boyden chamber assay
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Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1635-1635
Author(s):  
Georg Greiner ◽  
Nadine Witzeneder ◽  
Klaus Schmetterer ◽  
Theresia Popow-Kraupp ◽  
Wolfgang R Sperr ◽  
...  

Abstract Systemic mastocytosis (SM) is a myeloproliferative neoplasm (MPN) characterized by mast cell (MC) infiltration in the bone marrow (BM) and/or other organs. Most patients present with the activating KIT mutation D816V. Increased production of cytokines is typically found in patients with classical MPN and has been linked to alterations of the BM microenvironment (e.g. enhanced angiogenesis and fibrosis) that are also commonly found in SM. However, little is known about mechanisms contributing to microenvironment alterations in the BM in SM and the role of MC-derived cytokines. The aim of our study was to identify cytokines that are produced in neoplastic MC in a KIT-dependent manner and contribute to increased BM angiogenesis and fibrosis in SM. In a first step, we screened for KIT D816V-dependent production of cytokines relevant to inflammation and microenvironment alterations using growth factor-dependent human cell lines (TF-1 and Mo7e). In these experiments, expression of CCL-2, IL-8, OSM, and VEGF mRNA was induced by KIT D816V but not by wild type KIT. Based on its pleiotropic effects, we focused on CCL-2, also referred to as monocyte chemotactic protein 1 (MCP-1), a CC chemokine that recruits inflammatory cells to sites of inflammation and enhances angiogenesis. KIT D816V+ HMC-1.2 cell were found to express and secrete substantial amounts of CCL-2. Midostaurin, a multikinase inhibitor suppressing the kinase activity of KIT D816V, was found to reduce expression of CCL-2 similar to RNAi-mediated knockdown of KIT. Furthermore, knockdown of STAT5, a key transcription factor downstream of KIT D816V, reduced expression of CCL-2 in HMC-1.2 cells. These results confirmed that CCL-2 expression in neoplastic MC is dependent on KIT D816V. Since CCL-2 has been reported to promote angiogenesis, we analyzed effects of conditioned medium obtained from HMC-1.2 cells on human umbilical vein endothelial cells (HUVEC) known to express the CCL-2 receptor CCR-2. Indeed, MC-derived conditioned medium induced migration of HUVEC in a wound healing assay (214% ± 38% of control, mean ± SD, p<0.01) as well as in a boyden chamber assay (126% ± 8% of control, p<0.05). Moreover, pre-incubation with a neutralizing antibody against CCL-2 significantly reduced migratory responses (p<0.01), and RNAi-mediated knockdown of CCL-2 in neoplastic MC reduced the effect of conditioned medium to baseline levels (p<0.01). These results confirmed that the migration was CCL-2 dependent. Furthermore, patients with advanced SM often present with marked eosinophilia; and eosinophils are often located in the vicinity of, or even within, BM MC infiltrates. Therefore, we also studied the effect of CCL-2 on eosinophils. Normal human eosinophils as well as the eosinophilic cell line EOL-1 were found to express CCR-2, and to migrate against recombinant CCL-2 in a modified boyden chamber assay. Conditioned medium from KIT D816V+ MC also induced a migratory response in eosinophils (200% ± 30% of control, p<0.05), and a neutralizing antibody against CCL-2 reduced this effect to baseline levels (p<0.05). Finally, SM-patients were found to have significantly elevated serum CCL-2 levels (n=35, p=0.0048, 407.4 ± 42.3 pg/ml, mean ± SEM) compared to controls (274.3 ± 15.9 pg/ml). The highest serum levels of CCL-2 were detected in patients with advanced SM where tissue remodeling in the BM is often a prominent feature. Moreover, CCL-2 levels were found to correlate with the grade of MC infiltration in the BM (r=0.656, p=0.0002). In summary, KIT D816V induces the expression of various cytokines potentially involved in tissue remodeling and microenvironment alterations in SM. Moreover, we have identified CCL-2 as a critical, KIT D816V-dependent, cytokine-mediator that interacts with structural BM cells and thereby may be involved in disease evolution and progression in SM. Whether CCL-2 may also serve as a therapeutic target in SM is currently being examined. This study was supported by Austrian Science Fund (FWF) grant P26079-B13. Disclosures Sperr: Novartis: Honoraria. Valent:Pfizer: Honoraria; Celgene: Honoraria; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Novartis: Consultancy, Honoraria, Research Funding.



2006 ◽  
Vol 1 (12) ◽  
pp. 1934578X0600101
Author(s):  
Cláudia A. Simões-Pires ◽  
Fabianne M. Farias ◽  
Andrew Marston ◽  
Emerson F. Queiroz ◽  
Célia G. Chaves ◽  
...  

The alkaloid extract of the aerial parts of Psychotria myriantha (Rubiaceae) displayed antichemotactic activity on polymorphonuclear leukocytes (PMN) assessed by the Boyden chamber assay. On analysis of the crude extract by LC/APCI/MS and LC/UV/DAD, two major constituents could be detected. In order to rapidly identify the active compounds, a microfractionation was conducted during LC/UV/DAD analysis. By this means, both the collected compounds could be assayed separately in the Boyden chamber and were shown to inhibit PMN chemotaxis. Their isolation was performed by semi-preparative HPLC and their structures elucidated by classical spectroscopic methods, including UV, NMR, MS and HRMS. Both compounds showed characteristics of monoterpene indole glucoside alkaloids; one of them was identified as strictosidinic acid and the other was a new natural product, myrianthosine. The antichemotactic activity of the compounds may be related to an antiacute inflammation activity.



2005 ◽  
Vol 73 (1) ◽  
pp. 9-18 ◽  
Author(s):  
Christian Schichor ◽  
Siglinde Kerkau ◽  
Theresa Visted ◽  
Rudolf Martini ◽  
Rolf Bjerkvig ◽  
...  


2004 ◽  
pp. 015-022 ◽  
Author(s):  
Hong-Chen Chen


BioTechniques ◽  
2002 ◽  
Vol 33 (6) ◽  
pp. 1200-1204 ◽  
Author(s):  
Madhura R. Vipra ◽  
Jayant M. Chiplonkar




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