Search Results (932)

Background: Immune checkpoint inhibitor therapy in patients with lung cancer and metastatic melanoma is associated with exacerbation of autoimmune-related diseases. The efficacy of treatment targeting the programmed cell death receptor-1 (PD-1) checkpoint relies upon a feedback loop between interferon gamma (IFN-γ) and the interleukin-12 isoform, IL-12p40. Paradoxically, both cytokines and the anti-PD-1 antibody worsen psoriasis. We previously reported an immunomodulating peptide, designated IK14004, that inhibits progression of Lewis lung cancer in mice yet uncouples IFN-γ from IL-12p40 production in human immune cells. Methods: Immune cells obtained from healthy donors were exposed to IK14004 in vitro to further characterise the signalling pathways affected by this peptide. Using C57BL/6 immunocompetent mice, the effect of IK14004 was tested in models of lung melanoma and psoriatic skin. Results: Differential effects of IK14004 on the expression of IFN-α/β, the interleukin-15 (IL-15) receptor and signal transducers and activators of transcription were consistent with immune responses relevant to both cancer surveillance and immune tolerance. Moreover, both melanoma and psoriasis were inhibited by the peptide. Conclusions: Taken together, these findings suggest mechanisms underlying immune homeostasis that could be exploited in the setting of cancer and autoimmune pathologies. Peptide administered together with checkpoint blockers in relevant models of autoimmunity and cancer may offer an opportunity to gain further insight into how immune tolerance can be retained in patients receiving cancer immunotherapy. Full article

Human leukocyte antigen class I (HLA-I) molecules present intracellular peptides on the cell surface to enable CD8+ T cells to effectively control viral infections. Many viruses disrupt this antigen presentation pathway to evade immune detection. In this study, we demonstrate that SARS-CoV-2 Nsp1 impairs both the constitutive and interferon-γ (IFN-γ)-induced upregulation of HLA-I. Moreover, Nsp1 also blocks IFN-γ-induced expression of HLA-II. We found that, contrary to previously published work, the early SARS-CoV-2 B 1.1.7 Alpha variant lacking the accessory protein ORF8 retained full capacity to downregulate HLA-I, comparable to an ORF8-expressing wild-type isolate. While ectopic overexpression of ORF8 could reduce HLA-I surface levels, this effect was only observed at high expression levels. In contrast, moderate expression of the viral protein Nsp1 was sufficient to potently suppress both basal and IFN-γ-induced HLA-I, as well as HLA-II expression. To probe the underlying mechanism, we analyzed HLA-I-associated genes in previously published RNA-sequencing datasets and confirmed that Nsp1 reduces expression of components required for HLA-I biosynthesis and antigen processing. These findings identify Nsp1 as a key factor that impairs antigen presentation pathways, potentially contributing to the ability of SARS-CoV-2 to modulate immune recognition. Full article

Recent preclinical and clinical studies have confirmed the essential role of interferons in the host’s immune response against malignant cells. Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer strongly associated with Merkel cell polyomavirus (MCPyV). Despite progress in understanding MCC pathogenesis, the role of innate immune signaling, particularly interferon-γ (IFN γ) and its downstream pathways, remains underexplored. This review summarizes recent findings on IFN-γ in MCC, highlighting its dual role in promoting both antitumor immunity and immune evasion. IFN-γ enhances cytotoxic T cell responses, upregulates MHC class I/II expression, and induces tumor cell apoptosis. Transcriptomic studies have shown that IFN-γ treatment upregulates immune-regulatory genes including PD-L1, HLA-A/B/C, and IDO1 by over threefold; it also activates APOBEC3B and 3G, contributing to antiviral defense and tumor editing. Clinically, immune checkpoint inhibitors (ICIs) such as pembrolizumab and avelumab yield objective response rates of 30–56% and two-year overall survival rates exceeding 60% in advanced MCC. However, approximately 50% of patients do not respond, in part due to IFN-γ signaling deficiencies. This review further discusses IFN-γ’s crosstalk with the STAT1/3/5 pathways and emerging combination strategies aimed at restoring immune sensitivity. Understanding these mechanisms may inform personalized immunotherapeutic approaches and guide the development of IFN-γ–based interventions in MCC. Full article

Fecal microbiota transplantation (FMT) promotes growth performance and intestinal development in yellow-feathered broilers, but whether the virome and metabolites contribute to its growth-promoting effect remains unclear. This study removed the microbiota from FMT filtrate using a 0.45 μm filter membrane, retaining the virome and metabolites to perform fecal virome transplantation (FVT), aiming to investigate its regulatory role in broiler growth. Healthy yellow-feathered broilers with high body weights (top 10% of the population) were used as FVT donors. Ninety-six 8-day-old healthy male yellow-feathered broilers (95.67 ± 3.31 g) served as FVT recipients. Recipient chickens were randomly assigned to a control group and an FVT group. The control group was gavaged with 0.5 mL of normal saline daily, while the FVT group was gavaged with 0.5 mL of FVT solution daily. Growth performance, immune and antioxidant capacity, intestinal development and related gene expression, and microbial diversity were measured. The results showed that FVT improved the feed utilization rate of broilers (the feed conversion ratio decreased by 3%; p < 0.05), significantly increased jejunal length (21%), villus height (69%), and crypt depth (84%) (p < 0.05), and regulated the jejunal barrier: insulin-like growth factor-1 (IGF-1) (2.5 times) and Mucin 2 (MUC2) (63 times) were significantly upregulated (p < 0.05). FVT increased the abundance of beneficial bacteria Lactobacillales. However, negative effects were also observed: Immunoglobulin A (IgA), Immunoglobulin G (IgG), Immunoglobulin M (IgM), Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), and Interferon-gamma (IFN-γ) in broilers were significantly upregulated (p < 0.05), indicating immune system overactivation. Duodenal barrier-related genes Mucin 2 (MUC2), Occludin (OCLN), Claudin (CLDN1), and metabolism-related genes solute carrier family 5 member 1 (SLC5A1) and solute carrier family 7 member 9 (SLC7A9) were significantly downregulated (p < 0.05). The results of this trial demonstrate that, besides the microbiota, the gut virome and metabolites are also functional components contributing to the growth-promoting effect of FMT. The differential responses in the duodenum and jejunum reveal spatial heterogeneity and dual effects of FVT on the intestine. The negative effects limit the application of FMT/FVT. Identifying the primary functional components of FMT/FVT to develop safe and targeted microbial preparations is one potential solution. Full article

Type-I Interferon (IFN) is essential for antiviral immunity in both mice and humans; thus, we investigated whether LAT affects HSV-1 infectivity in the absence of IFN by infecting IFNαβR^−/− and wild-type control mice with HSV-1 McKrae (LAT-plus) and dLAT2903 (LAT-minus) viruses. IFNαβR^−/− mice survived ocular infection with the LAT-plus virus, while no infected mice survived infection with the LAT-minus virus. Increased death in infected mice correlated with a higher expression in the neurovirulence γ34.5 gene but not with gB expression. To determine the region of LAT that contributed to higher mortality, IFNαβR^−/− mice were infected with recombinant viruses expressing the first 1.5 kb or the first 811bp region of 1.5 kb LAT. Similar to LAT-plus infected mice, IFNαβR^−/− mice infected with LAT1.5kb were protected from death, while infection with the LAT811bp virus was similar to that of LAT-minus, suggesting that increased pathogenicity in the absence of LAT depends on the second half of 1.5 kb LAT. To confirm the in vivo upregulation of γ34.5 expression in the absence of LAT, rabbit skin and Neuro2A cells were infected with LAT-plus, LAT-minus, LAT1.5kb, or LAT811bp viruses. γ34.5 expression was significantly higher in LAT-minus- and LAT811bp-infected rabbit skin cells and Neuro2A cells than in LAT-plus- and LAT1.5kb-infected cells, suggesting that sequences after the 811bp of LAT contribute to γ34.5 upregulation. However, except for γ34.5 expression, ICP0, ICP4, and gB expression were not affected by the absence of LAT or truncated forms of LAT. To confirm that higher γ34.5 expression contributes to higher mortality in the absence of LAT, we infected IFNαβR^−/− mice with a recombinant virus lacking LAT and γ34.5 expression, and, in contrast to LAT-minus, all infected mice survived. Our results suggest that LAT controls γ34.5 expression and that higher γ34.5 expression and mortality in infected mice are associated with the second half of 1.5 kb LAT. Full article

Interferon (IFN) signaling plays vital roles in host defense against viral infection. However, a variety of observations have been reported in the literature regarding the roles of IFN signaling in COVID-19. Thus, it would be important to reach a clearer picture regarding the activation or suppression of IFN signaling in COVID-19. In this work, regulation of marker genes for IFN signaling was examined in natural infection, viral challenge, and vaccination based on 13 public transcriptome datasets. Three subsets of interferon-stimulated genes (ISGs) were selected for detailed examination, including one set of marker genes for type I IFN signaling (ISGa) and two sets of marker genes for type II IFN signaling (IFN-γ signaling, GBPs for the GBP gene cluster, and HLAd for the HLA-D gene cluster). In natural infection, activation of ISGa and GBPs was accompanied by the suppression of HLAd in hospitalized patients. Suppression of GBPs was also observed in certain critical conditions. The scale of regulation was much greater for ISGa than that of GBPs and HLAd. In addition, the suppression of HLAd was correlated with disease severity, and it took much longer for HLAd to return to the level of healthy controls than that for ISGa and GBPs. Upon viral challenge, the activation of ISGa and GBPs was similar to that of natural infection, while the suppression of HLAd was not observed. Moreover, GBPs’ return to the pre-infection level was at a faster pace than that of ISGa. Upon COVID-19 vaccination, activation was observed for all of these three gene sets, and the scale of activation was comparable for ISGa and GBPs. Notably, it took a much shorter time for GBPs and ISGa to return to the level of healthy controls than that in COVID-19 infection. In addition, the baseline values and transient activation of these gene sets were also associated with subsequent vaccination response. The intricate balance of IFN signaling was demonstrated in mild breakthrough infection, where attenuated response was observed in people with prior vaccination compared to that in vaccine-naïve subjects. Overall, distinctive temporal profiles of IFN signaling were observed in natural infection, viral challenge, and vaccination. The features observed in this work may provide novel insights into the disease management and vaccine development. Full article

Background/Objectives: Romania remains a tuberculosis (TB) hotspot in the European Union, yet host-derived factors of poor outcomes are poorly characterised. We quantified circulating pro-inflammatory cytokines and examined their interplay with behavioural risk factors, the nutritional status, and the clinical course in adults hospitalised with pulmonary TB. We analysed 80 adults with microbiologically confirmed pulmonary TB and 40 respiratory symptom controls; four TB patients (5%) died during hospitalisation, all within 10 days of admission. Methods: A retrospective analytical case–control study was conducted at the Constanța regional TB referral centre (October 2020—October 2023). Patients with smear- or culture-confirmed TB were frequency-matched by sex, 10-year age band, and BMI class to culture-negative respiratory controls at a 2:1 ratio. The patients’ serum interferon-γ (IFN-γ), interleukin-1α (IL-1α), interleukin-1β (IL-1β), and tumour-necrosis-factor-α (TNF-α) were quantified within 24 h of admission; the neutrophil/lymphocyte ratio (NLR) was extracted from full blood counts. Independent predictors of in-hospital mortality were identified by multivariable logistic regression; factors associated with the length of stay (LOS) were modelled with quasi-Poisson regression. Results: The median TNF-α (24.1 pg mL⁻¹ vs. 16.2 pg mL⁻¹; p = 0.009) and IL-1β (5.34 pg mL⁻¹ vs. 3.67 pg mL⁻¹; p = 0.008) were significantly higher in the TB cases than in controls. TNF-α was strongly correlated with IL-1β (ρ = 0.80; p < 0.001), while NLR showed weak concordance with multiplex cytokine patterns. Among the patients with TB, four early deaths (5%) exhibited a tripling of TNF-α (71.4 pg mL⁻¹) and a doubling of NLR (7.8) compared with the survivors. Each 10 pg mL⁻¹ rise in TNF-α independently increased the odds of in-hospital death by 1.8-fold (95% CI 1.1–3.0; p = 0.02). The LOS (median 29 days) was unrelated to the smoking, alcohol, or comorbidity load, but varied across BMI strata: underweight, 27 days; normal weight, 30 days; overweight, 23 days (Kruskal–Wallis p = 0.03). In a multivariable analysis, under-nutrition (BMI < 18.5 kg m⁻²) prolonged the LOS by 19% (IRR 1.19; 95% CI 1.05–1.34; p = 0.004) independently of the disease severity. Conclusions: A hyper-TNF-α/IL-1β systemic signature correlates with early mortality in Romanian pulmonary TB, while under-nutrition is the dominant modifiable determinant of prolonged hospitalisation. Admission algorithms that pair rapid TNF-α testing with systematic nutritional assessment could enable targeted host-directed therapy trials and optimise bed utilisation in high-burden settings. Full article

Background: Cerebral aneurysm (CA) is a focal or diffuse pathological dilation of the cerebral arterial wall that arises due to various etiological factors. It represents a serious vascular condition, particularly affecting the elderly, and carries a high risk of rupture and neurological morbidity. Clonidine (CL), an α2-adrenergic receptor agonist, has been reported to suppress aneurysm progression; however, its underlying molecular mechanisms, especially in relation to cerebral endothelial dysfunction, remain unclear. This study aimed to investigate the potential of CL to mitigate CA development by modulating apoptosis, inflammation, and oxidative stress in an Angiotensin II (Ang II)-induced endothelial injury model. Methods: Human brain microvascular endothelial cells (HBMECs) were used to establish an in vitro model of endothelial dysfunction by treating cells with 1 µM Ang II for 48 h. CL was administered 2 h prior to Ang II exposure at concentrations of 0.1, 1, and 10 µM. Cell viability was assessed using the MTT assay. Oxidative stress markers, including reactive oxygen species (ROS) and Nitric Oxide (NO), were measured using 2′,7′–dichlorofluorescin diacetate (DCFDA). Gene expression levels of vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP-2 and MMP-9), high mobility group box 1 (HMGB1), and nuclear factor kappa B (NF-κB) were quantified using RT-qPCR. Levels of proinflammatory cytokines; tumor necrosis factor-alpha (TNF-α), Interleukin-6 (IL-6), and interferon-gamma (IFN-γ); were measured using commercial ELISA kits. Results: Ang II significantly increased ROS production and reduced NO levels, accompanied by heightened proinflammatory cytokine release and endothelial dysfunction. MTT assay revealed a marked decrease in cell viability following Ang II treatment (34.18%), whereas CL preserved cell viability in a concentration-dependent manner: 44.24% at 0.1 µM, 66.56% at 1 µM, and 81.74% at 10 µM. CL treatment also significantly attenuated ROS generation and inflammatory cytokine levels (p < 0.05). Furthermore, the expression of VEGF, HMGB1, NF-κB, MMP-2, and MMP-9 was significantly downregulated in response to CL. Conclusions: CL exerts a protective effect on endothelial cells by reducing oxidative stress and suppressing proinflammatory signaling pathways in Ang II-induced injury. These results support the potential of CL to mitigate endothelial injury in vitro, though further in vivo studies are required to confirm its translational relevance. Full article

Military personnel deployed to Iraq and Afghanistan were exposed to emissions from open-air burn pits, where plastics, metals, and medical waste were incinerated. These exposures have been linked to deployment-related respiratory diseases (DRRD) and may also impact neurological health via the lung–brain axis. To investigate molecular mechanisms, adult male rats were exposed to filtered air, naphthalene (a representative volatile organic compound), or a combination of naphthalene and carbon black (surrogate for particulate matter; CBN) via whole-body inhalation (six hours/day, three consecutive days). Lung, brain, and plasma samples were collected 24 h after the final exposure. Pro-inflammatory biomarkers were assessed using multiplex electrochemiluminescence and western blot. Differentially expressed genes (DEGs) were identified by RNA sequencing, and elastic net modeling was used to define exposure-predictive gene signatures. CBN exposure altered inflammatory biomarkers across tissues, with activation of nuclear factor kappa B (NF-κB) signaling. In the lung, gene set enrichment revealed activated pathways related to proliferation and inflammation, while epithelial–mesenchymal transition (EMT) and oxidative phosphorylation were suppressed. In the brain, EMT, inflammation, and senescence pathways were activated, while ribosomal function and oxidative metabolism were downregulated. Elastic net modeling identified a lung gene signature predictive of CBN exposure, including Kcnq3, Tgfbr1, and Tm4sf19. These findings demonstrate that inhalation of a surrogate burn pit mixture induces inflammatory and metabolic gene expression changes in both lung and brain tissues, supporting the utility of this animal model for understanding systemic effects of airborne military toxicants and for identifying potential biomarkers relevant to DRRD and Veteran health. Full article

Anticytokine autoantibodies (AAbs), particularly anti-interferon-gamma (anti-IFN-γ) AAbs, disrupt cytokine functions, leading to infections, autoimmune-like diseases, and conditions resembling interleukin-12 (IL-12)/IFN-γ pathway defects. Advances in genetic testing have clarified overlaps between autoinflammatory, autoimmune disorders, and primary immunodeficiencies but reveal complex phenotypes and pathways. While these insights deepen our understanding of immune mechanisms, they also complicate diagnosis and treatment, with limited options for IFN-γ deficiencies caused by genetic mutations. The adult-onset immunodeficiency with disseminated lymphadenitis due to nontuberculous mycobacteria (NTM) and other opportunistic infections has been linked to high levels of anti-IFN-γ AAbs. This syndrome, initially identified in HIV-negative Asian patients, frequently affects individuals of Asian descent and may be associated with specific human leukocyte antigen (HLA) alleles. The presence of neutralizing anti-IFN-γ AAbs impairs the IFN-γ-dependent immune response, likely contributing to the persistent NTM infection. This study underscores the potential for late-onset anti-IFN-γ AAb syndrome to manifest with disseminated NTM (dNTM) infections, highlights the importance of timely diagnosis and considers rituximab as a potential therapeutic option. Full article

Background: To identify drug candidates that reduce cellular stress, linear peptides known as endomorphin (EM) analogs containing proline surrogates in position 2 were tested in in vitro injury models induced by corticosterone (CORT). Methods: In this study, neuroblastoma (SH-SY5Y) cells were treated with CORT and synthesized peptides, and then the cell viability and morphology, reactive oxygen species production (ROS), mitochondrial membrane potential (ΔΨm), adenosine triphosphate (ATP), and intracellular calcium ion [Ca²⁺]_i levels were evaluated. We also conducted an in-depth analysis of the apoptosis markers using quantitative real-time PCR (qPCR). Finally, we explore the brain-derived neurotrophic factor (BDNF) expression (qPCR) and protein levels (ELI-SA and Western blot). Results: The strongest neuroprotective effect in the CORT-induced stress model was shown by peptide 3 and peptide 7 (in the following sequence Tyr-Inp-Trp-Phe-NH2 and Tyr-Inp-Phe-Phe-NH2, respectively). These peptides significantly improved cell viability and reduced oxidative stress in CORT-treated cells. Conclusions: Their neuroprotective potential appears linked to anti-apoptotic effects, along with in-creased BDNF expression. Moreover, in the lipopolysaccharide (LPS)- and interferon-γ (IFN-γ)-induced damage model in macrophage RAW 264.7 cells, these two peptides reduced the secretion of inflammatory mediators nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Peptides exhibiting both neuroprotective and anti-inflammatory properties warrant further investigation as potential therapeutic agents. Full article

Previously, research adopted an ultrasound-assisted extraction method to isolate crude polysaccharide from blackened jujube, followed by preliminary structural identification of the purified polysaccharide (BJP). This manuscript analyzed the accurate structure and immunomodulatory activity of BJP. Further structural identification indicated that BJP was mainly composed of →3)-α-L-Araf-(1→, →3,5)-α-L-Araf-(1→, →3)-β-D-GalpA-(1→, →2,4)-β-D-Galp-(1→, →4)-β-D-GalpA-(1→, →3)-α-L-Rhap-(1→ and →3,4)-α-L-Rhap-(1→. The immunomodulatory effects of BJP were examined using a mouse model with immunosuppression induced by cyclophosphamide. The findings suggested that BJP could relieve the condition of immunosuppressed mice. BJP could inhibit decreases in the body weight and organ index of mice, and HE staining showed that BJP could alleviate the harm to spleen and thymus tissues. BJP enhanced the secretion of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), interleukin-6 (IL-6), immunoglobulin A (IgA), and immunoglobulin G (IgG) in serum. It also reduced liver oxidative stress by increasing superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) activities, while lowering malondialdehyde (MDA) levels. Moreover, BJP contributed to the maintenance of gut homeostasis by stimulating the generation of short-chain fatty acids in the cecal contents. The study aims to establish a solid basis for the comprehensive development of blackened jujube and furnish a theoretical framework for its polysaccharides’ role in immune modulation. Full article

Atherosclerosis (AS), a progressive inflammatory disease of coronary arteries, the aorta, and the internal carotid artery, is considered one of the main contributors to cardiovascular disorders. Blood flow is restricted by accumulating lipid-rich macrophages (foam cells), calcium, fibrin, and cellular debris into plaques on the intima of arterial walls. Butyrate maintains gut barrier integrity and modulates immune responses. Butyrate regulates G-protein-coupled receptor (GPCR) signaling and activates nuclear factor kappa-B (NF-κB), activator protein-1 (AP-1), and interferon regulatory factors (IFRs) involved in the production of proinflammatory cytokines. Depending on the inflammatory stimuli, butyrate may also inactivate NF-κB, resulting in the suppression of proinflammatory cytokines and the stimulation of anti-inflammatory cytokines. Butyrate modulates mitogen-activated protein kinase (MAPK) to promote or suppress macrophage inflammation, muscle cell growth, apoptosis, and the uptake of oxidized low-density lipoprotein (ox-LDL) in macrophages. Activation of the peroxisome proliferator-activated receptor γ (PPARγ) pathway plays a role in lipid metabolism, inflammation, and cell differentiation. Butyrate inhibits interferon γ (IFN-γ) signaling and suppresses NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) involved in inflammation and scar tissue formation. The dual role of butyrate in AS is discussed by addressing the interactions between butyrate, intestinal epithelial cells (IECs), endothelial cells (ECs) of the main arteries, and immune cells. Signals generated from these interactions may be applied in the diagnosis and intervention of AS. Reporters to detect early AS is suggested. This narrative review covers the most recent findings published in PubMed and Crossref databases. Full article

Background/Objectives: Prostate cancer is the second most common type of cancer diagnosed in men. Various treatments for this cancer, such as radiation therapy, surgery, and systemic therapy, can cause side effects in patients; therefore, there is a need to develop new treatment alternatives. One promising approach is virotherapy, which involves using oncolytic viruses (OVs), such as the recombinant Newcastle disease virus (rNDV). Methods: We used the lentogenic rNDV rLS1 strain (the control virus) as our backbone to develop two highly fusogenic rNDVs: rFLCF5nt (the parental virus) and rFLCF5nt-IFN-γ (rFLCF5nt expressing human interferon-gamma (IFN-γ)). We evaluated their oncolytic properties in a prostate cancer cell line (DU145). Results: The results showed the expression and stability of the IFN-γ protein, as confirmed using Western blotting after ten passages in specific pathogen-free chicken embryo eggs using the IFN-γ-expressing virus. Additionally, we detected a significantly high oncolytic activity in DU145 cells infected with the parental virus or the IFN-γ-expressing virus using MTS (a cell viability assay) and Annexin V-PE assays compared with the control virus (p < 0.0001 for both). Conclusions: In conclusion, our data show that IFN-γ-expressing virus can decrease cell viability and induce apoptosis in human prostate cancer in vitro. Full article

Indoleamine 2,3-dioxygenase 1 (IDO-1) and interferon-gamma (IFN-γ) are proteins that play a significant role in inflammatory conditions and tumor development. The detection of IDO1 and IFN-γ is crucial for understanding their interplay in immune responses. This study introduced a novel method for the simultaneous quantitative determination of IDO-1 and IFN-γ in different biological samples/materials. The method is based on an optical biosensor, with surface plasmon resonance detection carried out by the imaging version of the sensor (SPRi). Biotinylated antibodies immobilized on the surfaces of the linker and carboxymethylated dextran served as the recognition elements for the developed biosensor. Relevant studies were conducted to optimize the activities of the biosensor by employing appropriate reagent concentrations. Validation was performed for each protein separately; low detection and quantification limits were obtained (for IDO-1 LOD = 0.27 ng/mL, LOQ = 0.81 ng/mL; for IFN-γ LOD = 1.76 pg/mL and LOQ = 5.29 pg/mL). The sensor operating ranges were 0.001–10 ng/mL for IDO-1 and 0.1–1000 pg/mL for IFN-γ. The constructed biosensor demonstrated its sensitivity and precision when the appropriate analytical parameters were determined, based on the proposed method. It can also selectively capture IDO-1 and IFN-γ from a large sample matrix. The biosensor efficiency was confirmed by the determination of IDO-1 and IFN-γ in simultaneous measurements of the plasma and urine samples of patients diagnosed with bladder cancer and the control group. The outcomes were compared to those obtained using a certified ELISA test, demonstrating convergence between the two methodologies. The preliminary findings demonstrate the biosensor’s efficacy and suitability for comprehensive analyses of the examined biological samples. Full article