1 alleviated EMH of the spleen induced by CY. Unexpectedly, the proliferation activity of c-Kit+ HSPCs was only up-regulated in the spleen, but not in the bone marrow, after Rg1 treatment in CY-treated mice. We also found that a fraction of c-Kit+/CD45+ HSPCs was simultaneously increased in the circulation after Rg1 treatment. Interestingly, the effects of Rg1 on the elevation of HSPCs in bone marrow and in the peripheral blood were suppressed in CY-treated splenectomized mice. These results demonstrated that Rg1 improves myelosuppression induced by CY through its action on the proliferation of HSPCs in EMH of the spleen and migration of HSPCs from the spleen to the bone marrow. PMID:26153045

Ginsenoside Rg1 improves bone marrow haematopoietic activity via extramedullary haematopoiesis of the spleen.

PubMed

Liu, Hua-Hsing; Chen, Fei-Peng; Liu, Rong-Kai; Lin, Chun-Lin; Chang, Ko-Tung

2015-11-01

Cyclophosphamide (CY) is a chemotherapeutic agent used for cancer and immunological diseases. It induces cytotoxicity of bone marrow and causes myelosuppression and extramedullary haematopoiesis (EMH) in treated patients. EMH is characterized with the emergence of multipotent haematopoietic progenitors most likely in the spleen and liver. Previous studies indicated that a Chinese medicine, ginsenoside Rg1, confers a significant effect to elevate the number of lineage (Lin(-) ) Sca-1(+) c-Kit(+) haematopoietic stem and progenitor cells (HSPCs) and restore the function of bone marrow in CY-treated myelosuppressed mice. However, whether the amelioration of bone marrow by Rg1 accompanies an alleviation of EMH in the spleen was still unknown. In our study, the cellularity and weight of the spleen were significantly reduced after Rg1 treatment in CY-treated mice. Moreover, the number of c-Kit(+) HSPCs was significantly decreased but not as a result of apoptosis, indicating that Rg1 alleviated EMH of the spleen induced by CY. Unexpectedly, the proliferation activity of c-Kit(+) HSPCs was only up-regulated in the spleen, but not in the bone marrow, after Rg1 treatment in CY-treated mice. We also found that a fraction of c-Kit(+) /CD45(+) HSPCs was simultaneously increased in the circulation after Rg1 treatment. Interestingly, the effects of Rg1 on the elevation of HSPCs in bone marrow and in the peripheral blood were suppressed in CY-treated splenectomized mice. These results demonstrated that Rg1 improves myelosuppression induced by CY through its action on the proliferation of HSPCs in EMH of the spleen and migration of HSPCs from the spleen to the bone marrow. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

C-kit mutations in gastrointestinal stromal tumours.

PubMed

Morey, Adrienne L; Wanigesekera, G David; Hawkins, Nicholas J; Ward, Robyn L

2002-08-01

Gastrointestinal stromal tumours (GISTs), once assumed to be of smooth muscle origin, generally express CD117 and CD34, similar to the interstitial cells of Cajal. Assessment of malignant potential in GISTs is problematic, especially on small biopsies. Some recent data indicate that mutations in the juxtamembrane domain (exon 11) of the c-kit (CD117) proto-oncogene may be associated with a worse prognosis. In this study, the frequency of c-kit exon 11 mutations has been determined in a series of 18 gut stromal tumours. Immunophenotype was assessed by immunoperoxidase stains for smooth muscle actin, desmin, S100, CD34 and CD117, and each tumour classified as being of low, uncertain (intermediate) or high malignant potential based on standard histological criteria. DNA from each tumour was extracted from fresh (n = 5) or formalin-fixed, paraffin-embedded (n= 13) tissues using the direct lysis method. Exon 11 was amplified by PCR and sequencing of both sense and antisense strands was performed on two occasions using an ABI 377 sequencer. Mutations in exon 11 were detected in three of 14 confirmed GISTs, two being point mutations at codon 560 and one a 3-bp deletion resulting in the in-frame deletion of glutamine at codon 561. All three tumours were of high or intermediate malignant potential histologically. Three other 'high risk' primary GISTs and a metastatic GIST deposit were negative for exon 11 mutations. Data on this relatively small cohort of Australian patients indicate that c-kit exon 11 mutation analysis does not correlate well with histological assessment of malignant potential, and cannot be regarded as a reliable objective marker for poor prognosis in GISTs.

Long-pulse gastric electrical stimulation protects interstitial cells of Cajal in diabetic rats via IGF-1 signaling pathway.

PubMed

Li, Hai; Chen, Yan; Liu, Shi; Hou, Xiao-Hua

2016-06-21

To investigate the effects of different parameters of gastric electrical stimulation (GES) on interstitial cells of Cajal (ICCs) and changes in the insulin-like growth factor 1 (IGF-1) signal pathway in streptozotocin-induced diabetic rats. Male rats were randomized into control, diabetic (DM), diabetic with sham GES (DM + SGES), diabetic with GES1 (5.5 cpm, 100 ms, 4 mA) (DM + GES1), diabetic with GES2 (5.5 cpm, 300 ms, 4 mA) (DM + GES2) and diabetic with GES3 (5.5 cpm, 550 ms, 2 mA) (DM + GES3) groups. The expression levels of c-kit, M-SCF and IGF-1 receptors were evaluated in the gastric antrum using Western blot analysis. The distribution of ICCs was observed using immunolabeling for c-kit, while smooth muscle cells and IGF-1 receptors were identified using α-SMA and IGF-1R antibodies. Serum level of IGF-1 was tested using enzyme-linked immunosorbent assay. Gastric emptying was delayed in the DM group but improved in all GES groups, especially in the GES2 group. The expression levels of c-kit, M-SCF and IGF-1R were decreased in the DM group but increased in all GES groups. More ICCs (c-kit(+)) and smooth muscle cells (α-SMA(+)/IGF-1R(+)) were observed in all GES groups than in the DM group. The average level of IGF-1 in the DM group was markedly decreased, but it was up-regulated in all GES groups, especially in the GES2 group. The results suggest that long-pulse GES promotes the regeneration of ICCs. The IGF-1 signaling pathway might be involved in the mechanism underlying this process, which results in improved gastric emptying.

Function of oval cells in hepatocellular carcinoma in rats.

PubMed

Fang, Chi-Hua; Gong, Jia-Qing; Zhang, Wei

2004-09-01

To study oval cells' pathological characteristics and relationship with the occurrence of hepatocellular carcinoma (HCC); to observe the form and structural characteristics of oval cells; to explore the expression characteristics of C-kit, PCNA mRNA and c-myc gene during the occurrence and development of HCC and the effect of ulinastatin (UTI) on C-kit and PCNA expression. One hundred and twenty-five SD rats fed on 3,3'-diaminobenzidine (DAB) to construct HCC models were divided into control group, cancer-inducing group and UTI intervention group. In each group, rat liver samples were collected at weeks 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 respectively to study pathological distribution characteristics of oval cells in the process of carcinogenesis under optical microscope. Oval cells were separated by the methods of improved density gradient centrifugation and their structural characteristics were observed under optical microscope and electronic microscope respectively; the oval cells expressing C-kit and PCNA in the collected samples were observed by the methods of immunohistochemistry and image analysis and the expression of c-myc mRNA was also detected by reverse transcription polymerase chain reaction (RT-PCR). Oval cells proliferated firstly in the portal area then gradually migrated into hepatic parenchyma in the inducing group and intervention group. The oval cells distributed inside and outside the carcinoma nodes. The oval cells presented the characteristics of undifferentiated cells: a high ratio of nucleolus and cellular plasm and obvious nucleoli, rare organelle in plasm. Only a few mitochondria and endoplasmic reticulum and some villus-like apophysis on surface of cells could be seen. Cells stained with C-kit and PCNA antibody were mainly oval cells distributed in the portal area. The expression of c-myc mRNA increased with the progression of HCC. However, in the intervention group, UTI could retard its increase. Oval cells work throughout the development of HCC, and might play important roles in this process. c-myc gene may be a kind of promoter gene of HCC, and play a key role in hepatic injury and development of HCC. UTI could retard the occurrence of HCC. Copyright 2004 The WJG Press ISSN

Function of oval cells in hepatocellular carcinoma in rats

PubMed Central

Fang, Chi-Hua; Gong, Jia-Qing; Zhang, Wei

2004-01-01

AIM: To study oval cells pathological characteristics and relationship with the occurrence of hepatocellular carcinoma (HCC); to observe the form and structural characteristics of oval cells; to explore the expression characteristics of C-kit, PCNA mRNA and c-myc gene during the occurrence and development of HCC and the effect of ulinastatin (UTI) on C-kit and PCNA expression. METHODS: One hundred and twenty-five SD rats fed on 3,3'-diaminobenzidine (DAB) to construct HCC models were divided into control group, cancer-inducing group and UTI intervention group. In each group, rat liver samples were collected at weeks 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 respectively to study pathological distribution characteristics of oval cells in the process of carcinogenesis under optical microscope. Oval cells were separated by the methods of improved density gradient centrifugation and their structural characteristics were observed under optical microscope and electronic microscope respectively; the oval cells expressing C-kit and PCNA in the collected samples were observed by the methods of immunohistochemistry and image analysis and the expression of c-myc mRNA was also detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Oval cells proliferated firstly in the portal area then gradually migrated into hepatic parenchyma in the inducing group and intervention group. The oval cells distributed inside and outside the carcinoma nodes. The oval cells presented the characteristics of undifferentiated cells: a high ratio of nucleolus and cellular plasm and obvious nucleoli, rare organelle in plasm. Only a few mitochondria and endoplasmic reticulum and some villus-like apophysis on surface of cells could be seen. Cells stained with C-kit and PCNA antibody were mainly oval cells distributed in the portal area. The expression of c-myc mRNA increased with the progression of HCC. However, in the intervention group, UTI could retard its increase. CONCLUSION: Oval cells work throughout the development of HCC, and might play important roles in this process. c-myc gene may be a kind of promoter gene of HCC, and play a key role in hepatic injury and development of HCC. UTI could retard the occurrence of HCC. PMID:15300889

75 FR 70962 - General Motors Corporation, Grant of Petition for Decision of Inconsequential Noncompliance

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-19

... passenger cars built from April 2008 through November 12, 2008. Paragraph S4.3 of FMVSS No. 110 requires in... inflator kit serves the purpose of getting back on the road. Since the inflator kit is located in the same... Pontiac G5 passenger cars did not fully comply with paragraphs S4.3(c) and S4.3(d) of 49 CFR 571.110...

Food and medical sample freezer kit concept for Shuttle

NASA Technical Reports Server (NTRS)

Copeland, R. J.; Jaax, J. R.; Proctor, B. W.

1977-01-01

A variety of food and storage of samples can be provided by a Space Shuttle Orbiter Freezer Kit. The proposed concept is an integrated package consisting of four -23 C (-10 F) storage compartments and a Stirling cycle refrigeration unit. The Stirling cycle mechanical refrigeration was selected over alternative systems for superior efficiency and safety. The trade-offs and a conceptual design of the system are presented.

Validation of an improved Anaplasma antibody competitive ELISA for detection of Anaplasma ovis antibody in domestic sheep.

PubMed

Mason, Kathleen L; Gonzalez, Michael V; Chung, Chungwon; Mousel, Michelle R; White, Stephen N; Taylor, Joshua B; Scoles, Glen A

2017-09-01

An accurate and simple-to-perform new version of a competitive ELISA (cELISA) kit that became commercially available in 2015 for testing of cattle for antibody to Anaplasma marginale was validated for detection of Anaplasma ovis antibody in domestic sheep. True positives and negatives were identified using nested PCR (nPCR) as the gold standard. Negative bovine control sera supplied with the kit were used to calculate % inhibition (%I), designated bovine control ELISA (BcELISA), and this was compared to %I calculated from negative ovine sera derived from hand-raised, pathogen-free sheep, designated ovine control ELISA (OcELISA). The receiver operating characteristics area under the curve was 1.0 with a p value <0.001 regardless of the source of the control sera. The cutoff values for negative BcELISA and OcELISA were <30%I and <27%I, respectively. Our work confirmed that this Anaplasma antibody cELISA kit version 2 can be used with the serum controls supplied in the kit to test for A. ovis antibody in domestic sheep. Furthermore, this work confirmed the historically high infection prevalence (>93%) at the U.S. Sheep Experiment Station (Dubois, Idaho), in spite of efforts to reduce the possibility for iatrogenic transmission there, suggesting high levels of tick-borne transmission.

IL-4 downregulates expression of the target receptor CD30 in neoplastic canine mast cells

PubMed Central

Bauer, K.; Hadzijusufovic, E.; Cerny-Reiterer, S.; Hoermann, G.; Reifinger, M.; Pirker, A.; Valent, P.; Willmann, M.

2018-01-01

CD30 is a novel therapeutic target in human mast cell (MC) neoplasms. In this ‘comparative oncology’ study, we examined CD30 expression and regulation in neoplastic canine MC using a panel of immunomodulatory cytokines [interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-13 and stem cell factor (SCF)] and the canine mastocytoma cell lines NI-1 and C2. Of all cytokines tested IL-4 was found to downregulate expression of CD30 in NI-1 and C2 cells. We also found that the CD30-targeting antibody-conjugate brentuximab vedotin induces growth inhibition and apoptosis in both MC lines. Next, we asked whether IL-4-induced downregulation of CD30 interferes with brentuximab vedotin-effects. Indeed, pre-incubation of NI-1 cells with IL-4 decreased responsiveness towards brentuximab vedotin. To overcome IL-4-mediated resistance, we applied drug combinations and found that brentuximab vedotin synergizes with the Kit-targeting drugs masitinib and PKC412 in inhibiting growth of NI-1 and C2 cells. In summary, CD30 is a new marker and IL-4-regulated target in neoplastic canine MC. PMID:27507155

Evaluation of six serological ELISA kits available in Italy as screening tests for equine infectious anaemia surveillance.

PubMed

Nardini, Roberto; Autorino, Gian Luca; Issel, Charles J; Cook, R Frank; Ricci, Ida; Frontoso, Raffaele; Rosone, Francesca; Scicluna, Maria Teresa

2017-04-14

ELISAs are known to have a higher diagnostic sensitivity than the agar gel immunodiffusion (AGID) when employed for serological diagnosis of equine infectious anaemia (EIA). For this purpose, an "in-house" and five commercial ELISAs available in Italy were assessed by the National Reference Centre for EIA for their analytic specificity (Sp); precocity, defined as capability of detecting first antibodies produced during a new infection; precision based on repeatability and reproducibility, estimated from the coefficient of variation (CV); accuracy, estimated from multiple K and relative Sp and sensitivity (Se). Two serum panels, positive for non-equine retroviruses and the most frequent equine viruses, were employed to measure analytic Sp. ELISA precocity was also compared to that of one "in-house" and three commercial AGID kits, employing a panel of sera, collected weekly from horses infected with modified EIA viruses. Precision and accuracy were defined using results of a panel containing positive and negative sera examined in an inter-laboratory trial with the participation of the ten Official Laboratories. Furthermore, a questionnaire was used to assess the appropriateness of each kit for routine use. Analytic Sp was 100%, while the 75th percentile of CVs for positive sera varied from 0.4% to 12.73% for repeatability and from 1.6% to 44.87% for reproducibility. Although CV of the negative serum was constantly high, its outcome was unaltered. Relative Se ranged from 98.2% to 100%, relative Sp was constantly 100% and multiple K ranged from 0.95 to 1. Precocity differed among the assays: three kits detected 4.8% and 42.9% positive samples on 21 days post infection (dpi), all assays detected positive samples on 28 dpi, between 47.6% and 95.2%. Precocity of ELISAs was superior to that of the AGIDs except for two assays. In view of the feedback obtained from the questionnaires, all kits were considered appropriate for routine use. All ELISAs having high Se and precocity are preferable as a screening test in EIA surveillance programmes to the AGID tests examined. These two tests can be incorporated in a serial diagnostic pathway to improve the efficacy of a surveillance plan.

Dysregulation of Aldosterone Secretion in Mast Cell-Deficient Mice.

PubMed

Boyer, Hadrien-Gaël; Wils, Julien; Renouf, Sylvie; Arabo, Arnaud; Duparc, Céline; Boutelet, Isabelle; Lefebvre, Hervé; Louiset, Estelle

2017-12-01

Resident adrenal mast cells have been shown to activate aldosterone secretion in rat and man. Especially, mast cell proliferation has been observed in adrenal tissues from patients with aldosterone-producing adrenocortical adenoma. In the present study, we show that the activity of adrenal mast cells is stimulated by low-sodium diet and correlates with aldosterone synthesis in C57BL/6 and BALB/c mice. We have also investigated the regulation of aldosterone secretion in mast cell-deficient C57BL/6 Kit W-sh/W-sh mice in comparison with wild-type C57BL/6 mice. Kit W-sh/W-sh mice submitted to normal sodium diet had basal plasma aldosterone levels similar to those observed in wild-type animals. Conversely, low-sodium diet unexpectedly induced an exaggerated aldosterone response, which seemed to result from an increase in adrenal renin and angiotensin type 1 receptor expression. Severe hyperaldosteronism was associated with an increase in systolic blood pressure and marked hypokalemia, which favored polyuria. Adrenal renin and angiotensin type 1 receptor overexpression may represent a compensatory mechanism aimed at activating aldosterone production in the absence of mast cells. Finally, C57BL/6 Kit W-sh/W-sh mice represent an unexpected animal model of primary aldosteronism, which has the particularity to be triggered by sodium restriction. © 2017 American Heart Association, Inc.

Evaluation of a toxoid for protection of rabbits against enterotoxaemia experimentally induced by trypsin-activated supernatant of Clostridium spiroforme.

PubMed

Ellis, T M; Gregory, A R; Logue, G D

1991-06-01

Investigations were conducted into an enterotoxaemia caused by Clostridium spiroforme responsible for significant losses in commercial rabbit farms in Western Australia. Two trials using laboratory and farm bred rabbits were performed to evaluate the protective value of a toxoid prepared from the supernatant of C. spiroforme cultures against intraperitoneal challenge with the trypsin-activated toxin of C. spiroforme. The trials showed clearly that a single vaccination at weaning (four weeks) was protective against toxin but more complete and lasting protection was conferred following a second vaccination administered 14 days after the first. Adults likewise showed similar levels of protective antibodies but did not appear to pass on this protection to their kits although ELISA results indicated levels of antibody in kits from unvaccinated mother to be lower than progeny from vaccinated mothers. However antibody levels in kits from vaccinated mothers were very low and did not protect against challenge with toxin.

Cyclic 3',5'-adenosine monophosphate (cAMP) signaling in the anterior pituitary gland in health and disease.

PubMed

Hernández-Ramírez, Laura C; Trivellin, Giampaolo; Stratakis, Constantine A

2018-03-05

The cyclic 3',5'-adenosine monophosphate (cAMP) was the first among the so-called "second messengers" to be described. It is conserved in most organisms and functions as a signal transducer by mediating the intracellular effects of multiple hormones and neurotransmitters. In this review, we first delineate how different members of the cAMP pathway ensure its correct compartmentalization and activity, mediate the terminal intracellular effects, and allow the crosstalk with other signaling pathways. We then focus on the pituitary gland, where cAMP exerts a crucial function by controlling the responsiveness of the cells to hypothalamic hormones, neurotransmitters and peripheral factors. We discuss the most relevant physiological functions mediated by cAMP in the different pituitary cell types, and summarize the defects affecting this pathway that have been reported in the literature. We finally discuss how a deregulated cAMP pathway is involved in the pathogenesis of pituitary disorders and how it affects the response to therapy. Copyright © 2017. Published by Elsevier B.V.

Individual and family factors associated with self-esteem in young people with epilepsy: A multiple mediation analysis.

PubMed

Chew, Judith; Haase, Anne M; Carpenter, John

2017-01-01

As young people experience added demands from living with epilepsy, which may lead to poor psychosocial adjustment, it is essential to examine mechanisms of change to provide practitioners with knowledge to develop effective interventions. The aim of this study was to examine individual and family-level factors - stress and illness perceptions, coping behaviors and family resilience - that promote or maintain young people's self-esteem. From November 2013 to August 2014, young people attending a neurology clinic in KK Women's and Children's Hospital, Singapore, participated in a cross-sectional survey (n=152; 13-16years old). Multiple mediation analyses were conducted to evaluate whether these variables mediated the relationship between illness severity (i.e., low, moderate, high) and self-esteem. Multiple mediation analyses demonstrated that illness severity had a direct effect on young people's self-esteem. Compared to those with moderate illness severity (reference group), young people with low severity had significantly higher self-esteem (c=3.42, p<0.05); while those with high severity had a more negative view of themselves (c=-3.93, p<0.001). Illness severity also had an indirect influence on self-esteem through its effects on mediators, such as perceived stress, illness perceptions and family resilience (D 1 : Total ab=3.46, 95% CI 1.13, 5.71; D 2 : Total ab=-2.80, 95% CI -4.35, -1.30). However, young people's coping levels did not predict their self-esteem, when accounting for the effects of other variables. The continued presence of seizure occurrences is likely to place greater demands on young people and their families: in turn, increased stress and negative illness perceptions negatively affected family processes that promote resilience. As the mediating effect of these modifiable factors were above and beyond the contributions of illness characteristics and young people's levels of coping, this has implications for developing individual and family interventions aimed to support young people living with epilepsy. Copyright © 2016 Elsevier Inc. All rights reserved.

Autoantigen La promotes efficient RNAi, antiviral response, and transposon silencing by facilitating multiple-turnover RISC catalysis

PubMed Central

Liu, Ying; Tan, Huiling; Tian, Hui; Liang, Chunyang; Chen, She; Liu, Qinghua

2011-01-01

SUMMARY The effector of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). C3PO promotes the activation of RISC by degrading Argonaute2 (Ago2)-nicked passenger strand of duplex siRNA. Active RISC is a multiple-turnover enzyme that uses the guide strand of siRNA to direct Ago2-mediated sequence-specific cleavage of complementary mRNA. How this effector step of RNAi is regulated is currently unknown. Here, we used human Ago2 minimal RISC system to purify Sjögren’s syndrome antigen B (SSB)/autoantigen La as an activator of the RISC-mediated mRNA cleavage activity. Our reconstitution studies showed that La could promote multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from RISC. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC. PMID:22055194

A Frameshift Mutation in KIT is Associated with  White Spotting in the Arabian Camel.

PubMed

Holl, Heather; Isaza, Ramiro; Mohamoud, Yasmin; Ahmed, Ayeda; Almathen, Faisal; Youcef, Cherifi; Gaouar, Semir; Antczak, Douglas F; Brooks, Samantha

2017-03-09

While the typical Arabian camel is characterized by a single colored coat, there are rare populations with white spotting patterns. White spotting coat patterns are found in virtually all domesticated species, but are rare in wild species. Theories suggest that white spotting is linked to the domestication process, and is occasionally associated with health disorders. Though mutations have been found in a diverse array of species, fewer than 30 genes have been associated with spotting patterns, thus providing a key set of candidate genes for the Arabian camel. We obtained 26 spotted camels and 24 solid controls for candidate gene analysis. One spotted and eight solid camels were whole genome sequenced as part of a separate project. The spotted camel was heterozygous for a frameshift deletion in KIT (c.1842delG, named KITW1 for White spotting 1), whereas all other camels were wild-type (KIT+/KIT+). No additional mutations unique to the spotted camel were detected in the EDNRB, EDN3, SOX10, KITLG, PDGFRA, MITF, and PAX3 candidate white spotting genes. Sanger sequencing of the study population identified an additional five kITW1/KIT+ spotted camels. The frameshift results in a premature stop codon five amino acids downstream, thus terminating KIT at the tyrosine kinase domain. An additional 13 spotted camels tested KIT+/KIT+, but due to phenotypic differences when compared to the KITW1/KIT+ camels, they likely represent an independent mutation. Our study suggests that there are at least two causes of white spotting in the Arabian camel, the newly described KITW1 allele and an uncharacterized mutation.

Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

PubMed Central

Peinado, Héctor; Alečković, Maša; Lavotshkin, Simon; Matei, Irina; Costa-Silva, Bruno; Moreno-Bueno, Gema; Hergueta-Redondo, Marta; Williams, Caitlin; García-Santos, Guillermo; Nitadori-Hoshino, Ayuko; Hoffman, Caitlin; Badal, Karen; Garcia, Benjamin A.; Callahan, Margaret K.; Yuan, Jianda; Martins, Vilma R.; Skog, Johan; Kaplan, Rosandra N.; Brady, Mary S.; Wolchok, Jedd D.; Chapman, Paul B.; Kang, Yibin; Bromberg, Jacqueline; Lyden, David

2013-01-01

Tumor-derived exosomes are emerging mediators of tumorigenesis with tissue-specific addresses and messages. We explored the function of melanoma-derived exosomes in the formation of primary tumor and metastases in mouse and human subjects. Exosomes from highly metastatic melanoma increased the metastatic behavior of primary tumors by permanently “educating” bone marrow (BM) progenitors via the MET receptor. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites, and reprogrammed BM progenitors towards a c-Kit+Tie2+Met+ pro-vasculogenic phenotype. Reducing Met expression in exosomes diminished the pro-metastatic behavior of BM cells. Importantly, MET expression was elevated in circulating CD45−C-KITlow/+TIE2+ BM progenitors from metastatic melanoma subjects. RAB1a, RAB5b, RAB7, and RAB27a were highly expressed in melanoma cells and Rab27a RNA interference decreased exosome production, preventing BM education, tumor growth and metastasis. Finally, we identified an exosome-specific “melanoma signature” with prognostic and therapeutic potential, comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. PMID:22635005

77 FR 24211 - Agency Information Collection Activities: Proposed Collection; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2012-04-23

... through the use of automated collection techniques or other forms of information technology. Proposed... designated OTP site History (Screening) of Hepatitis C exposure Results of Rapid Hepatitis C Testing (Kit...

Evaluation of Scat Deposition Transects versus Radio Telemetry for Developing a Species Distribution Model for a Rare Desert Carnivore, the Kit Fox.

PubMed

Dempsey, Steven J; Gese, Eric M; Kluever, Bryan M; Lonsinger, Robert C; Waits, Lisette P

2015-01-01

Development and evaluation of noninvasive methods for monitoring species distribution and abundance is a growing area of ecological research. While noninvasive methods have the advantage of reduced risk of negative factors associated with capture, comparisons to methods using more traditional invasive sampling is lacking. Historically kit foxes (Vulpes macrotis) occupied the desert and semi-arid regions of southwestern North America. Once the most abundant carnivore in the Great Basin Desert of Utah, the species is now considered rare. In recent decades, attempts have been made to model the environmental variables influencing kit fox distribution. Using noninvasive scat deposition surveys for determination of kit fox presence, we modeled resource selection functions to predict kit fox distribution using three popular techniques (Maxent, fixed-effects, and mixed-effects generalized linear models) and compared these with similar models developed from invasive sampling (telemetry locations from radio-collared foxes). Resource selection functions were developed using a combination of landscape variables including elevation, slope, aspect, vegetation height, and soil type. All models were tested against subsequent scat collections as a method of model validation. We demonstrate the importance of comparing multiple model types for development of resource selection functions used to predict a species distribution, and evaluating the importance of environmental variables on species distribution. All models we examined showed a large effect of elevation on kit fox presence, followed by slope and vegetation height. However, the invasive sampling method (i.e., radio-telemetry) appeared to be better at determining resource selection, and therefore may be more robust in predicting kit fox distribution. In contrast, the distribution maps created from the noninvasive sampling (i.e., scat transects) were significantly different than the invasive method, thus scat transects may be appropriate when used in an occupancy framework to predict species distribution. We concluded that while scat deposition transects may be useful for monitoring kit fox abundance and possibly occupancy, they do not appear to be appropriate for determining resource selection. On our study area, scat transects were biased to roadways, while data collected using radio-telemetry was dictated by movements of the kit foxes themselves. We recommend that future studies applying noninvasive scat sampling should consider a more robust random sampling design across the landscape (e.g., random transects or more complete road coverage) that would then provide a more accurate and unbiased depiction of resource selection useful to predict kit fox distribution.