Monitoring induced gene expression of single cells in a multilayer microchip.

We present a microfluidic system that facilitates long-term measurements of single cell response to external stimuli. The difficulty of addressing cells individually was overcome by using a two-layer microfluidic device. The top layer is designed for trapping and culturing of cells while the bottom layer is employed for supplying chemical compounds that can be transported towards the cells in defined concentrations and temporal sequences. A porous polyester membrane that supports transport and diffusion of compounds from below separates the microchannels of both layers. The performance and potential of the device are demonstrated using human embryonic kidney cells (HEK293) transfected with an inducible gene expression system. Expression of a fluorescent protein (ZsGreen1-DR) is observed while varying the concentration and exposure time of the inducer tetracycline. The study reveals the heterogeneous response of the cells as well as average responses of tens of cells that are analyzed in parallel. The microfluidic platform enables systematic studies under defined conditions and is a valuable tool for general single cell studies to obtain insights into mechanisms and kinetics that are not accessible by conventional macroscopic methods.

Initial observations on the efficacy of highly active antiretroviral therapy in the treatment of HIV-associated autoimmune thrombocytopenia.

BACKGROUND: Immune thrombocytopenic purpura (ITP) occurs in as many as 40% of patients infected with the human immunodeficiency virus (HIV). We sought to evaluate the effect of highly active antiretroviral therapy (HAART) on platelet counts in such patients. METHODS: Data collected from 11 homosexual men with HIV-associated ITP and < or = 50 x 10(9) platelets were analyzed after they were placed on HAART. At initial evaluation, 7 patients were antiretroviral naive, 2 were taking zidovudine alone, and 2 were receiving combination antiretroviral therapy for known HIV infection. For 6 patients with <30 x 10(9) platelets, prednisone was initially coadministered with HAART. The primary outcome measure was the platelet count response to HAART, which was measured weekly until counts had normalized on 3 consecutive occasions, then every 3 months while on HAART. Secondary outcome measures were HIV-viral RNA levels and CD4+ cell counts. RESULTS: One month after the initiation of HAART, 10 evaluable patients had an increase in mean platelet count. This improvement was sustained at 6 and 12 months' follow-up for 9 of 10 evaluable patients. Increases in mean platelet count at 6 and 12 months of the 9 responders were statistically significant. The range of follow-up in the 9 responders is 21 to 46 months (median, 30 months), with no thrombocytopenic relapses. The 9 long-term platelet responders have been maintained on HAART and at 12 months had a mean reduction of > 1.5 log10 in HIV viral RNA serum levels and a marked improvement in CD4+ T-lymphocyte cell count. CONCLUSION: HAART seems to be effective in improving platelet counts in the setting of HIV-associated ITP, enhancing CD4+ cell counts, and reducing HIV viral loads.