Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells

This study aimed to demonstrate that microspheres, used as delivery vehicle of DNA-Hsp65/TDM [plasmid DNA encoding heat shock protein 65 (Hsp65) coencapsulated with trehalose dimycolate (TDM) into PLGA microspheres], are widely spread among several organs after intramuscular administration in BALB/c mice. In general, we showed that these particles were phagocytosed by antigen presenting cells, such as macrophages and dendritic cells. Besides, it was demonstrated herein that draining lymph node cells presented a significant increase in the number of cells expressing costimulatory molecules (CD80 and CD86) and MHC class II, and also that the administration of the DNA-Hsp65/TDM and vector/TDM formulations resulted in the up-regulation of CD80, CD86 and MHC class II expression when compared to control formulations (vector/TDM and empty). Regarding the intracellular trafficking we observed that following phagocytosis, the microspheres were not found in the late endosomes and/or lysosomes, until 15 days after internalization, and we suggest that these constructions were hydrolysed in early compartments. Overall, these data expand our knowledge on PLGA [poly (lactic-co- glycolic acid)] microspheres as gene carriers in vaccination strategies, as well as open perspectives for their potential use in clinical practice

Serum Amyloid P Therapeutically Attenuates Murine Bleomycin-Induced Pulmonary Fibrosis via Its Effects on Macrophages

Macrophages promote tissue remodeling but few mechanisms exist to modulate their activity during tissue fibrosis. Serum amyloid P (SAP), a member of the pentraxin family of proteins, signals through Fcγ receptors which are known to affect macrophage activation. We determined that IPF/UIP patients have increased protein levels of several alternatively activated pro-fibrotic (M2) macrophage-associated proteins in the lung and monocytes from these patients show skewing towards an M2 macrophage phenotype. SAP therapeutically inhibits established bleomycin-induced pulmonary fibrosis, when administered systemically or locally to the lungs. The reduction in aberrant collagen deposition was associated with a reduction in M2 macrophages in the lung and increased IP10/CXCL10. These data highlight the role of macrophages in fibrotic lung disease, and demonstrate a therapeutic action of SAP on macrophages which may extend to many fibrotic indications caused by over-exuberant pro-fibrotic macrophage responses

Fluorescent PLGA microspheres were captured by antigen presenting cells after intramuscular administration

<p><b>Copyright information:</b></p><p>Taken from "Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells"</p><p>http://www.gvt-journal.com/content/5/1/9</p><p>Genetic Vaccines and Therapy 2007;5():9-9.</p><p>Published online 20 Sep 2007</p><p>PMCID:PMC2042973.</p><p></p> To determine the leukocyte subsets involved in the uptake process, mice received a single intramuscular injection of 6-coumarin labeled microspheres containing DNA-Hsp65/TDM, vector/TDM or empty microspheres (without plasmid DNA and TDM). After 7 days, the draining lymph nodes cells were collected for FACS analysis (CD80, CD86, CD11b, CD11c and class II MHC). Control group was injected with saline. (A): Number of cells positive for cell surface molecules CD80, CD86 and MHC class II; *p < 0.05 versus control formulations (vector/DMT and empty). (B): MFI increase expressed as a percentage from pcDNA3-Hsp65/TDM and pcDNA3/TDM formulations, normalized to empty formulation. The values represented in the graphs are the mean ± SD of three experiments

Tissue distribution of PLGA microspheres after intramuscular administration

<p><b>Copyright information:</b></p><p>Taken from "Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells"</p><p>http://www.gvt-journal.com/content/5/1/9</p><p>Genetic Vaccines and Therapy 2007;5():9-9.</p><p>Published online 20 Sep 2007</p><p>PMCID:PMC2042973.</p><p></p> Mice received a single intramuscular injection of fluorescent microspheres (DNA-Hsp65/TDM). At various time points (between 1 day and 120 days) the total cells from several tissues (draining lymph node, spleen, thymus, lung, liver, kidney, testicle and single-cell suspension of bone marrow) were obtained for FACS analysis (for each analysis, 100.000 events were acquired). Each group consisted of three or five animals. The results were shown as the median of positive values for each time. ×: in this time the results were negative

Intracellular compartmentalization of fluorescent PLGA microspheres inside peritoneal macrophages

<p><b>Copyright information:</b></p><p>Taken from "Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells"</p><p>http://www.gvt-journal.com/content/5/1/9</p><p>Genetic Vaccines and Therapy 2007;5():9-9.</p><p>Published online 20 Sep 2007</p><p>PMCID:PMC2042973.</p><p></p> Confocal images of peritoneal macrophages incubated with fluorescent microspheres (green) for 1 day (A-B), 5 days (C-D), 8 days (E-F) and 10 days (G-H). Texas Red dextran was used as late endossome/lysosomal marker (red). Microspheres did not co-localize with Texas Red dextran in the endo-lysosomal compartment at any of the analyzed time (A, C, E and G). (B, D, F and H): Differential interference contrast microscopy of peritoneal macrophages incubated with microspheres plus Texas Red dextran. Arrows show intracellular hydrolysis of microspheres inside macrophages

Enhanced M2 monocyte/macrophage phenotype in IPF/UIP.

<p>(A–D) Levels of macrophage-associated mediators measured in lung tissue biopsies taken from IPF/UIP patients (filled bars) or from the normal margins of lung tumors following resection (open bars). M2 macrophage-associated IL13 (A), IL1RA (B) and resistin (C); and M1-associated fractalkine/CX3CL1 (D) from non-fibrotic (<i>n</i> = 11) or IPF/UIP (<i>n</i> = 13) lung tissue were assessed using Luminex technology. *<i>P</i>≤0.05 significance using Mann Whitney analysis.</p

Local and abbreviated SAP treatment significant reduces bleomycin-induced collagen generation and deposition.

<p>Mice were challenged with intratracheal bleomycin (0.03 U) on day 0 and dosed with SAP (6 mg/kg) every other day on days 11–19 intraperitoneally (i.p., filled bar) or intranasally (i.n., down-hatched bar); or dosed with SAP (10 mg/kg, i.p.) on days 11,12,13 only (cross-hatched bar); or dosed with control (P5S, i.n., open bar) every other day on days 11–19. Collagen was quantitated biochemically (A) and visualized histologically (B). Original magnification, ×20. Bars represent the mean ± s.e.m. of a minimum of <i>n</i> = 5 per group. *<i>P</i>≤0.05 significance using Mann Whitney t-test.</p

Attenuated bleomycin-induced lung fibrosis and M2 macrophage phenotype with SAP treatment.

<p>Mice challenged with intratracheal bleomycin (0.05 U) on day 0 and dosed with SAP (6 mg/kg, i.p. filled bars) or control (PBS, i.p., open bars) on days 11–20 were evaluated at day 21 for gross morphological changes visualized in H&E stained lung sections (A); collagen generation quantitated by hydroxyproline analysis (B); procollagen IaI and procollagen III gene expression quantitated and normalized to β-actin by branched DNA technology (C). Macrophage phenotype in the lung assessed following SAP treatment by immunolocalizing IL13Rα2 (D), gene transcript levels determined by branched DNA technology at day 21 for NOS2, CCL2, oncostatin (OSM) and MARCO (E), and FIZZ1 and ST2 (F). (G) Plasma IP10/CXCL10 levels determined by Luminex technology. Bars represent the mean ± s.e.m. of a minimum of <i>n</i> = 4 per group. Original magnification, ×20. *<i>P</i>≤0.05 significance using Student's t-test.</p