Search Results (33)

Background: Tuberculosis (TB) remains a global health priority, with current interventions like the Bacille Calmette–Guérin (BCG) vaccine lacking efficacy against latent infection and drug-resistant strains. Novel vaccines targeting both latent and active TB are urgently needed. Objective: This study aims to design a multi-epitope vaccine (MEV) and evaluate its immunogenicity, structural stability, and interactions with toll-like receptor 2/4 (TLR-2/4) via computational biology approaches. Methods: We designed MEV using bioinformatics tools, prioritizing immunodominant epitopes from Mycobacterium tuberculosis antigens. Structural stability was optimized through disulfide engineering, and molecular docking/dynamics simulations were used to analyze interactions and conformational dynamics with TLR-2/4. Antigenicity, immunogenicity, population coverage, and immune responses were computationally assessed. Results: The MEV candidate, CP91110P, exhibited 86.18% predicted global human leukocyte antigen (HLA)-I/II coverage, high antigenicity (VaxiJen: 0.8789), and immunogenicity (IEDB: 4.40091), with favorable stability (instability index: 33.48) and solubility (0.485). Tertiary structure analysis indicated that 98.34% residues were located in favored regions. Molecular docking suggested strong TLR-2 (−1535.9 kcal/mol) and TLR-4 (−1672.5 kcal/mol) binding. Molecular dynamics simulations indicated stable TLR-2 interactions (RMSD: 6–8 Å; Rg: 38.50–39.50 Å) and flexible TLR-4 binding (RMSD: 2–6 Å; Rg: 33–36 Å). Principal component analysis, free energy landscapes, and dynamic cross-correlation matrix analyses highlighted TLR-2’s structural coherence versus TLR-4’s adaptive flexibility. Immune simulations predicted potential robust natural killer cell activation, T helper 1 polarization (interferon-gamma/interleukin-2 dominance), and elevated IgM/IgG levels. Conclusions: CP91110P is predicted to stably bind to TLR-2 and flexibly interact with TLR-4, with prediction of its high antigenicity and broad coverage across immune populations. However, this conclusion requires confirmation through experimental validation. Therefore, it may provide a promising candidate for experimental validation in the development of tuberculosis vaccines. Full article

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

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

Background/Objectives: Esophageal squamous cell carcinoma (ESCC) is a common form of esophageal cancer with a poor prognosis and limited treatment options. Epidermal growth factor receptor (EGFR), an overexpressed oncogenic gene in all ESCC patients, is an attractive target for developing therapies against ESCC. There is an extremely urgent need to develop immunotherapy tools targeting EGFR for the treatment of ESCC. Methods: In this study, we developed human Interleukin-21 (hIL-21)-armed, chimeric-antigen-receptor-modified T (CAR-T) cells targeting EGFR as a new therapeutic approach. The CAR contains a variable domain of the llama heavy chain of heavy-chain antibodies (VHHs), also known as nanobodies (Nbs), as a promising substitute for the commonly used single-chain variable fragment (ScFv) for CAR-T development. Results: We show that nanobody-derived, EGFR-targeting CAR-T cells specifically kill EGFR-positive esophageal cancer cells in vitro and in animal models. Human IL-21 expression in CAR-T cells further improved their expansion and antitumor ability and were observed to secrete more interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and Interleukin-2 (IL-2) when co-cultured with ESCC cell lines in vitro. More CD8⁺ CAR-T cells and CD3⁺CD8⁺CD45RO⁺CD62L⁺ central memory T cells were detected in CAR-T cells expressing hIL-21 cells. Notably, hIL-21-expressing CAR-T cells showed superior antitumor activity in vivo in a KYSE-150 xenograft mouse model. Conclusions: Our results show that hIL-21-armed, nanobody-derived, EGFR-specific CAR-T cell therapy is a highly promising option for treating ESCC patients. Full article

The spore-forming probiotic Bacillus coagulans JBI-YZ6.3 interacts with the gut epithelium via its secreted metabolites as well as its cell walls, engaging pattern-recognition receptors on the epithelium. We evaluated its effects on human T84 gut epithelial cells using in vitro co-cultures, comparing metabolically active germinated spores to the isolated metabolite fraction and cell wall fraction under unstressed versus inflamed conditions. Germinated spores affected epithelial communication via chemokines interleukin-8, interferon gamma-induced protein-10, and macrophage inflammatory protein-1 alpha and beta after 2 and 24 h of co-culture. Non-linear dose responses confirmed that bacterial density affected the epigenetic state of the epithelial cells. In contrast, the cell wall fraction increased cytokine and chemokine levels under both normal and inflamed conditions, demonstrating that the intact bacterium had anti-inflammatory properties, regulating pro-inflammatory signals from its cell walls. During recovery from mechanical wounding, germinated spores accelerated healing, both in the absence and presence of LPS-induced inflammation; both the metabolite and cell wall fractions contributed to this effect. The release of zonulin, a regulator of tight junction integrity, was reduced by germinated spores after 2 h. These findings suggest that B. coagulans JBI-YZ6.3 modulates epithelial chemokine signaling, supports barrier integrity, and enhances epithelial resilience, highlighting its potential as an efficacious multi-faceted probiotic for gut health. Full article

Natural killer (NK) and CD8⁺ T cell function is compromised in human immunodeficiency virus type 1 (HIV-1) infection by increased expression of inhibitory receptors such as TIGIT (T cell immunoreceptor with Ig and ITIM domains). Blocking inhibitory receptors or their ligands with monoclonal antibodies (mAb) has potential to improve antiviral immunity in general and facilitate HIV eradication strategies. We assessed the impact of TIGIT engagement and blockade on cytotoxicity, degranulation, and interferon-gamma (IFN-γ) production by CD8⁺ T cells from persons living with HIV (PLWH). The effect of TIGIT engagement on non-specific anti-CD3-redirected cytotoxicity was assessed in redirected cytotoxicity assays, and the effect of TIGIT blockade on HIV-specific CD8⁺ T cell responses was assessed by flow cytometry. In 14/19 cases where peripheral blood mononuclear cells (PBMC) mediated >10% redirected cytotoxicity, TIGIT engagement reduced the level of cytotoxicity to <90% of control values. We selected PLWH with >1000 HIV Gag or Nef-specific IFN-γ spot forming cells per million PBMC to quantify the effects of TIGIT blockade on HIV-specific CD8⁺ T cell responses by flow cytometry. Cell surface TIGIT expression decreased on CD8⁺ T cells from 23/40 PLWH following TIGIT blockade and this loss was associated with increased anti-TIGIT mAb fluorescence on monocytes. In total, 6 of these 23 PLWH had enhanced HIV-specific CD8⁺ T cell degranulation and IFN-γ production with TIGIT blockade, compared to 0/17 with no decrease in cell surface TIGIT expression. Reduced CD8⁺ T cell TIGIT expression with TIGIT blockade involved trogocytosis by circulating monocytes, suggesting that an effector monocyte population and intact fragment crystallizable (Fc) functions are required for mAb-based TIGIT blockade to effectively enhance HIV-specific CD8⁺ T cell responses. Full article

Breast cancer is a global health issue that, when in the metastasis stage, is characterized by the lack of estrogen receptor-α, the progesterone receptor, and human epidermal growth receptor expressions. The present study analyzed the differential gene expression related to the immune system affected by ionizing radiation and estrogen in cell lines derived from an experimental breast cancer model that was previously developed; where the immortalized human breast epithelial cell line MCF-10F, a triple-negative breast cancer cell line, was exposed to low doses of high linear energy transfer α particle radiation (150 keV/μm), it subsequently grew in the presence or absence of 17β-estradiol. Results indicated that interferon-related developmental regulator 1 gene expression was affected in the estrogen-treated cell line; this interferon, as well as the Interferon-Induced Transmembrane protein 2, and the TNF alpha-induced Protein 6 gene expression levels were higher than the control in the Alpha3 cell line. Furthermore, the interferon-related developmental regulator 1, the Interferon-Induced Transmembrane protein 2, the TNF alpha-induced Protein 6, the Nuclear Factor Interleukin 3-regulated, and the Interferon-Gamma Receptor 1 showed high expression levels in the Alpha5 cell line, and the Interferon Regulatory Factor 6 was high in the Tumor2 cell line. Additionally, to further strengthen these data, publicly available datasets were analyzed. This analysis was conducted to assess the correlation between estrogen receptor alpha expression and the genes mentioned above in breast cancer patients, the differential gene expression between tumor and normal tissues, the immune infiltration level, the ER status, and the survival outcome adjusted by the clinical stage factor. It can be concluded that the genes of the interferon family and Tumor Necrosis factors can be potential therapeutic targets for breast cancer, since they are active before tumor formation as a defense of the body under radiation or estrogen effects. Full article

Influenza A virus (IAV) is known for causing seasonal epidemics ranging from flu to more severe outcomes like pneumonia, cytokine storms, and acute respiratory distress syndrome. The innate immune response and inflammasome activation play pivotal roles in sensing, preventing, and clearing the infection, as well as in the potential exacerbation of disease progression. This study examines the complex relationships between donor-specific characteristics and cytokine responses during H3N2 IAV infection using an ex vivo model. At 24 h post infection in 31 human lung explant tissue samples, key cytokines such as interleukin (IL)-6, IL-10, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) were upregulated. Interestingly, a history of lung cancer did not impact the acute immune response. However, cigarette smoking and programmed death-ligand 1 (PD-L1) expression on macrophages significantly increased IL-2 levels. Conversely, age inversely affected IL-4 levels, and diabetes mellitus negatively influenced IL-6 levels. Additionally, both diabetes mellitus and programmed cell death protein 1 (PD-1) expression on CD3⁺/CD4⁺ T cells negatively impacted TNF-α levels, while body mass index was inversely associated with IFN-γ production. Toll-like receptor 2 (TLR2) expression emerged as crucial in mediating acute innate and adaptive immune responses. These findings highlight the intricate interplay between individual physiological traits and immune responses during influenza infection, underscoring the importance of tailored and personalized approaches in IAV treatment and prevention. Full article

Natural killer (NK) cells can be effective immunotherapeutic anti-cancer agents due to their ability to selectively target and kill tumor cells. This activity is modulated by the interaction of NK cell receptors with inhibitory ligands on the surface of target cells. NK cell inhibitory ligands can be upregulated on tumor cell surfaces in response to interferon-gamma (IFN-γ), a cytokine which is produced by activated NK cells. We hypothesized that the resistance of tumor cells to NK cell killing could be overcome by expression of the parainfluenza virus 5 (PIV5) V protein, which has known roles in blocking IFN-γ signaling. This was tested with human PM21-NK cells produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Infection of human SK-N-SH neuroblastoma cells with PIV5 blocked IFN-γ-mediated upregulation of three NK cell inhibitory ligands and enhanced in vitro killing of these tumor cells by PM21-NK cells. SK-N-SH cells transduced to constitutively express the V protein alone were resistant to IFN-γ-mediated increases in cell surface expression of NK cell inhibitory ligands. Real-time in vitro cell viability assays demonstrated that V protein expression in SK-N-SH cells was sufficient to increase PM21-NK cell-mediated killing. Toward a potential therapeutic application, transient lentiviral delivery of the V gene also enhanced PM21-NK cell killing in vitro. Our results provide the foundation for novel therapeutic applications of V protein expression in combination with ex vivo NK cell therapy to effectively increase the killing of tumor cells. Full article

Olfactory perception is an important physiological function for human well-being and health. Loss of olfaction, or anosmia, caused by viral infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has received considerable attention, especially in persistent cases that take a long time to recover. This review discusses the integration of different components of the olfactory epithelium to serve as a structural and functional unit and explores how they are affected during viral infections, leading to the development of olfactory dysfunction. The review mainly focused on the role of receptors mediating the disruption of olfactory signal transduction pathways such as angiotensin converting enzyme 2 (ACE2), transmembrane protease serine type 2 (TMPRSS2), neuropilin 1 (NRP1), basigin (CD147), olfactory, transient receptor potential vanilloid 1 (TRPV1), purinergic, and interferon gamma receptors. Furthermore, the compromised function of the epithelial sodium channel (ENaC) induced by SARS-CoV-2 infection and its contribution to olfactory dysfunction are also discussed. Collectively, this review provides fundamental information about the many types of receptors that may modulate olfaction and participate in olfactory dysfunction. It will help to understand the underlying pathophysiology of virus-induced anosmia, which may help in finding and designing effective therapies targeting molecules involved in viral invasion and olfaction. To the best of our knowledge, this is the only review that covered all the receptors potentially involved in, or mediating, the disruption of olfactory signal transduction pathways during COVID-19 infection. This wide and complex spectrum of receptors that mediates the pathophysiology of olfactory dysfunction reflects the many ways in which anosmia can be therapeutically managed. Full article

Recent studies highlight the integral role of the interferon gamma receptor (IFNγR) pathway in T cell–mediated cytotoxicity against solid but not liquid tumors. IFNγ not only directly facilitates tumor cell death by T cells but also indirectly promotes cytotoxicity via myeloid phagocytosis in the tumor microenvironment. Meanwhile, full human ex vivo immune checkpoint drug screening remains challenging. We hypothesized that an engineered gamma interferon activation site response element luciferase reporter (GAS-Luc2) can be utilized for immune checkpoint drug screening in diverse ex vivo T cell–solid tumor cell co-culture systems. We comprehensively profiled cell surface proteins in ATCC’s extensive collection of human tumor and immune cell lines, identifying those with endogenously high expression of established and novel immune checkpoint molecules and binding ligands. We then engineered three GAS-Luc2 reporter tumor cell lines expressing immune checkpoints PD-L1, CD155, or B7-H3/CD276. Luciferase expression was suppressed upon relevant immune checkpoint–ligand engagement. In the presence of an immune checkpoint inhibitor, T cells released IFNγ, activating the JAK-STAT pathway in GAS-Luc2 cells, and generating a quantifiable bioluminescent signal for inhibitor evaluation. These reporter lines also detected paracrine IFNγ signaling for immune checkpoint-targeted ADCC drug screening. Further development into an artificial antigen-presenting cell line (aAPC) significantly enhanced T cell signaling for superior performance in these ex vivo immune checkpoint drug screening platforms. Full article

Renal tumors comprise ~7% of all malignant pediatric tumors. Approximately 90% of pediatric kidney tumors comprise Wilms tumors, and the remaining 10% include clear cell sarcoma of the kidney, malignant rhabdoid tumor of the kidney, renal cell carcinoma and other rare renal tumors. Over the last 30 years, the role of cytokines and their receptors has been considerably investigated in both cancer progression and anti-cancer therapy. However, more effective immunotherapies require the cytokine profiling of each tumor type and comprehensive understanding of tumor biology. In this study, we aimed to investigate the activation of signaling pathways in response to cytokines in three pediatric kidney tumor cell lines, in WT-CLS1 and WT-3ab cells (both are Wilms tumors), and in G-401 cells (a rhabdoid kidney tumor, formerly classified as Wilms tumor). We observed that interferon-alpha (IFN-α) and interferon-gamma (IFN-γ) very strongly induced the activation of the STAT1 protein, whereas IL-6 and IFN-α activated STAT3 and IL-4 activated STAT6 in all examined tumor cell lines. STAT protein activation was examined by flow cytometry and Western blot using phospho-specific anti-STAT antibodies which recognize only activated (phosphorylated) STAT proteins. Nuclear translocation of phospho-STAT proteins upon activation with specific cytokines was furthermore confirmed by immunofluorescence. Our results also showed that both IFN-α and IFN-γ caused upregulation of major histocompatibility complex (MHC) class I proteins, however, these cytokines did not have any effect on the expression of MHC class II proteins. We also observed that pediatric kidney tumor cell lines exhibit the functional expression of an additional cytokine signaling pathway, the tumor necrosis factor (TNF)-α-mediated activation of nuclear factor kappa B (NF-κB). In summary, our data show that human pediatric renal tumor cell lines are responsive to stimulation with various human cytokines and could be used as in vitro models for profiling cytokine signaling pathways. Full article

Raynaud’s Phenomenon (RP) leading to repetitive ischemia and reperfusion (IR) stress, is the first recognizable sign of systemic sclerosis (SSc) leading to increased oxidative stress. High-mobility group box-1 (HMGB1) is a nuclear factor released by apoptotic and necrotic cells after oxidative stress. Since HMGB1 can signal through the receptor for advanced glycation end products (RAGE), we investigated whether an RP attack promotes the release of HMGB1, leading to fibroblast activation and the upregulation of interferon (IFN)-inducible genes. A cold challenge was performed to simulate an RP attack in patients with SSc, primary RP (PRP), and healthy controls. We measured levels of HMGB1 and IFN gamma-induced Protein 10 (IP-10) at different time points in the serum. Digital perfusion was assessed by photoplethysmography. In vitro, HMGB1 or transforming growth factor (TGF-β1) (as control) was used to stimulate healthy human dermal fibroblasts. Inflammatory, profibrotic, and IFN-inducible genes, were measured by RT-qPCR. In an independent cohort, sera were obtained from 20 patients with SSc and 20 age- and sex-matched healthy controls to determine HMGB1 and IP-10 levels. We found that HMGB1 levels increased significantly 30 min after the cold challenge in SSc compared to healthy controls. In vitro stimulation with HMGB1 resulted in increased mRNA expression of IP-10, and interleukin-6 (IL-6) while TGF-β1 stimulation promoted IL-6 and Connective Tissue Growth Factor (CTGF). In serum, both HMGB1 and IP-10 levels were significantly higher in patients with SSc compared to healthy controls. We show that cold challenge leads to the release of HMGB1 in SSc patients. HMGB1 induces IP-10 expression in dermal fibroblasts partly through the soluble RAGE (sRAGE) axis suggesting a link between RP attacks, the release of HMGB1 and IFN-induced proteins as a putative early pathogenetic mechanism in SSc. Full article

Interferon gamma (IFNγ) is central to the inflammatory immune response, such as that entrained by BCG immunotherapy for bladder cancer. However, immune-mediated tumour cell killing is subject to modulation by immunoinhibitory “checkpoint” receptors such as PD-L1. We investigated the effects of IFNγ on barrier-forming in vitro-differentiated normal human urothelium using mRNA-sequencing, and showed canonical upregulation of MHC class I/II and de novo expression of the T cell tropic CXCL9-11 chemokines. Normal urothelium constitutively expressed immunoinhibitory B7 family member VSIR (VISTA), while CD274 (PD-L1) expression was induced/upregulated by IFNγ. We generated a urothelial IFNγ response gene signature. When applied to the unsupervised clustering of non-muscle-invasive bladder cancers, the IFNγ-signature predicted longer recurrence-free survival. In muscle-invasive cancers, the IFNγ-signature split the basal/squamous consensus subtype, with significantly worse overall survival when weak or absent. This study offers novel insights into strategies to enhance immunotherapy via the IFNγ and VISTA/PD-L1 nexus. Full article

Human interferon-gamma (hIFN$\gamma$) is a crucial signaling molecule with an important role in the initialization and development of the immune response of the host. However, its aberrant activity is also associated with the progression of a multitude of autoimmune and other diseases, which determines the need for effective inhibitors of its activity. The development of such treatments requires proper understanding of the interaction of hIFN$\gamma$ to its cell-surface receptor hIFNGR1. Currently, there is no comprehensive model of the mechanism of this binding process. Here, we employ molecular dynamics simulations to study on a microscopic level the process of hIFN$\gamma$–hIFNGR1 complex formation in different scenarios. We find that the two molecules alone fail to form a stable complex, but the presence of heparan-sulfate-like oligosaccharides largely facilitates the process by both demobilizing the highly flexible C-termini of the cytokine and assisting in the proper positioning of its globule between the receptor subunits. An antiproliferative-activity assay on cells depleted from cell-surface heparan sulfate (HS) sulfation together with the phosphorylation levels of the signal transducer and activator of transcription STAT1 confirms qualitatively the simulation-based multistage complex-formation model. Our results reveal the key role of HS and its proteoglycans in all processes involving hIFN$\gamma$ signalling. Full article