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Background

Cardiovascular toxicity is a well-known complication of chemotherapy, especially doxorubicin (DXR), and irradiation of the mediastinum for classical Hodgkin lymphoma (cHL). Due to the excellent prognosis in cHL, the mortality rate in late toxicity historically exceeds that of relapse of lymphoma. This highlights the need for strategies to minimize toxicity.Our aim was to characterize the prevalence of cardiovascular diseases (CVDs) in our cohort of cHL patients treated with DXR with or without radiotherapy according to standard practice and to identify any plasma protein associations with preexisting or emerging CVD posttreatment.Methods

We analyzed 182 different proteins in plasma samples from 56 cHL patients and 60 controls using Olink multiplex protein panels Oncology II and Cardiovascular III. The analysis was supplemented with separate analyses of N-terminal pro-brain natriuretic peptide (NTpro-BNP), troponin I and C-reactive protein (CRP). The patient samples were prospectively collected prior to, during and after treatment.Results

Our analysis revealed a statistically significant association between the compound endpoint of heart failure and ischemic heart disease and the protein biomarkers cysteine rich protein 61 (CYR61), glycoprotein nonmetastatic melanoma protein B (GPNMB) and activated leukocyte cell adhesion molecule (ALCAM) in samples collected after treatment for cHL.Conclusion

This exploratory study identified three new biomarkers reflecting different biological processes associated with CVD in patients treated for cHL. Adding biomarkers to risk prediction in this population has the potential to identify patients with a high risk of cardiovascular events who need focused follow-up.

An electrochemical biosensor based on modified carbon screen-printed electrodes was developed for the detection of hemagglutinin of influenza A H1N1 virus (H1). Gold nanoflowers were electrodeposited on the electrode to increase conductivity and surface area. The electrochemical signal was amplified by functionalization of the gold nanoflowers with 4-aminothiophenol, which resulted in a 100-fold decrease of the charge transfer resistance due to a tunneling effect. Subsequently, monoclonal antibodies against H1 were immobilized on the surface via covalent amide bond formation, followed by blocking with bovine serum albumin to minimize nonspecific hydrophobic binding. The electrodes were characterized by cyclic voltammetry and electrochemical impedance spectroscopy experiments in the presence of [Fe(CN)6]3-/4-. Differential pulse voltammetry was used to measure the change in current across the electrode as a function of H1 concentration. This was performed on a series of samples of artificial saliva containing H1 protein in a clinically relevant concentration range. In these experiments, the biosensor showed a limit of detection of 19 pg/mL. Finally, the biosensor platform was coupled to an automated microfluidics system, and no significant decrease of the electrochemical signal was observed.

Introduction: Pregnant women in sub-Saharan Africa face heightened susceptibility to Plasmodium falciparum malaria, with placental sequestration driving adverse outcomes. The infection may lead to pregnancy-associated malaria (PAM) because of the sequestration of Plasmodium falciparum-infected erythrocytes in the placental intervillous space. Although there are several tools for diagnosing malaria infection during pregnancy, including blood smear microscopic examination, rapid diagnostic tests, and PCR, there are no tools for detecting placental infection and, by extension, any dysfunction associated with PAM. Thus, PAM, specifically placental infection, can only be confirmed via postnatal placental histopathology. Therefore, there is an urgent need for specific plasma biomarkers of PAM.

Methods: Here, we used the high throughput proximity extension assay to screen plasma from malaria-exposed pregnant women for differentially expressed proteins that may serve as candidate biomarkers of Plasmodium falciparum infection during pregnancy, with future potential to inform diagnosis of PAM or adverse malaria outcomes. Such biomarkers may also elucidate the pathophysiology of PAM.

Results: Using proximity extension assay (PEA), we identified elevated IgG Fc receptor IIb (FCGR2B) and heme oxygenase-1 (HO-1) in malaria-positive pregnancies, while neurturin (NRTN) and IL-20 were downregulated.

Discussion: IL-20 emerged as a top candidate biomarker, warranting validation in large cohorts with placental histopathology.

Background

Gastric cancer (GC), the world’s fifth most common malignancy, remains a major cause of death, motivating minimally invasive protein measurements. Small extracellular vesicles (sEVs) mirror cellular state but are difficult to profile from limited material. Proximity extension assay (PEA) enables multiplex, sensitive detection. We developed a PEA-centred workflow to profile GC cell-line sEVs, benchmarked against cells and an EV-rich control, and nominate hypothesis-generating candidates.

Methodss

EVs from four GC cell lines (Kato III, AGS, MKN45, MKN7) were isolated by differential centrifugation/filtration and ultracentrifugation; prostasomes (seminal-fluid EVs; ethics-approved) were purified by ultracentrifugation, Superdex 200 size-exclusion, and sucrose gradient. EVs were characterized by TEM, NTA, and Western blot (CD9/CD63/CD81; Calnexin). Protein profiling used Olink PEA across five Target 96 panels, two commercial (Oncology, Inflammation) and three experimental (Cancer, Cellular Pathways, Neurology) with availability-based selection; single replicate per condition. NPX values were background/LOD-corrected in R, with PCA. STRING PPI used medium confidence (0.4) with FDR-adjusted enrichment. Selected proteins were validated by SP-PLA (qPCR), EV-PLA (flow cytometry), and WB.

Resultss

EVs and prostasomes were purified and characterized per MISEV2023 (TEM/NTA; CD9/CD63/CD81+, calnexin–). Using five Olink PEA panels, 460 proteins were profiled; principal component analysis separated sEVs from parental cells. We identified 71 proteins enriched in GC sEVs, including EphA2, cMet, IL-6, SCF, AXIN1, NOTCH1, and CAV1; 17 were unique to Kato III sEVs. STRING showed a highly connected network, with top pathways: Pathways in cancer, Cytokine–cytokine receptor interaction, and PI3K–Akt. Orthogonal assays (SP-PLA, EV-PLA, WB densitometry) confirmed the presence of EphA2 and cMet on GC sEVs.

Discussions

PEA-based profiling of GC cell-line sEVs nominates 71 enriched proteins (e.g., EphA2, cMet, NOTCH1, CAV1) as hypothesis-generating candidates that may reflect cell-type–specific cargo and tumor–microenvironment signaling. Key limitations include the in-vitro design, single replicates, preselected PEA panels, and the use of prostasomes rather than healthy plasma sEVs, which may affect generalizability and specificity. Translational relevance requires validation in patient biofluids with orthogonal assays and cohort-level evaluation.

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.

This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C-type lectin-like receptor 2 and combining all thrombin-collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor-induced PEVs.

Our findings underscore that PEVs are tunable through receptor-mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative.

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.

METHODS: CD4⁺ T cells that were isolated from blood samples from healthy individuals and from patients with type 1 diabetes (T1D) were activated in vitro. We carried out metabolic assays, multiple proximity extension assay (PEA), ELISA, qPCR, immunoblotting, immunofluorescence staining, flow cytometry analysis, MS-based proteomics, as well as electrophysiology and live-cell Ca²⁺ imaging.

FINDINGS: We demonstrate that GABA-mediated reduction of metabolic activity and the release of inflammatory proteins, including IFNγ and IL-10, were abolished in human CD4⁺ T cells from healthy individuals and patients with T1D when the glucose concentration was elevated above levels typically observed in healthy people. Insulin increased GABA_A receptor-subunit ρ2 expression, enhanced the GABA_A receptors-mediated currents and Ca²⁺ influx. GABA decreased, whereas insulin sustained, hexokinase activity and glycolysis in a glucose concentration-dependent manner.

INTERPRETATION: These findings support that metabolic factors, such as glucose and insulin, influence the GABA-mediated immunomodulation of human primary T cells effector functions.

FUNDING: The Swedish Children's Diabetes Foundation, The Swedish Diabetes Foundation, The Swedish Research Council 2018-02952, EXODIAB, The Ernfors Foundation, The Thurings Foundation and the Science for Life Laboratory.

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. TK1 is usually elevated in cancer patients' serum, making the enzyme a valuable tumor proliferation biomarker that strongly correlates with cancer stage and metastatic capabilities. Here, we investigated the presence of TK1 in EVs derived from three prostate cancer cell lines with various p53 mutation statuses (LNCaP, PC3, and DU145), EVs from the normal prostate epithelial cell line RWPE-1 and EVs isolated from human seminal fluid (prostasomes). We measured the TK1 activity by a real-time assay for these EVs. We demonstrated that the TK1 enzyme activity is higher in EVs derived from the malignant cell lines, with the highest activity from cells deriving from the most aggressive cancer, compared to the prostasomes and RWPE-1 EVs. The measurement of TK1 activity in EVs may be essential in future prostate cancer studies.

Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from & oslash;1.2 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. To detect isotype-specific antibody reactivity, one antigen probe was replaced with oligonucleotide-conjugated antibodies specific for antibody isotypes. Binding of pairs of oligonucleotide-conjugated probes to antibodies in patient samples brings oligonucleotides in proximity. An added DNA polymerase uses a transient hybridization between the oligonucleotides to prime synthesis of a DNA strand, which serves as a DNA amplicon that is quantified by real-time PCR. The S1-RBD-specific IgG, IgM, and IgA antibodies in DBS samples collected over the course of a first and second vaccination exhibited kinetics consistent with previous reports. Both DBS and DSS collected from 42 individuals in the autumn of 2023 showed significant level of total S1-RBD antibodies with a correlation of R = 0.70. However, levels in DSS were generally 10 to 100-fold lower than in DBS. Anti-S1-RBD IgG and IgA in DSS demonstrated a correlation of R = 0.6.

Infectious bronchitis virus (IBV) causes high morbidity and mortality and large economic losses in the poultry industry. The virus has the ability to genetically mutate into new IBV strains, causing devastating disease and outbreaks. 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. Two of these aptamers, AptIBV5 and AptIBV2, were used to establish homogenous and solid-phase proximity ligation assays (PLAs). The developed assays were evaluated for their sensitivity and specificity using collected field samples and then compared to the newly developed sandwich enzyme-linked aptamer assay (ELAA) and reverse transcription-quantitative PCR (qRT-PCR), as the gold-standard method. The solid-phase PLA showed a lower limit of detection and a broader dynamic range than the two other assays. The developed technique may serve as an alternative assay for the diagnosis of IBV, with the potential to be extended to the detection of other important animal or human viruses.IMPORTANCE Infectious bronchitis virus (IBV) causes high morbidity and mortality and large economic losses in the poultry industry. The virus has the ability to genetically mutate into new IBV strains, causing devastating disease and outbreaks. To better monitor the emergence of this virus, the development of a rapid and highly sensitive diagnostic method should be implemented. For this, we generated aptamers with high affinity and specificity to the IBV in an ssDNA library. Using two high-affinity aptamers, we developed a sandwich ELAA and a very sensitive aptamer-based proximity ligation assay (PLA). The new assay showed high sensitivity and specificity and was used to detect IBV in farm samples. The PLA was compared to the newly developed sandwich ELAA and qRT-PCR, as the gold-standard technique.