A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery.

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

The impact of representative payee services on medication adherence among unstably housed people living with HIV/AIDS.

Rates of viral suppression among people living with HIV/AIDS remain low, especially within marginalized populations such as people who are unstably housed. Representative payee is a service in which the US Social Security Administration appoints an individual or an organization to provide financial management for vulnerable individuals who are unable to manage their finances including housing payments. Little or no published research examines the association between financial management services such as representative payee and HIV clinical adherence. We conducted a pilot study with 18 unstably housed participants living with HIV/AIDS to examine the impact of representative payee services on viral suppression. Of the 11 participants who were not virally suppressed at baseline, 9 (81.8%) of them had achieved viral suppression at six-month follow-up (p = .004). Our findings suggest that providing unstably housed people living with HIV/AIDS with representative payee services may help them to improve their housing stability and clinical adherence. Additional research is needed to fully explore correlations between representative payee services and viral suppression.

Oxidative stress promotes polarization of human T cell differentiation toward a T helper 2 phenotype.

These studies were conducted to determine the effects of oxidative stress on human T cell differentiation and polarization into Th1 or Th2 phenotypes. Highly purified naive CD4+ T cells were isolated from PBMC of healthy, nonatopic donors. CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAb in the presence or absence of oxidative stress as supplied by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which generates a low level of superoxide anion. Increases in cellular superoxide were observed by exposure to DMNQ. Exposure of unpolarized CD4+ T cells to IL-12 or IL-4 resulted in a Th1 or Th2 phenotype, respectively. T cells stimulated in the absence of polarizing cytokines secreted modest amounts of IFN-gamma and TNF-alpha. Cells stimulated in the continuous presence of 5 microM DMNQ, displayed a marked up-regulation in Th2 cytokines, including IL-4, IL-5, and IL-13, but not the Th1 cytokine IFN-gamma. Th2 responses were blunted by concomitant exposure to thiol antioxidants. Long-term exposure of T cells to DMNQ resulted in growth of cells expressing CCR4, and a decrease in cells expressing CXCR3, indicating phenotypic conversion to Th2 cells. These results suggest that oxidative stress favors a Th2-polarizing condition.

Gamma-glutamyl transpeptidase activity alters the T cell response to oxidative stress and Fas-induced apoptosis.

The ectoenzyme gamma-glutamyl transpeptidase (GGT) is absent on resting naive peripheral blood T cells, highly expressed upon stimulation and intermediate on resting memory T cells. In other tissues, GGT is essential for the recapture of the antioxidant glutathione (GSH). T cells with different levels of GGT activity were examined for their ability to withstand oxidative stress. To create a model system that reflected the level of GGT seen on naive and memory T cells, Jurkat T cells were cloned by limiting dilution and their GGT expression analyzed. Jurkat expressing GGT at levels comparable to resting memory T cells have levels of intracellular reactive oxygen species (ROS) that are only 65% that seen in Jurkat that have low levels of GGT (similar to naive T cells). Treatment of the cells with H(2)O(2) increases ROS in both cells, although the level seen in the GGT(high) Jurkat is less than half that in the GGT(low) variant. Despite protection from oxidative stress, the GGT(high) Jurkat were found to be 2- to 3-fold more sensitive to Fas-induced apoptosis. The redox-regulated NF-kappaB pathway is activated in GGT(low) cells, resulting in higher levels of cIAP-1/2 proteins that limit caspase activity. The GGT(low) cells were found to have higher levels of NF-kappaB in the nucleus as well as lower levels of IkappaB-alpha. The GGT(low) cells also express higher levels of the caspase inhibitors cIAP-1/2 and have lower levels of caspase activity. These findings suggest that GGT expression regulates ROS in T lymphocytes and modulates Fas-induced killing by altering NF-kappaB activity.

Evidence for the locations of distinct steroid and Vinca alkaloid interaction domains within the murine mdr1b P-glycoprotein.

P-glycoproteins (P-gp) cause the efflux of a wide variety of unrelated hydrophobic compounds out of cells. However, the locations of the sites at which different classes of molecules initially interact with the protein are not well defined. A unique system was developed to search for P-gp drug-interaction domains using mutational analysis. The strategy is based upon identifying mutations that cause a decrease in the activity of P-gp inhibitors, which are structurally related to chemotherapeutic drugs transported by P-gps. Evidence of distinct steroid and taxane interaction domains has already been presented. The work reported here extends the study of the steroid interaction domain and presents evidence for a separate vinblastine interaction domain. A total of 10 steroid-related mutations, involving seven amino acids that are confined within transmembrane segments (TMS) 4 to 6, have been characterized. The location of these mutations indicates that steroids interact with the transporter within the inner leaflet of the plasma membrane. Four previously unidentified, Vinca-related mutations, involving three amino acids, have also been found. Unexpectedly, these mutations are clustered within an eight-amino acid segment proximal to the TMS-4 region. This portion of the protein is thought to be within the cytoplasmic compartment of the cell. Thus, the results suggest that at least part of the initial interaction between P-gp and Vinca alkaloids occurs in the cytoplasm. The steroid interaction domain does not extend into this region of the protein. However, this cytoplasmic section of the protein is likely to play an important role in promoting steroid transport.