Beyond basic characterization and omics: Immunomodulatory roles of platelet-derived extracellular vesicles unveiled by functional testing.

Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor-mediated activation and environmental cues.

Monitoring SARS-CoV-2 IgA, IgM and IgG antibodies in dried blood and saliva samples using antibody proximity extension assays (AbPEA).

Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from ø1.2 mm discs cut from dried blood and saliva spots (DBS and DSS) without the need for elution. For total Ig detection, antigen probes were prepared by conjugating recombinant spike protein subunit 1 (S1-RBD) to a pair of oligonucleotides.

Increased levels of thymidine kinase 1 in malignant cell-derived extracellular vesicles.

Extracellular vesicles (EVs), whose main subtypes are exosomes, microparticles, and apoptotic bodies, are secreted by all cells and harbor biomolecules such as DNA, RNA, and proteins. They function as intercellular messengers and, depending on their cargo, may have multiple roles in cancer development. Thymidine kinase 1 (TK1) is a cell cycle-dependent enzyme used as a biomarker for cell proliferation.

GABA-mediated inhibition of human CD4 T cell functions is enhanced by insulin but impaired by high glucose levels.

Background: γ-aminobutyric acid (GABA), known as the main inhibitory neurotransmitter in the brain, exerts immunomodulatory functions by interaction with immune cells, including T cells. Metabolic programs of T cells are closely linked to their effector functions including proliferation, differentiation, and cytokine production. The physiological molecules glucose and insulin may provide environmental cues and guidance, but whether they coordinate to regulate GABA-mediated T cell immunomodulation is still being examined.

OxLDL sensitizes platelets for increased formation of extracellular vesicles capable of finetuning macrophage gene expression.

Platelet extracellular vesicles (PEVs) generated upon platelet activation may play a role in inflammatory pathologies such as atherosclerosis. Oxidized low-density lipoprotein (oxLDL), a well-known contributor to atherogenesis, activates platelets and presensitizes them for activation by other agonists. We studied the effect of oxLDL on the secretion, composition, and inflammatory functions of PEVs using contemporary EV analytics.

Aptamer-Assisted Proximity Ligation Assay for Sensitive Detection of Infectious Bronchitis Coronavirus.

Infectious bronchitis virus (IBV) is a coronavirus responsible for major health problems in the poultry industry. New virus strains continue to appear, causing large economic losses. To develop a rapid and accurate new quantitative assay for diagnosis of the virus without DNA extraction, we selected highly specific single-stranded DNA (ssDNA) aptamers with a high affinity to IBV, using the systematic evolution of ligands by exponential enrichment (SELEX) technology for aptamer screening, followed by high-throughput sequencing technology.

Surface protein profiling of prostate-derived extracellular vesicles by mass spectrometry and proximity assays.

Extracellular vesicles (EVs) are mediators of intercellular communication and a promising class of biomarkers. Surface proteins of EVs play decisive roles in establishing a connection with recipient cells, and they are putative targets for diagnostic assays. Analysis of the surface proteins can thus both illuminate the biological functions of EVs and help identify potential biomarkers.

Detection of SARS-CoV-2 antibodies in serum and dried blood spot samples of vaccinated individuals using a sensitive homogeneous proximity extension assay.

A homogeneous PCR-based assay for sensitive and specific detection of antibodies in serum or dried blood spots (DBS) is presented and the method is used to monitor individuals infected with or vaccinated against SARS-CoV-2. Detection probes were prepared by conjugating the recombinant spike protein subunit 1 (S1), containing the receptor binding domain (RBD) of SARS-CoV-2, to each of a pair of specific oligonucleotides. The same was done for the nucleocapsid protein (NP).

Monitoring drug-target interactions through target engagement-mediated amplification on arrays and in situ.

Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes.

Plasma protein biomarker profiling reveals major differences between acute leukaemia, lymphoma patients and controls.

Aiming to accommodate the unmet need for easily accessible biomarkers with a focus on biological differences between haematological diseases, the diagnostic value of plasma proteins in acute leukaemias and lymphomas was investigated. A multiplex proximity extension assay (PEA) was used to analyze 183 proteins in diagnostic plasma samples from 251 acute leukaemia and lymphoma patients and compared with samples from 60 healthy controls. Multivariate modelling using partial least square discriminant analysis revealed highly significant differences between distinct disease subgroups and controls.

Mitochondrial Respiration-Dependent ANT2-UCP2 Interaction.

Adenine nucleotide translocases (ANTs) and uncoupling proteins (UCPs) are known to facilitate proton leak across the inner mitochondrial membrane. However, it remains to be unravelled whether UCP2/3 contribute to significant amount of proton leak . Reports are indicative of UCP2 dependent proton-coupled efflux of C4 metabolites from the mitochondrial matrix.

Platelet-Derived PDGFB Promotes Recruitment of Cancer-Associated Fibroblasts, Deposition of Extracellular Matrix and Tgfβ Signaling in the Tumor Microenvironment.

Platelets constitute a major reservoir of platelet-derived growth factor B (PDGFB) and are continuously activated in the tumor microenvironment, exposing tumors to the plethora of growth factors contained in platelet granules. To address the specific role of platelet-derived PDGFB in the tumor microenvironment, we have created a mouse model with conditional knockout of PDGFB in platelets (pl-PDGFB KO). Lack of PDGFB in platelets resulted in 10-fold lower PDGFB concentration in the tumor microenvironment, fewer cancer-associated fibroblasts and reduced deposition of the extracellular matrix (ECM) molecules fibronectin and collagen I in the orthotopic RIP1-Tag2 model for pancreatic neuroendocrine cancer.

Immune-Proteome Profiling in Classical Hodgkin Lymphoma Tumor Diagnostic Tissue.

In classical Hodgkin Lymphoma (cHL), immunoediting via protein signaling is key to evading tumor surveillance. We aimed to identify immune-related proteins that distinguish diagnostic cHL tissues (=diagnostic tumor lysates, = 27) from control tissues (reactive lymph node lysates, = 30). Further, we correlated our findings with the proteome plasma profile between cHL patients ( = 26) and healthy controls ( = 27).

Image-based high-throughput mapping of TGF-β-induced phosphocomplexes at a single-cell level.

Protein interactions and posttranslational modifications orchestrate cellular responses to e.g. cytokines and drugs, but it has been difficult to monitor these dynamic events in high-throughput.

Analysis of blood group antigens on MUC5AC in mucinous ovarian cancer tissues using in situ proximity ligation assay.

MUC5AC has been indicated to be a marker for mucinous ovarian cancer (OC). We investigated the use of in situ proximity ligation assay (PLA) for blood group ABH expressing MUC5AC to differentiate between serous and mucinous OC, to validate preceding observations that also MUC5AC ABH expression is increased in mucinous OC. We developed PLA for anti-A, B, and H/anti-MUC5AC and a PLA using a combined lectin Ulex europaeus agglutinin I (UEA I)/anti-MUC5AC assay.

Accurate detection of Newcastle disease virus using proximity-dependent DNA aptamer ligation assays.

Detecting viral antigens at low concentrations in field samples can be crucial for early veterinary diagnostics. Proximity ligation assays (PLAs) in both solution and solid-phase formats are widely used for high-performance protein detection in medical research. However, the affinity reagents used, which are mainly poly- and monoclonal antibodies, play an important role in the performance of PLAs.

Plasma proteome profiling of cardiotoxicity in patients with diffuse large B-cell lymphoma.

Background: Cardiovascular toxicity is a notorious complication of doxorubicin (DXR) therapy for diffuse large B-cell lymphoma (DLBCL). Although surveillance of well-known biological markers for cardiovascular disease (CVD) as NTproBNP and Troponins may be helpful, there are no established markers to monitor for evolving CVD during treatment. New possibilities have arisen with the emergence of newer techniques allowing for analysis of plasma proteins that can be associated with cardiovascular disease.

Generation of ssDNA aptamers as diagnostic tool for Newcastle avian virus.

Aptamers are short single-stranded DNA (ssDNA), RNA or synthetic XNA molecules, which are used as a class of affinity binders recognizing target molecules with a very high affinity and specificity. The aim of this study was to generate and characterize ssDNA aptamers for the detection of Newcastle disease virus (NDV). These aptamers were selected using systematic evolution of ligands by exponential enrichment (SELEX) in combination with quantitative high-throughput DNA sequencing.

Human proteins incorporated into tick-borne encephalitis virus revealed by in situ proximity ligation.

Host proteins incorporated into virus particles have been reported to contribute to infectivity and tissue-tropism. This incorporation of host proteins is expected to be variable among viral particles, however, protein analysis at single-virus levels has been challenging. We have developed a method to detect host proteins incorporated on the surface of virions using the in situ proximity ligation assay (isPLA) with rolling circle amplification (RCA), employing oligonucleotide-conjugated antibody pairs.

Early increment of soluble triggering receptor expressed on myeloid cells 2 in plasma might be a predictor of poor outcome after ischemic stroke.

Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) is derived from cleavage of TREM2, which is expressed on the cell surface of microlgia and other tissue-specific macrophages. In the present study, the changes in the sTREM2 levels after ischemic stroke (IS) and their association with clinical outcomes were evaluated. A total of 43 patients diagnosed with non-cardioembolic IS between June 2011 and May 2014 were consecutively included in this study.

Depression, GABA, and Age Correlate with Plasma Levels of Inflammatory Markers.

Immunomodulation is increasingly being recognised as a part of mental diseases. Here, we examined whether levels of immunological protein markers changed with depression, age, or the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). An analysis of plasma samples from patients with a major depressive episode and control blood donors (CBD) revealed the expression of 67 inflammatory markers.

Profiling surface proteins on individual exosomes using a proximity barcoding assay.

Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical.

Reduced sialyl-Lewis on salivary MUC7 from patients with burning mouth syndrome.

We analysed and compared MUC7 O-glycosylation and inflammatory biomarkers in saliva from female patients with burning mouth syndrome (BMS) and gender/age-matched controls. Oligosaccharides from salivary MUC7 from BMS and controls were released. Inflammatory mediators were measured by multiplex proximity extension assay.

Multiplex plasma protein profiling identifies novel markers to discriminate patients with adenocarcinoma of the lung.

Background: The overall prognosis of non-small cell lung cancer (NSCLC) is poor, and currently only patients with localized disease are potentially curable. Therefore, preferably non-invasively determined biomarkers that detect NSCLC patients at early stages of the disease are of high clinical relevance. The aim of this study was to identify and validate novel protein markers in plasma using the highly sensitive DNA-assisted multiplex proximity extension assay (PEA) to discriminate NSCLC from other lung diseases.