[Show abstract][Hide abstract]ABSTRACT: Supplementary methods SPION synthesis Aminopropylsilane (APS)-and aminodextran (AD)-SPIONs were prepared by coprecipitation of iron (II) and (III) salts in a basic ammonium solution. A mixture of 3.98 g FeCl 2 ·4H 2 O and 9.74 g FeCl 3 ·6H 2 O was dissolved in 50 ml of deoxygenated water at a Fe 2+ /Fe 3+ ratio of 1:1.8. When iron salts were completely dissolved, the solution was slowly added to a 400 ml solution of ammonium hydroxide in deoxygenated water (35% v/v) and magnetically stirred. A black precipitate formed immediately and the reaction was maintained for 5 h with stirring. For AD-SPION, Fe salts were precipitated in a basic ammonium solution containing 4.2 g DEAE-dextran (diethylaminoethyl dextran), leading to smaller particle size (6.8 nm) and larger hydrodynamic size (150 nm). For APS-SPION, the precipitate was acid-treated (nitric acid heated to boiling ) to improve colloidal properties, then coated by slow addition (10 ml.s-1) of 1.22 ml APS (0.005 mol) to a mixture of 10 ml particles (28 g Fe 2 O 3 /L) and 10 ml methanol, with strong stirring (12 h). Methanol was eliminated using a rotary evaporator . In this case, larger particles are obtained (8.3 nm) with smaller hydrodynamic sizes (54 nm). Dimercaptosuccinic acid (DMSA)-SPIONs were synthesised by thermal decomposition of iron oleate (4.5 g) in a solution of oleic acid (0.7 g) in octadecene (50 ml). The solution was heated under nitrogen flow to 80ºC, then to reflux (315ºC) for 4 h in a 100 ml flask with mechanical stirring. The final solution was cooled to room temperature and washed several times with ethanol. Ethanol was evaporated and particles (10 nm diameter) were resuspended in toluene. The particle surface was modified with DMSA by mechanical stirring of a mixture of 50 mg particles, 90 mg DMSA, 5 ml DMSO (dimethylsulphoxide) and 20 ml toluene (72 h). DMSA-coated particles were then washed three times with ethanol and redispersed in water. NaOH was added to yield pH 10. Hydrodynamic size is 65 nm. All
[Show abstract][Hide abstract]ABSTRACT: NK cells are a major component of the immune system, and alterations in their activity are cor-related with various autoimmune diseases. In the present work, we observed an increased ex-pression of the NKG2D ligand MICA in SLE patients' kidneys but not healthy subjects. We also show glomerulus-specific expression of the NKG2D ligands Rae-1 and Mult-1 in various murine SLE models, which correlated with a higher number of glomerular-infiltrating NK cells. As the role of NK cells in the immunopathogenesis of SLE is poorly un-derstood, we explored NK cell differentiation and activity in tissues and organs in SLE-prone murine models by use of diseased and prediseased MRL/MpJ and MRL/lpr mice. We report here that phenotypically iNK cells accumulate only in the spleen but not in BM or kidneys of diseased mice. Infiltrating NK cells in kidneys undergoing a lupus nephritic process showed a more mature, activated phenotype com-pared with kidney, as well as peripheral NK cells from prediseased mice, as determined by IFN-g and STAT5 analysis. These findings and the presence of glomerulus-specific NKG2D ligands in lupus-prone mice identify a role for NK cells and NKG2D ligands in the lupus nephritic process, which could aid in understanding their role in human SLE.
Full-text · Article · Jan 2015 · Journal of Leukocyte Biology
[Show abstract][Hide abstract]ABSTRACT: The role of p110δ PI3K in lymphoid cells has been studied extensively, showing its importance in immune cell differentiation, activation and development. Altered T cell localization in p110δ-deficient mouse spleen suggested a role for p110δ in non-hematopoietic stromal cells, which maintain hematopoietic cell segregation. We tested this hypothesis using p110δ(WT/WT) mouse bone marrow to reconstitute lethally irradiated p110δ(WT/WT) or p110δ(D910A/D910A) (which express catalytically inactive p110δ) recipients, and studied localization, number and percentage of hematopoietic cell subsets in spleen and lymph nodes, in homeostatic conditions and after antigen stimulation. These analyses showed diffuse T cell areas in p110δ(D910A/D910A) and in reconstituted p110δ(D910A/D910A) mice in homeostatic conditions. In these mice, spleen CD4(+) and CD8(+) T cell numbers did not increase in response to antigen, suggesting that a p110δ(D910A/D910A) stroma defect impedes correct T cell response. FACS analysis of spleen stromal cell populations showed a decrease in the percentage of gp38(-)CD31(+) cells in p110δ(D910A/D910A) mice. qRT-PCR studies detected p110δ mRNA expression in p110δ(WT/WT) spleen gp38(-)CD31(+) and gp38(+)CD31(+) subsets, which was reduced in p110δ(D910A/D910A) spleen. Lack of p110δ activity in these cell populations correlated with lower LTβR, CCL19 and CCL21 mRNA levels; these molecules participate in T cell localization to specific spleen areas. Our results could explain the lower T cell numbers and more diffuse T cell areas found in p110δ(D910A/D910A) mouse spleen, as well as the lower T cell expansion after antigen stimulation in p110δ(D910A/D910A) compared with p110δ(WT/WT) mice.
[Show abstract][Hide abstract]ABSTRACT: Intracellular cAMP levels in p110γ+/− BMM increase after forskolin stimulation. Western blot of BMM extracts from p110γ+/− mice, to detect protein-bound cAMP after forskolin (FSK) stimulation (0, 24 and 48 h).
[Show abstract][Hide abstract]ABSTRACT: Proliferation of
GM-CSF-stimulated macrophages is unaffected by p110γ deficiency. Percentage of BMM in cell cycle S phase at various times post-GM-CSF stimulation in p110γ+/− and p110γ−/− BMM (n = 2 experiments, each with a pool of 3 mice/genotype).
[Show abstract][Hide abstract]ABSTRACT: Foxp3+ regulatory T cells infiltrate in atherosclerotic lesions from LDLR−/−p110γ+/− and LDLR-/−p110γ−/− mice. Quantitative analysis of Foxp3+ cells per aortic sinus section of indicated mice (n = 8/genotype). Results show mean ± SD.
[Show abstract][Hide abstract]ABSTRACT: Immune cell populations and M-CSF concentration in peripheral blood from LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice. Peripheral blood was extracted from LDLR−/−p110γ+/− and LDLR−/−p110γ −/− mice before (t = 0) and after (t = 2 months) on a high-fat diet. Flow cytometry staining was used to detect T cells (CD3+) (A), inflammatory monocytes (Ly6Chi) (B) and granulocytes (Gr1+) (C) (n = 7 mice/genotype, t = 0; n = 4 mice/genotype, t = 2 months). Mean ± SD, Student’s t-test, p<0.05 and p<0.01. (D) In serum from peripheral blood obtained as above, M-CSF levels were determined by ELISA using the Milliplex Kit (Millipore). Mean ± SD. Student’s t-test.
[Show abstract][Hide abstract]ABSTRACT: Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR(-/-)p110γ(+/-) and LDLR(-/-)p110γ(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ(+/-) and p110γ(-/-) mice. Lack of p110γ in LDLR(-/-) mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR(-/-)p110γ(-/-) mice were smaller than in LDLR(-/-)p110γ(+/-) controls, which coincided with decreased macrophage proliferation in LDLR(-/-)p110γ(-/-) mouse lesions. This proliferation defect was also observed in p110γ(-/-) bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR(-/-)p110γ(-/-) mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR(-/-) mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.
[Show abstract][Hide abstract]ABSTRACT: Mice lacking LDLR and PI3K p110γ show smaller atherosclerotic lesions than controls. Lesion progression was studied in LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice before (t = 0) and after (t = 2 months) high-fat diet treatment. (A) Total serum cholesterol, HDL- and LDL-cholesterol and triglycerides were measured. t = 0, n = 10 mice/genotype; t = 2 months, n = 6 mice/genotype. Mean ± SD. Student’s t-test, p<0.05 (B) Representative photomicrographs of hematoxylin/eosin-stained aortic sinus sections from LDLR−/−p110γ+/− and LDLR−/−p110γ−/− female mice. Lesion area is delimited. Bar = 200 μm. (C) Quantitative analysis of lesion size in the aortic sinus of LDLR−/−p110γ+/− (n = 6) and LDLR−/−p110γ−/− mice (n = 6) using ImageJ. Mean ± SD. Student’s t-test, p<0.05. (D) Mac-3+ area per aortic sinus section, quantitated with ImageJ. Mean ± SD; Student’s t-test, p<0.05. (E) Absolute numbers of lesion CD3+ cells per aortic sinus section, quantitated with ImageJ. Mean ± SD; Poisson test, p<0.01.
[Show abstract][Hide abstract]ABSTRACT: Although iron oxide magnetic nanoparticles (MNP) have been proposed for numerous biomedical applications, little is known about their biotransformation and long-term toxicity in the body. Dimercaptosuccinic acid (DMSA)-coated magnetic nanoparticles have been proven efficient for in vivo drug delivery, but these results must nonetheless be sustained by comprehensive studies of long-term distribution, degradation and toxicity. We studied DMSA-coated magnetic nanoparticles effects in vitro on NCTC 1469 non-parenchymal hepatocytes, and analyzed their biodistribution and biotransformation in vivo in C57BL/6 mice. Our results indicate that DMSA-coated magnetic nanoparticles have little effect on cell viability, oxidative stress, cell cycle or apoptosis on NCTC 1469 cells in vitro. In vivo distribution and transformation was studied by alternating current magnetic susceptibility measurements, a technique that permits distinction of MNP from other iron species. Our results show that DMSA-coated MNP accumulate in spleen, liver and lung tissues for extended periods of time, in which nanoparticles undergo a process of conversion from superparamagnetic iron oxide nanoparticles to other non-superparamagnetic iron forms, with no significant signs of toxicity. This work provides first evidence of DMSA-coated magnetite nanoparticles biotransformation in vivo.
No preview · Article · Jul 2013 · Journal of Controlled Release
[Show abstract][Hide abstract]ABSTRACT: As radio- and chemotherapy-based cancer treatments affect both tumors and healthy tissue, cancer immunotherapy attempts to specifically enhance the natural immune response to tumor cells. In mouse models of cancer, we tested uniform dimercaptosuccinic acid (DMSA)-coated monodisperse magnetic nanoparticles as a delivery system for the anti-tumorigenic cytokine IFN-γ. IFN-γ-adsorbed DMSA-coated magnetic nanoparticles were targeted to the tumor site by application of an external magnetic field. We analyzed nanoparticle biodistribution before and after IFN-γ conjugation, as well as the efficiency of nanoparticle accumulation in tumors, IFN-γ release in the area of interest, and the effects of both on tumor development. At the tumor site, we observed a high degree of nanoparticle accumulation and of cytokine delivery, which led to increased T cell and macrophage infiltration and promoted an anti-angiogenic effect. The combined action led to a notable reduction in tumor size. Our findings indicate that IFN-γ-adsorbed DMSA-coated magnetite nanoparticles can be used as an efficient in vivo drug delivery system for tumor immunotherapy.
[Show abstract][Hide abstract]ABSTRACT: Uptake, cytotoxicity and interaction of improved superparamagnetic iron oxide nanoparticles were studied in cells, tissues and organs after single and multiple exposures.
We prepared dimercaptosuccinic acid-coated iron oxide nanoparticles by thermal decomposition in organic medium, resulting in aqueous suspensions with a small hydrodynamic size (< 100 nm), high saturation magnetization and susceptibility, high nuclear magnetic resonance contrast and low cytotoxicity.
In vitro and in vivo behavior showed that these nanoparticles are efficient carriers for drug delivery to the liver and brain that can be combined with MRI detection.