4 research outputs found
DNA Analysis Using an Integrated Microchip for Multiplex PCR Amplification and Electrophoresis for Reference Samples
A system
that automatically performs the PCR amplification and microchip electrophoretic
(ME) separation for rapid forensic short tandem repeat (STR) forensic
profiling in a single disposable plastic chip is demonstrated. The
microchip subassays were optimized to deliver results comparable to
conventional benchtop methods. The microchip process was accomplished
in sub-90 min compared with >2.5 h for the conventional approach.
An infrared laser with a noncontact temperature sensing system was
optimized for a 45 min PCR compared with the conventional 90 min amplification
time. The separation conditions were optimized using LPA-<i>co</i>-dihexylacrylamide block copolymers specifically designed for microchip
separations to achieve accurate DNA size calling in an effective length
of 7 cm in a plastic microchip. This effective separation length is
less than half of other reports for integrated STR analysis and allows
a compact, inexpensive microchip design. This separation quality was
maintained when integrated with microchip PCR. Thirty samples were
analyzed conventionally and then compared with data generated by the
microfluidic chip system. The microfluidic system allele calling was
100% concordant with the conventional process. This study also investigated
allelic ladder consistency over time. The PCR-ME genetic profiles
were analyzed using binning palettes generated from two sets of allelic
ladders run three and six months apart. Using these binning palettes,
no allele calling errors were detected in the 30 samples demonstrating
that a microfluidic platform can be highly consistent over long periods
of time
video_1_Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.mov
<p>If misregulated, macrophage (Mϕ)–T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF)- and Mϕ colony-stimulating factor (M-CSF)-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor β (FRβ), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRβ<sup>+</sup>CD39<sup>+</sup>CD73<sup>+</sup> Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.</p
presentation_1_Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.PDF
<p>If misregulated, macrophage (Mϕ)–T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF)- and Mϕ colony-stimulating factor (M-CSF)-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor β (FRβ), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRβ<sup>+</sup>CD39<sup>+</sup>CD73<sup>+</sup> Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.</p
video_2_Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.mov
<p>If misregulated, macrophage (Mϕ)–T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF)- and Mϕ colony-stimulating factor (M-CSF)-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor β (FRβ), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRβ<sup>+</sup>CD39<sup>+</sup>CD73<sup>+</sup> Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.</p