Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR’s specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.

Celiac disease (CeD) is a conditional autoimmune disorder with T cell-mediated immune response to gluten coupled with antibody production to gliadin and the self-protein tissue transglutaminase (TG2). TG2 contributes to the CeD pathomechanism by deamidating gliadin, thereby generating more immunogenic peptides. Anti-gliadin antibodies may appear before the autoantibody production. The scope of this study was to dissect these early antibody responses by investigating serum samples collected during the PreventCD prospective double-blind study, where infants with high CeD risk were randomized to 200 mg daily gluten intake or placebo from 4 to 6 months of age, followed by frequent blood testing on regular gluten consumption in both groups. After primary gluten intake, children with or without later CeD produced IgA and IgG antibodies which preferentially recognized non-deamidated gliadin peptides. At CeD development with anti-TG2 seroconversion, there was a significant increase in the antibody reaction toward deamidated gliadin peptides (DGP), with maturation in the binding strength for the PEQPFP gamma-gliadin core peptide. The earliest produced autoantibodies targeted TG2’s celiac epitope 2. Our results reveal a qualitative change in the gliadin-directed humoral immune response at the time when anti-TG2 antibodies appear, but anti-DGP antibodies in the absence of anti-TG2 antibodies are not disease-predictive.

During exercise, increased oxygen consumption results in elevated production of reactive oxygen species (ROS). If the antioxidant system is unable to counteract this surge in ROS, oxidative stress occurs. Physical activity modulates both the generation and clearance of ROS through dynamic interactions between metabolic and antioxidant systems, and also influences the oxidative burst activity of phagocytes, a key component of the innate immune response. To investigate the acute physiological responses to high-intensity interval training (HIIT), we assessed the effects of a single HIIT session on oxidative stress markers and the oxidative burst activity of phagocytes in young professional athletes and non-athlete individuals. Blood samples were collected before and after a HIIT session from eleven male athletes (mean age: 22.1 ± 4.5 years) and ten male non-athlete university students (mean age: 21.6 ± 2.3 years). Participants performed a single treadmill HIIT session of ten 45-s intervals at 75–85% of heart rate reserve, separated by 45-s low-intensity recovery periods, with target intensities individualized using the Karvonen formula. Total antioxidant capacity, activities of catalase, superoxide dismutase and glutathione peroxidase enzymes, total serum nitrite/nitrate levels, lipid peroxidation products, and oxidative burst activity of phagocytes were evaluated before and after exercise. In athletes, a significant increase was observed in the activity of superoxide dismutase (from a median of 2.09 to 2.21 U/mL; p = 0.037) and catalase (from a median of 32.94 to 45.45 nmol/min/mL; p = 0.034) after exercise, whereas no significant changes were found in the control group. Total serum nitrite/nitrate levels significantly increased in both groups after exercise (athletes: from a median of 8.70 to 9.95 µM; p = 0.029; controls: from a median of 10.20 to 11.50 µM; p = 0.016). Oxidative burst capacity of peripheral blood phagocytes was significantly higher in athletes both before (median: 10,422 vs. 6766; p = 0.029) and after (median: 9365 vs. 7370; p = 0.047) the HIIT session compared to controls. Our findings demonstrate that training status markedly influences oxidative stress responses, with athletes exhibiting more effective long-term antioxidant adaptations. These results emphasize the necessity of tailoring exercise regimens to baseline fitness levels in order to optimize oxidative stress management across different populations.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease characterized by autoantibody production and multi-organ involvement. Anifrolumab, a monoclonal antibody targeting the type I interferon (IFN) receptor, has been approved for the treatment of SLE. Our aim was to investigate the long-term effects of inhibited type I IFN signaling on circulating follicular helper T subsets (T_FH), follicular regulatory T cells (T_FR), and B lymphocyte subpopulations, reflecting the ongoing germinal center reactions in SLE patients. Peripheral blood samples were obtained from ten SLE patients before the initiation of anifrolumab treatment, and at months 6 and 12 of the intervention period. Flow cytometry analysis was performed to assess the frequencies of circulating T_FH cell subsets, T_FR cells, and certain B cell subpopulations. Serological parameters, including autoantibody levels and complement components, were determined as part of the routine diagnostic evaluation. We observed a significant and sustained reduction in the percentage of activated circulating T_FH cells. Notably, the frequency of CXCR3⁻CCR6⁺ T_FH17 cells decreased, whereas the proportion of CXCR3⁺CCR6⁻ T_FH1 cells increased significantly. Furthermore, the proportion of the IgD⁻CD27⁻ double-negative B lymphocytes was also significantly reduced. These findings suggest that anifrolumab therapy attenuates T_FH cell activation, which may contribute to its clinical efficacy by modulating germinal center responses in SLE.

Background/Objectives: Age-related increases in oxidative stress and chronic low-grade inflammation contribute to skeletal muscle deterioration, impaired physical performance, and loss of functional independence in older adults. Regular exercise may counteract these processes by improving both musculoskeletal function and systemic redox homeostasis; however, the effects of multimodal functional exercise on antioxidant defence mechanisms and innate immune redox regulation remain incompletely understood. Therefore, the aim of the present study was to investigate changes associated with a six-week multimodal functional exercise program in body composition, muscle strength, physical performance, antioxidant defence and phagocyte oxidative burst activity in older women. Methods: Seventeen physically inactive women aged over 65 years participated in a six-week functional training program consisting of aerobic, resistance, balance, coordination, and proprioceptive exercises performed twice weekly. Body composition, handgrip strength, and Short Physical Performance Battery (SPPB) scores were assessed before and after the intervention. In addition, serum antioxidant enzyme activities, total antioxidant capacity, lipid peroxidation markers, nitrite/nitrate concentrations, and phagocyte oxidative burst activity were determined. Results: Following the intervention, improvements were observed in skeletal muscle mass, muscle percentage, handgrip strength, and SPPB scores, while body fat mass and body fat percentage decreased. Catalase activity increased, whereas superoxide dismutase activity, glutathione peroxidase activity, and lipid peroxidation markers remained unchanged. Serum nitrite/nitrate concentrations increased following the intervention. Although neutrophil counts did not change, phagocyte oxidative burst activity decreased significantly. Conclusions: These findings indicate that a six-week multimodal functional exercise program was associated with favourable changes in body composition, enhanced muscle strength, and improved functional performance in previously inactive older women. In parallel, favourable changes in antioxidant defence, nitric oxide metabolism, and immune-related redox regulation were also observed following the intervention, supporting the potential role of multimodal functional training as an effective non-pharmacological strategy for promoting healthy ageing.