Levels of human proteins in plasma associated with acute paediatric malaria.

BACKGROUND: The intimate interaction between the pathophysiology of the human host and the biology of the Plasmodium falciparum parasite results in a wide spectrum of disease outcomes in malaria. Development of severe disease is associated with a progressively augmented imbalance in pro- and anti-inflammatory responses to high parasite loads and sequestration of parasitized erythrocytes. Although these phenomena collectively constitute common denominators for the wide variety of discrete severe malaria manifestations, the mechanistic rationales behind discrepancies in outcome are poorly understood. Exploration of the human pathophysiological response by variations in protein profiles in plasma presents an excellent opportunity to increase the understanding. This is ultimately required for better prediction, prevention and treatment of malaria, which is essential for ongoing elimination and eradication efforts. RESULTS: An affinity proteomics approach was used to analyse 541 paediatric plasma samples collected from community controls and patients with mild or severe malaria in Rwanda. Protein profiles were generated with an antibody-based suspension bead array containing 255 antibodies targetting 115 human proteins. Here, 57 proteins were identified with significantly altered levels (adjusted p-values < 0.001) in patients with malaria compared to controls. From these, the 27 most significant proteins (adjusted p-values < 10-14) were selected for a stringent analysis approach. Here, 24 proteins showed elevated levels in malaria patients and included proteins involved in acute inflammatory response as well as cell adhesion. The remaining three proteins, also implicated in immune regulation and cellular adhesivity, displayed lower abundance in malaria patients. In addition, 37 proteins (adjusted p-values < 0.05) were identified with increased levels in patients with severe compared to mild malaria. This set includes, proteins involved in tissue remodelling and erythrocyte membrane proteins. Collectively, this approach has been successfully used to identify proteins both with known and unknown association with different stages of malaria. CONCLUSION: In this study, a high-throughput affinity proteomics approach was used to find protein profiles in plasma linked to P. falciparum infection and malaria disease progression. The proteins presented herein are mainly involved in inflammatory response, cellular adhesion and as constituents of erythrocyte membrane. These findings have a great potential to provide increased conceptual understanding of host-parasite interaction and malaria pathogenesis.

A vertical flow paper-microarray assay with isothermal DNA amplification for detection of Neisseria meningitidis.

Paper-based biosensors offer a promising technology to be used at the point of care, enabled by good performance, convenience and low-cost. In this article, we describe a colorimetric vertical-flow DNA microarray (DNA-VFM) that takes advantage of the screening capability of DNA microarrays in a paper format together with isothermal amplification by means of Recombinase Polymerase Amplification (RPA). Different assay parameters such as hybridization buffer, flow rate, printing buffer and capture probe concentration were optimized. A limit of detection (LOD) of 4.4 nM was achieved as determined by tabletop scanning. The DNA-VFM was applied as a proof of concept for detection of Neisseria meningitidis, a primary cause of bacterial meningitis. The LOD was determined to be between 38 and 2.1 × 106 copies/VFMassay, depending on the choice of DNA capture probes. The presented approach provides multiplex capabilities of DNA microarrays in a paper-based format for future point-of-care applications.

Health care workers' perceptions of point-of-care testing in a low-income country-A qualitative study in Southwestern Uganda.

BACKGROUND: Point-of-care (POC) tests have become increasingly available and more widely used in recent years. They have been of particular importance to low-income settings, enabling them with clinical capacities that had previously been limited. POC testing programs hold a great potential for significant improvement in low-income health systems. However, as most POC tests are developed in high-income countries, disengagement between developers and end-users inhibit their full potential. This study explores perceptions of POC test end-users in a low-income setting, aiming to support the development of novel POC tests for low-income countries. METHODS: A qualitative study was conducted in Mbarara District, Southwestern Uganda, in October 2014. Fifty health care workers were included in seven focus groups, comprising midwives, laboratory technicians, clinical and medical officers, junior and senior nurses, and medical doctors. Discussions were audio-recorded and transcribed verbatim. Transcripts were coded through a data-driven approach for qualitative content analysis. RESULTS: Nineteen different POC tests were identified as currently being in use. While participants displayed being widely accustomed to and appreciative of the use of POC tests, they also assessed the use and characteristics of current tests as imperfect. An ideal POC test was characterized as being adapted to local conditions, thoughtfully implemented in the specific health system, and capable of improving the care of patients. Tests for specific medical conditions were requested. Opinions differed with regard to the ideal distribution of POC tests in the local health system. CONCLUSION: POC tests are commonly used and greatly appreciated in this study setting. However, there are dissatisfactions with current POC tests and their use. To maximize benefit, stakeholders need to include end-user perspectives in the development and implementation of POC tests. Insights from this study will influence our ongoing efforts to develop POC tests that will be particularly usable in low-income settings.

Regulation of myeloid cells by activated T cells determines the efficacy of PD-1 blockade.

Removal of immuno-suppression has been reported to enhance antitumor immunity primed by checkpoint inhibitors. Although PD-1 blockade failed to control tumor growth in a transgenic murine neuroblastoma model, concurrent inhibition of colony stimulating factor 1 receptor (CSF-1R) by BLZ945 reprogrammed suppressive myeloid cells and significantly enhanced therapeutic effects. Microarray analysis of tumor tissues identified a significant increase of T-cell infiltration guided by myeloid cell-derived chemokines CXCL9, 10, and 11. Blocking the responsible chemokine receptor CXCR3 hampered T-cell infiltration and reduced antitumor efficacy of the combination therapy. Multivariate analysis of 59 immune-cell parameters in tumors and spleens detected the correlation between PD-L1-expressing myeloid cells and tumor burden. In vitro, anti-PD-1 antibody Nivolumab in combination with BLZ945 increased the activation of primary human T and NK cells. Importantly, we revealed a previously uncharacterized pathway, in which T cells secreted M-CSF upon PD-1 blockade, leading to enhanced suppressive capacity of monocytes by upregulation of PD-L1 and purinergic enzymes. In multiple datasets of neuroblastoma patients, gene expression of CD73 correlated strongly with myeloid cell markers CD163 and CSF-1R in neuroblastoma tumors, and associated with worse survival in high-risk patients. Altogether, our data reveal the dual role of activated T cells on myeloid cell functions and provide a rationale for the combination therapy of anti-PD-1 antibody with CSF-1R inhibitor.

An 8 minute colorimetric paper-based reverse phase vertical flow serum microarray for screening of hyper IgE syndrome.

Reverse phase microarrays are useful tools for affinity-based detection in hundreds of samples simultaneously. However, current methods typically require long assay times and fluorescent detection. Here we describe a paper-based Vertical Flow Microarray (VFM) assay as a rapid 8-minute colorimetric alternative for reverse phase microarray analysis. The VFM platform was optimized for detection of IgE with a detection limit of 1.9 µg mL(-1) in whole serum. Optimized conditions were then used to screen 113 serum samples simultaneously for hyper IgE syndrome (hIgE), a rare primary immunodeficiency characterized by elevated levels of IgE. The same set of samples were then analysed with a conventional planar microarray with fluorescent detection for head-to-head testing. Both assays found elevated levels in three out of four hIgE patient samples, whereas no control samples displayed elevated levels in either method. The comparison experiments showed a good correlation between the two assays, as determined from a linear correlation study (Pearson's r = 0.76). Further, the assay-time reduction and reproducibility (intra assay CV = 12.4 ± 4.11%) demonstrate the applicability of the VFM platform for high throughput reverse phase screening.