Correlates of risk of TB disease in infants with differential response to BCG vaccination

Includes bibliographical references.Studying prospective immune correlates of risk of TB disease following BCG vaccination is an important first step towards determining correlates of protection against TB, which can be identified only in a placebo-controlled randomized controlled trial (RCT) of an effective vaccine. To study correlates of risk of TB disease, we collected and stored blood from healthy 10-week old infants vaccinated with BCG at birth. During two years of follow up, infants who developed lung TB were defined as cases, while those who did not develop TB disease were defined as controls. We measured Th1/Th17 cytokine production by BCG-specific T cells, release of pro- and anti-inflammatory mediators, cytotoxic T cell potential and proliferation in response to BCG as potential correlates of risk of TB disease but none of these outcomes were different between cases and controls. However, transcriptional profiling of PBMC revealed two clusters of infants and interestingly, the gene expression profiles from cases and controls in the two clusters were in opposite directions. Based on this, we hypothesised that analysing the two clusters of infants separately will allow discovery of correlates of risk of TB, which were absent when clustering was not taken into account

High content, high-throughput screening for small molecule inducers of NF-κB translocation.

NF-κB is an important mediator of immune activity and its activation is essential in mounting immune response to pathogens. Here, we describe the optimization and implementation of a high-throughput screening platform that utilizes high content imaging and analysis to monitor NF-κB nuclear translocation. We screened 38,991 compounds from three different small molecule libraries and identified 103 compound as hits; 31% of these were active in a dose response assay. Several of the molecules lacked cytotoxicity or had a selectivity index of more than 2-fold. Our image-based approach provides an important first step towards identifying small molecules with immunomodulatory activity

Serine and proline-rich ligands enriched via phage-display technology show preferential binding to BCR/ABL expressing cells

Background and objectives: Despite the use of targeted therapy, chronic myelogenous leukemia (CML) currently remains incurable with drug therapy, with patients requiring life-long treatment. Developing either a vaccine to prevent the disease or another novel drug to specifically target and eradicate the CML cell will require the identification of CML-associated cell-surface markers and molecules that can bind specifically to the cell surface. In an attempt to discover peptides that bind specifically to cells in the early chronic phase of the disease, we used phage-display technology to identify heptapeptides that bind specifically to the surface of BCR/ABL-expressing fibroblasts. Methods: An in vitro system using NIH3T3 stably transfected with pGD210 (BCR/ABL) was used as a model for the chronic phase of the disease. The cells were panned using a linear heptapeptide phage library (Ph.D 7.0) in a negative/positive panning strategy with NIH3T3 containing only the plasmid vector as the wild type control. Results: We identified four novel peptides that were enriched through this technique. These peptides contained either multiple proline residues or serine/threonine–proline pairs and showed a confirmed binding preference for BCR/ABL+ fibroblasts. The peptide Y-R-A-P-W-P-P also showed a binding affinity for granulocytes from untreated CML patients. Conclusion: We have identified several novel peptides that can be used in future studies to identify specific CML cell-surface antigens or provide a novel drug-delivery mechanism. Keywords: Phage-display, BCR/ABL, CML, Fibroblast, Cell-surfac

Hits from NF-κB nuclear translocation assay.

<p>Hits from NF-κB nuclear translocation assay.</p

Screen validation.

<p>HUVECs were seeded overnight, stimulated with 100 ng/mL TNF-α and stained for NF-κB p65. NF-κB p65 average pixel intensity was measured in both the nucleus and cytoplasm of each cell and a translocation value obtained by calculating the NUC/CYT. Screen validation was performed as three independent experiments, each with duplicate plates of negative control (DMSO), positive control (100 ng/mL TNF-α) and dose response (10-point, 3-fold dilutions of TNF-α). (A-C) are data from 1 plate of each run for positive and negative controls. (A) Run 1 (B) Run 2 (C) Run 3. (D) Dose response curves from all three days. NUC/CYT was normalized and plotted using non- linear 4-parameter fit in order to calculate the EC₅₀ = effective concentration 50%, defined as the concentration at which 50% of the maximum response is seen.</p

Optimizing activation stimuli for NF-κB nuclear translocation.

<p>HUVECs were seeded overnight, stimulated with TNF-α for 30 min and stained for NF-κB. (A) Images from the DAPI and FITC channel. White arrows indicate non-stained nuclei in FITC channel; green arrows showing stained nuclei in FITC channel (B) Time course of translocation. HUVECs were stimulated with 10 ng/mL TNF-α. (C) Translocation in response to different inducers. HUVECs were stimulated for 30 min with 10 ng/mL TNF-α, 100 ng/mL lipopolysaccharide (LPS), 10 μM phorbol myristate acetate (PMA), 10 μM prostratin, or 10 μM calcimycin (D) Nuclear translocation in response to TNF-α. HUVECs were stimulated for 30 min. The average pixel intensity was measured in both the nucleus and cytoplasm of each cell and a translocation value obtained calculating the NUC/CYT. Results are shown in boxes and whiskers. Boxes denote the interquartile range with the median represented by line inside the boxes. Whiskers show maximum and minimum values. Differences between conditions tested was assessed using the Kruskal- Wallis test. Data are a representative of two different experiments (n = 2) with at least 8 replicates per condition tested.</p

Activity for confirmed hits.

<p>EC₅₀ = Activity in HUVECs, IC₅₀ = Cytotoxicity in HepG2, D = Unique compound identification number. Data are mean ± SD of three independent experiments for hit confirmation (for * compounds only N = 2 was run) and two independent experiments for cytotoxicity. The selectivity index (SI) was determined as IC₅₀/EC₅₀.</p

Small molecule library screen.

<p><b>(</b>A) Plate lay out for single point compound screen at 10 μM. Positive control: 100 ng/mL TNF-α in columns 1 and 24. Negative control: 1% DMSO in columns 2 and 23. Screening data from (B) MyriaScreen II. (C) TimTec. (D) ChemBridge libraries. Red line denotes cutoff for hit identification (NUC/CYT ≥ 1.3).</p