Category: Important Info

Posted on

Leptin Stimulates Rat Aortic Smooth Muscle Cell Proliferation and Migration

Leptin, a peptide secreted from adipose tissue, plays an important role in the regulation of food intake and energy expenditure. In obese patients, plasma leptin levels are elevated and obesity is one of the major risk factors for cardiovascular diseases. Therefore, in this study, we investigated the effect of leptin on vascular smooth muscle cell (VSMC) functions. Cultured rat aortic VSMC expressed 130-kDa short form of leptin receptor. Leptin stimulated both proliferation and migration of VSMC. Leptin stimulated phosphorylation and activation of mitogen-activated protein (MAP) kinases, and also increased phosphatidylinositol (PI) 3-kinase activity. Further, two distinct PI 3-kinase inhibitors, wortmannin and LY294002 inhibited the migratory effect of leptin. These results demonstrate that leptin is a proliferative and migratory factor for VSMC, implying that leptin may play a role in the formation and development of vascular lesions.

link to pdf at http://www.ncbi.nlm.nih.gov/pubmed/11729375

Posted on

Neutrophil Chemotactic Activity of Sputum From Patients With COPD

Neutrophilic inflammation is a major feature of COPD.  Several factors in bronchial secretions have been identified as chemoattractants for neutrophils. The present study was designed to assess the contribution of interleukin (IL)-8 and leukotriene B4 (LTB4) to neutrophil chemotaxis evoked by sputum obtained from patients with established COPD.
Design: Sputum supernatant of 20 patients with COPD was used as chemoattractant in a 96-well
chemotaxis chamber, with subsequent quantification of migrated cells by a luminescence assay.
The contribution of IL-8 and LTB4 to chemotaxis was determined by addition of a neutralizing
antibody and a selective receptor antagonist, respectively.
Measurements and results: COPD sputum caused neutrophil chemotaxis in a concentration dependent manner, with a maximum response evoked with a 10-fold dilution of the original
sample. Pretreatment of sputum or neutrophils with either an anti–IL-8 antibody or the LTB4
antagonist, SB 201146, led to a concentration-dependent inhibition of sputum-induced neutrophil chemotaxis, with a maximum suppression (mean  SEM) of 29.2  4.9% (p < 0.001) from baseline by 100 ng/mL of anti–IL-8 antibody, and 45.6  7% (p < 0.02) by 10 mol/L of SB
201146. The combination of the anti–IL-8 antibody and SB 201146 inhibited neutrophil
chemotaxis, but this was not significantly greater than the effect of SB 201146 or anti–IL-8 alone.
Conclusions: These data confirm the importance of IL-8 and LTB4 as chemoattractants for
neutrophils in bronchial secretions from patients with COPD, and suggest that specific inhibitors
may have therapeutic potential in COPD.

http://journal.publications.chestnet.org/article.aspx?articleid=1081469

Posted on

Murine Astrocytes Express a Functional Chemokine Receptor

Elevated levels of chemokines have been observed in various diseases of the CNS. Little is known, however, about how these chemokines affect parenchymal cells of the CNS. The  current studies examine astrocyte chemotaxis to the mouse chemokine macrophage inflammatory protein-1 (MIP-1a). Murine astrocytes demonstrate directed migration along a chemical gradient in response to 10e-10 – 10e-8M MIP-1a. Peak chemotactic responses are noted at 1e-09M. MIP-1a-induced astrocyte migration is specifically inhibitable with pertussis toxin, suggesting a role for G1a proteins in the signaling process. RT-PCR and insitu hybridization were used to identify expression of the murine CCR1MIP-1a receptor on astrocytes. Astrocytes contain mRNA for CCR1, but messages for CCR4 and the orphan chemokine receptorMIP-1aR-like #1 were not detected. The combined results suggest that a functional chemokine receptor is expressed on resident cells of the CNS. We speculate that the interactions of chemokines with astrocytes are involved in inflammatory reactions of the CNS.

www.jneurosci.org/content/17/17/6522.full.pdf

Posted on

Activation of CD8 T cells induces expression of CD4, which functions as a chemotactic receptor

It was previously shown that costimulation of CD8+ lymphocytes results in de novo expression of CD4. This study expanded on this observation to investigate the function of CD4 on CD8 cells. The ability of costimulated CD8 cells to respond to interleukin 16 (IL-16), a ligand that binds CD4 and induces cellular chemotaxis, was examined. IL-16–mediated ligation of CD4 expressed on CD8 T cells was found to induce an intracellular signal that directs migration of these cells in vitro. Thus, expression of CD4 on a CD8 lymphocyte has functional importance and may serve to control distribution of newly activated CD8 T cells in vivo.

http://bloodjournal.hematologylibrary.org/content/99/1/207.long

Posted on

A Bovine Whey Protein Extract Can Enhance Innate Immunity by Priming Normal Human Blood Neutrophils

Bovine milk-derived products, in particular whey proteins, exhibit beneficial properties for human health, including the acquired immune response. However, their effects on innate immunity have received little attention. Neutrophils are key cells of innate defenses through their primary functions of chemotaxis, phagocytosis, oxidative burst, and degranulation.
A whey protein extract (WPE) purified from bovine lactoserum was evaluated for its direct and indirect effects on these primary functions of normal human blood neutrophils in vitro. Although WPE had no direct effects on primary functions, a 24-h pretreatment of neutrophils with WPE was associated with a significant and dose-dependent increase of their chemotaxis, superoxide production, and degranulation in response to N-formyl-methionine-leucine-phenylalanine, as well as of their phagocytosis of bioparticles. The pretreatment increased the surface expression of CD11b, CD16B, and CD32A receptors. The major WPE protein components b-lactoglobulin (b-LG) and a-lactalbumin (a-LA) were the main active fractions having an additive effect on human neutrophils that became more responsive to a subsequent stimulation. This effect on NADPH oxidase activity was associated with translocation of p47phox to plasma membrane. Glycomacropeptide, a peptide present in measurable amounts in WPE products, was able to enhance the individual effect of b-LG or a-LA on neutrophils. The present data suggest that WPE, through b-LG and a-LA, has the capacity to enhance or ‘‘prime’’ human neutrophil responses to a subsequent stimulation, an effect that could be associated with increased innate defenses in vivo.

jn.nutrition.org/content/early/2008/12/23/jn.108.098459.full.pdf

Posted on

Rapid fluorescence-based measurement of neutrophil migration in vitro

We have standardized a new chemotaxis chamber that uses fluorescence as the cellular marker for the measurement of leukocyte migration in vitro in disposable 96-well microplates. This new fluorescence-based assay is a robust assay because filter pore size, cell density, filter composition, and filter thickness do not affect PMN migration towards interleukin-8 or the complement fragment, C5a. When compared to two separate chemotaxis assays in which the migrated cells are counted
visually, the fluorescence-based assay was more rapid, less labor intensive, and more sensitive. This new assay is a significant advance in the measurement of leukocyte migration in vitro.

Frevert, Wong, Goodman, Goodwin, and Martin. “Rapid Fluorescence-based Measurement of Neutrophil Migration in Vitro.” 1998, Journal of Immunological Methods, 213, 41-52.

Posted on

Slit2 involvement in glioma cell migration is mediated by Robo1 receptor

Slit and Robo proteins are evolutionarily conserved molecules whose interaction underlies axon guidance and neuronal precursor cell migration. During development secreted Slit proteins mediate chemorepulsive signals on cells expressing Robo receptors. Because similar molecular mechanisms may be utilized in glioma cell invasion and neuroblast migration, we studied the expression of Slit2 and its transmembrane receptor Robo1 as well as their functional role in migration in glioma cells. qRTPCR and immunohistochemistry of human specimens revealed that Slit2 was distinctly expressed by non-neoplastic neurons, but at only very low levels in fibrillary astrocytoma and glioblastoma. Robo1 also was mainly restricted to neurons in the normal brain, whereas astrocytic tumor cells in situ as well as glioblastoma cell lines overexpressed Robo1 at mRNA and protein levels.
Recombinant human Slit2 in a concentration of 0.45 nM was repulsive for glioma cell lines in a modified Boyden chamber assay. RNAi-mediated knockdown of Robo1 in glioma cell lines neutralized the repulsive effect of Slit2, demonstrating that Robo1 served as the major Slit2
receptor. Our findings suggest that a chemorepulsive effect mediated by interaction of Slit2 and Robo1 participates in glioma cell guidance in the brain.

available as a pdf from med.stanford.edu/nbc/articles/Bowen%20Slite2.pdf

Posted on

Enhanced migration of fibroblasts derived from lungs with fibrotic lesions

The migration and proliferation of fibroblasts may be important in the pathogenesis  of pulmonary fibrosis.
Considerable data are available on the proliferation of fibroblasts, but very few on their migration.
Methods – The migratory activity of fibroblasts obtained from lung biopsy specimens from 11 patients with idiopathic pulmonary fibrosis (IPF) was studied using a 96-well chemotaxis chamber.
Fibroblasts from eight normal controls, seven patients with interstitial fibrosis associated with a collagen vascular disease (IP-CVD), and 13 patients with sarcoidosis were also examined. Migratory
activity was tested in a serum-free medium in the presence and absence of platelet derived growth factor (PDGF), 30 ng/mL, as a chemoattractant.

http://thorax.highwire.org/content/50/9/984.abstract

Posted on

Ramatroban (BAY u3405), Inhibits Prostaglandin D2-Induced Eosinophil Migration in Vitro

Ramatroban (Baynas, BAY u3405), a thromboxane A2(TxA2) antagonist marketed for allergic rhinitis, has been shown to partially attenuate prostaglandin (PG)D2-induced bronchial hyperresponsiveness in humans, as well as reduce antigen-induced early- and late-phase inflammatory responses in mice, guinea pigs, and rats. PGD2 is known to induce eosinophilia following intranasal administration, and to induce eosinophil activation in vitro. In addition to the TxA2 receptor, PGD2 is known as a ligand for the PGD2receptor, and the newly identified G-protein-coupled chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). To fully characterize PGD2-mediated inflammatory responses relevant to eosinophil activation, further analysis of the mechanism of action of ramatroban has now been performed. PGD2-stimulated human eosinophil migration was shown to be mediated exclusively through activation of CRTH2, and surprisingly, these effects were completely inhibited by ramatroban. This is also the first report detailing an orally bioavailable small molecule CRTH2 antagonist. Our findings suggest that clinical efficacy of ramatroban may be in part mediated through its action on this Th2-, eosinophil-, and basophil-specific chemoattractant receptor.

http://jpet.aspetjournals.org/content/305/1/347.full

Posted on

In vitro anti-angiogenic properties of LGD1069

LGD1069 (Targretin®) is a selective retinoid X receptor (RXR) ligand, which is used in patients for cutaneous T-cell lymphoma. Our published study reported that LGD1069 inhibited tumor-induced angiogenesis in non-small cell lung cancer. In present study, we found that LGD1069 suppressed the proliferation, adhesion, invasion and migration of endothelial cells directly, and affected the expression of vegf and some matrix genes.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3120806/