Xpression levels of naive mice (means of 3 mice per group).Visualization of gold-liposomesTo visualize the uptake of gold-containing liposomes, knee joints were removed from mice 24 hours after treatment with goldliposomes and were decalcified in ECTA/PVP (polyvinylpyrrilodine) in TRIS buffer for 2 weeks. Knee joints were then frozen in order PHA-739358 liquid nitrogen and sections were cut on a cryostat (Microm HM500M, Waldorf, Germany) and mounted on Superfrost microscopic slides (Menzel Glaser, Germany). Silver enhancement ?of colloidal gold was performed with Sigma silver enhancement kit (Sigma, St. Louis, MO, USA) and terminated by incubating with a 0.5 sodium thiosulphate solution in distilled water. Sections were counterstained with hematoxylin.Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)The RT-PCR was performed using the ABI Prism 7000 Sequence Detection system (Applied Biosystems) for quantification with SYBR Green and melting curve analysis. Primers were purchase Defactinib designed with Primer Express Version 2.0 (Applied Biosystems). PCR conditions were as follows: 2 minutes at 50uC and 10 minutes at 95 50uC, followed by 40 cycles of 15 seconds at 95uC and 1 minute at 60uC. Primer concentrations were 300 nM. All PCR’s were performed in a total volume of 20 ml. Data are presented as expression levels relative to the house-keeping gene GAPDH (means of 5 mice per group).Macrophage 26001275 cultureTo obtain murine macrophages in vitro, bone marrow cells from femoral shafts of C57Bl/6 mice were cultured in DMEM mediumPLP Liposomes Inhibit M1 Macrophage ActivationStatistical analysisDifferences between treatment groups was tested for statistical significance with Student’s t-test using GraphPad Prism 5 software. Results are expressed as mean +/2 S.D.Targeting the inflamed synovium with glucocorticoid liposomes strongly suppresses experimental arthritisMice with established antigen-induced arthritis (AIA) expressing a strong M1 signature at day 3, were treated with a single intravenous injection of Lip-PLP (10 mg/kg) and showed a strong suppression of joint swelling as measured by 99MTc-uptake by 74 within 1 day when compared to saline controls and by 61 when compared to free PLP treatment (Fig. 2A). At day 5 after treatment, Lip-PLP had almost completely suppressed joint swelling and histological examination of frontal knee joint sections showed a mean suppression of the synovial infiltrate of 29 at day 1 and of 80 at day 5 after treatment (Fig. 2B+C).Results Inflamed synovium strongly expresses a dominant M1 signature during the course of antigen-induced arthritisTo characterize the expression of M1 and M2 markers in the inflamed synovium during experimental arthritis, we isolated messenger RNA from synovial biopsies in a standard manner [21] at various time points (days 1, 3, 5 and 7) after induction of antigen-induced arthritis (AIA). Gene expression in inflamed synovium was determined by microarray as described earlier [10] and compared with control synovium obtained from normal mouse knee joints. Microarray analysis showed that various M1 markers (IL-1b, IL-6, FccRI and CD86) were strongly upregulated at day 1 after induction of arthritis up to day 7 (Fig. 1A). The majority of M2 markers (IL-1RII, CD163, CD206, Arg1 and Ym1) were also somewhat upregulated during the course of arthritis, although in lesser extent than the M1 markers, with the exception of Arg1 and Ym1 which were especially high at day 1 after induction of AIA (Fig. 1B). Altog.Xpression levels of naive mice (means of 3 mice per group).Visualization of gold-liposomesTo visualize the uptake of gold-containing liposomes, knee joints were removed from mice 24 hours after treatment with goldliposomes and were decalcified in ECTA/PVP (polyvinylpyrrilodine) in TRIS buffer for 2 weeks. Knee joints were then frozen in liquid nitrogen and sections were cut on a cryostat (Microm HM500M, Waldorf, Germany) and mounted on Superfrost microscopic slides (Menzel Glaser, Germany). Silver enhancement ?of colloidal gold was performed with Sigma silver enhancement kit (Sigma, St. Louis, MO, USA) and terminated by incubating with a 0.5 sodium thiosulphate solution in distilled water. Sections were counterstained with hematoxylin.Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)The RT-PCR was performed using the ABI Prism 7000 Sequence Detection system (Applied Biosystems) for quantification with SYBR Green and melting curve analysis. Primers were designed with Primer Express Version 2.0 (Applied Biosystems). PCR conditions were as follows: 2 minutes at 50uC and 10 minutes at 95 50uC, followed by 40 cycles of 15 seconds at 95uC and 1 minute at 60uC. Primer concentrations were 300 nM. All PCR’s were performed in a total volume of 20 ml. Data are presented as expression levels relative to the house-keeping gene GAPDH (means of 5 mice per group).Macrophage 26001275 cultureTo obtain murine macrophages in vitro, bone marrow cells from femoral shafts of C57Bl/6 mice were cultured in DMEM mediumPLP Liposomes Inhibit M1 Macrophage ActivationStatistical analysisDifferences between treatment groups was tested for statistical significance with Student’s t-test using GraphPad Prism 5 software. Results are expressed as mean +/2 S.D.Targeting the inflamed synovium with glucocorticoid liposomes strongly suppresses experimental arthritisMice with established antigen-induced arthritis (AIA) expressing a strong M1 signature at day 3, were treated with a single intravenous injection of Lip-PLP (10 mg/kg) and showed a strong suppression of joint swelling as measured by 99MTc-uptake by 74 within 1 day when compared to saline controls and by 61 when compared to free PLP treatment (Fig. 2A). At day 5 after treatment, Lip-PLP had almost completely suppressed joint swelling and histological examination of frontal knee joint sections showed a mean suppression of the synovial infiltrate of 29 at day 1 and of 80 at day 5 after treatment (Fig. 2B+C).Results Inflamed synovium strongly expresses a dominant M1 signature during the course of antigen-induced arthritisTo characterize the expression of M1 and M2 markers in the inflamed synovium during experimental arthritis, we isolated messenger RNA from synovial biopsies in a standard manner [21] at various time points (days 1, 3, 5 and 7) after induction of antigen-induced arthritis (AIA). Gene expression in inflamed synovium was determined by microarray as described earlier [10] and compared with control synovium obtained from normal mouse knee joints. Microarray analysis showed that various M1 markers (IL-1b, IL-6, FccRI and CD86) were strongly upregulated at day 1 after induction of arthritis up to day 7 (Fig. 1A). The majority of M2 markers (IL-1RII, CD163, CD206, Arg1 and Ym1) were also somewhat upregulated during the course of arthritis, although in lesser extent than the M1 markers, with the exception of Arg1 and Ym1 which were especially high at day 1 after induction of AIA (Fig. 1B). Altog.
