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Butyrophilin 3A1 (BTN3A1) binds small phosphorous-containing molecules, which initiates transmembrane signaling and activates butyrophilin-responsive cells. We synthesized several phosphinophosphonates and their corresponding tris-pivaloyloxymethyl prodrugs and examined their effects on BTN3A1. An analog of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) bound to BTN3A1 with intermediate affinity, which was enthalpy-driven. Docking studies revealed binding to the basic surface pocket and interactions between the allylic hydroxyl group and the BTN3A1 backbone. The phosphinophosphonate stimulated proliferation of Vγ9Vδ2 T cells with moderate activity (EC50 = 26 µM). Cellular potency was enhanced >600-fold in the tris-POM prodrug (EC50 = 0.041 µM). The novel prodrug also induced T cell mediated leukemia cell lysis. Analysis of dose response data reveals HMBPP-induced Hill coefficients of 0.69 for target cell lysis and 0.68 in interferon secretion. Together, tris-POM prodrugs enhance the cellular activity of phosphinophosphonates, reveal structure-activity relationships of butyrophilin ligands, and support a negatively cooperative model of cellular butyrophilin activation.

Cell-cleavable protecting groups often enhance cellular delivery of species that are charged at physiological pH. Although several phosphonate protecting groups have achieved clinical success, it remains difficult to use these prodrugs in live cells to clarify biological mechanisms. Here, we present a strategy that uses a 7-methoxycoumarin-3-carboxylic acid ester as a fluorescent protecting group. This strategy was applied to synthesis of an (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) analogue to assess cellular uptake and human Vγ9Vδ2 T cell activation. The fluorescent ester displayed low cellular toxicity (IC50 >100 μm) and strong T cell activation (EC50 =0.018 μm) relative to the unprotected anion (EC50 =23 μm). The coumarin-derived analogue allowed no-wash analysis of biological deprotection, which revealed rapid internalization of the prodrug. These results demonstrate that fluorescent groups can be applied both as functional drug delivery tools and useful biological probes of drug uptake.

SummaryPhosphoantigen-sensitive Vγ9Vδ2 T cells are important responders to infections and malignancy. However, the mechanisms by which phosphoantigens stimulate Vγ9Vδ2 T cells are unclear. Here, we synthesized phosphoantigen prodrugs and used them to demonstrate that intracellular delivery of phosphoantigens is required for their activity. The pivaloyloxymethyl prodrug is the most potent phosphoantigen described to date, with stronger stimulation of Vγ9Vδ2 T cells from human peripheral blood and greater ability to induce lysis of Daudi lymphoma cells relative to the previously most potent compound, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP). We demonstrate high binding affinity between phosphoantigens and the intracellular region of butyrophilin 3A1 (BTN3A1), localized to the PRY/SPRY (B30.2) domain, but also affecting the membrane proximal region. Our findings promote a phosphoantigen prodrug approach for cancer immunotherapy and unravel fundamental aspects of the mechanisms of Vγ9Vδ2 T cell activation.

We propose a scale-out solution for deep packet processing (DPP) appliances, which uses a standard Ethernet switch in combination with a load balancing controller. The majority of the data-plane traffic is distributed via the switch's built-in traffic distribution function and no connection state is kept. If the load balancing controller detects a skew in the load of the DPP appliances, it updates the traffic rules in the switch to redirect new connections to less busy DPP appliances. This adaptive solution is beneficial for load-balancing at high data rates because state is kept for only the redirected connections. For typical traffic patterns, our solution reduces the packet drops with minimal connection redirection. We show an example capture, where we are able to improve the packet drop rate by 94.8% with only 3% of the connections being redirected.

Gene expression data from microarrays are being applied to predict preclinical and clinical endpoints, but the reliability of these predictions has not been established. In the MAQC-II project, 36 independent teams analyzed six microarray data sets to generate predictive models for classifying a sample with respect to one of 13 endpoints indicative of lung or liver toxicity in rodents, or of breast cancer, multiple myeloma or neuroblastoma in humans. In total, >30,000 models were built using many combinations of analytical methods. The teams generated predictive models without knowing the biological meaning of some of the endpoints and, to mimic clinical reality, tested the models on data that had not been used for training. We found that model performance depended largely on the endpoint and team proficiency and that different approaches generated models of similar performance. The conclusions and recommendations from MAQC-II should be useful for regulatory agencies, study committees and independent investigators that evaluate methods for global gene expression analysis.

Comparison and integration of expression data derived from diverse microarray platforms is challenging. Factors affecting cross-platform comparison of microarray data include the choice of normalization method used, annotation differences, presence of splice variants, RNA degradation and probe distance from 3' end. A thorough evaluation of two commercial microarray platforms to determine an appropriate methodology for making cross-platform correlations is described here.

Colloid osmotic pressure (COP) was measured simultaneously with cardiorespiratory measurements in 103 surgical patients suspected of having circulatory problems. In a small subset of 28 patients, measurements were taken before, during, and after surgical operations. Similarly, data sets were taken before, during, and after infusions of colloids and crystalloids to assess the interactions of these variables during the stress of surgery and the administration of fluid therapy. COP was found to decrease during and shortly after surgical operations despite reasonably well-maintained pressure, volume, and flow variables. Concentrated (25%) albumin and plasma protein fraction (PPF) increased COP, cardiac index (CI), CVP, pulmonary capillary wedge pressure (WP), and blood volume, whereas crystalloids transiently increased CI, CVP, and WP but did not significantly change COP and blood volume. Low COP values were weakly related to survival, and COP-WP differences less than or equal to 3 mm Hg were roughly related to ARDS and pulmonary edema.