Search Results (1,056)

IBD pathogenesis is underpinned by an imbalance between excess inflammation caused by effector T-cells and inadequate suppression by regulatory T-cells (Tregs). Interleukin-37 (IL-37) is a potent, anti-inflammatory cytokine that signals via its receptors IL-1R5 and IL-1R8. Hence, augmenting anti-inflammatory mechanisms that drive IL-37 expression is a strategy to control IBD-associated inflammation. However, the role of IL-37 and its receptors in T-cells remains incompletely understood. Here, we investigated T-cell expression profiles of IL-37 and its receptors to understand the drivers of dysregulated T-cell responses in IBD and develop novel, more effective therapies. T-cell subsets from healthy control (HC), Crohn’s disease (CD) and ulcerative colitis (UC) peripheral blood mononuclear cells (PBMC) and lamina propria mononuclear cells (LPMC) were assessed for expression of IL-37 and its receptors by flow cytometry. CD3+IL-1R8+ T-cell transcriptomes underwent RNA sequencing. The phenotype and suppressive capacity of Tregs supplemented with IL-37 was assessed in vitro. Our results indicate that IL-37 and its receptors were differentially expressed among PBMC and LPMC T-cell subsets in IBD patients compared to HC. Transcription signatures unique to IBD were revealed, particularly histone and mitochondrial pathways. Remarkably, culturing Tregs with IL-37 preserved FOXP3 expression and suppressiveness at a level comparable to treatment with the well-established Treg stabilizing agent rapamycin. Altogether, our study identified differences in T-cells expressing IL-37 and its receptors that are indicative of T-cell dysfunction in IBD. These findings highlight a novel and promising avenue for restoring immune homeostasis in IBD by targeting and boosting the IL-37 signalling pathway. Full article

SLAMF7, also known as CD319, a SLAM (signaling lymphocytic activation molecule) family receptor, is relatively weakly expressed on chronic lymphocytic leukemia (CLL) B cells. This study evaluated the ability of elotuzumab (E), an anti-SLAMF7/CD319 antibody, to induce antibody-dependent cellular cytotoxicity (ADCC) against CLL cell lines (MEC-1, MEC-2, CI, HG-3, PGA-1, WA-OSEL). ADCC was assessed by flow cytometry using E (100 μg/mL), rituximab (R, 100 μg/mL), and their combination (E + R). CLL lines served as targets (T), while peripheral blood mononuclear cells (PBMCs) or NK cells from healthy donors served as effectors (E) at an 8:1 E:T ratio for 4 h. With PBMCs, E-induced ADCC ranged from 1.3 ± 1.2% (PGA-1) to 14.6 ± 8.1% (MEC-1); R-induced ADCC ranged from 9.2 ± 4.6% (PGA-1) to 16.6 ± 9.4% (WA-OSEL). With NK cells, E-induced ADCC ranged from 1.8 ± 3.7% (PGA-1) to 27.3 ± 4.7% (MEC-1); R-induced ADCC ranged from 5.1 ± 4.3% (PGA-1) to 27.5 ± 13.6% (CI). E outperformed R in MEC-1, while R was superior elsewhere. Cell lines with higher SLAMF7/CD319 expression displayed increased sensitivity to E. Cell lines with del17p showed higher SLAMF7/CD319 expression. The combination of E + R showed no significant synergy over monotherapies. In conclusion, elotuzumab induced significant ADCC in CLL cells, warranting further therapeutic evaluation. Full article

The authors hypothesize that idiopathic Parkinson’s disease may result from an alteration in microvascular flow at a “critical point” in the nervous system that is characterized by pigmented cells that express neuromelanin and/or lipofuscin. “Critical points” include the olfactory epithelium/bulb, the autonomic nervous system, the enteric nervous system, the prefrontal–cortico-pontine network, and the amygdala. Hypoxia–ischemia following blood flow disturbance would recruit and activate leukocytes and induce the infiltration of peripheral immune cells into neural tissue. The excess of toxic factors produced by hyperactive immune cells, such as myeloperoxidase and its derivatives, would cause the oxidation of lipids, proteins, and biogenic monoamines such as dopamine, which in turn would facilitate the accumulation and precipitation of neuromelanin, lipofuscin, and alpha-synuclein. In addition, neuromelanin and lipofuscin precipitates may accentuate the misfolding and aggregation of alpha-synuclein. This “amplification” mechanism could help explain the crucial role of pigmented neurons in the onset of Parkinson’s disease pathology, triggering abnormal neurotoxic alpha-synuclein spread throughout the nervous system from the “critical point” of origin, and enabling a self-perpetuating degenerative process. The proposed hypothesis may have implications for the identification of new therapeutic targets, early prevention strategies, and the development of vascular and/or immune biomarkers. Full article

Background: Neuroblastoma (NB) is the most common extracranial paediatric solid cancer, with a 50% survival rate for high-risk patients. Circulating tumour cells (CTCs) are malignant cells shed by the primary tumour and metastatic sites that circulate in the bloodstream. CTCs form clusters with themselves (homotypic) or other cell types (heterotypic). Objectives: To use previously generated ImageStreamX Imaging Flow Cytometer data from blood samples from 24 patients across all NB risk groups, to examine for the presence of CTC clusters. Methods: Immunofluorescence and brightfield morphology were used to identify clusters followed by analysis using IDEAS image analysis software. Results: The mean number of clusters detected per sample was 87 (range, 0–725). Of the clusters detected, 1967/2094 (93.9%) were heterotypic and only 127/2094 (6.1%) were homotypic and found in 6/24 patients. Interestingly, in 3/24 patients, at least one cluster (median, 2 and range, 1–18) was found, but no single CTCs were detected. Clusters mostly comprised two cells (62.8%), with the maximum number of cells in homotypic and heterotypic clusters being four and eight, respectively. Conclusions: These results highlight that imaging flow cytometry can be used to detect and characterise CTC clusters in peripheral blood samples from NB patients, leading to further research exploring the composition and role of CTC clusters in NB metastasis. Full article

Background/Objectives: Interleukin-17A (IL-17A) is a key pathogenic cytokine in autoimmune arthropathies. Current monoclonal antibody inhibitors targeting the IL-17/IL-17RA axis demonstrate clinical efficacy but face significant limitations, including immunogenicity, the loss of therapeutic response, and cold-chain storage. Our study evaluated oligonucleotide aptamers targeting IL-17A and its receptor as an alternative to monoclonal antibodies to suppress an IL-17A-induced inflammatory response in cell models relevant to immunoinflammatory rheumatic diseases. Methods: We examined three aptamers: 2′-F-RNA aptamers Apt21-2 and Apt3-4 specific to IL-17A and DNA aptamer RA10-6 targeting the receptor of IL-17A. Their ability to suppress IL-17A functional activity was assessed in peripheral blood mononuclear cells (PBMCs) from healthy donors and personalized fibroblast-like synoviocytes (FLSs) from patients with axial spondyloarthritis (axSpA) and rheumatoid arthritis (RA). Inhibition was measured by quantifying IL-6 and MMP-13 secretion using ELISA and flow cytometry, using secukinumab as a reference control. Results: In PBMC, all aptamers suppressed IL-17A-stimulated IL-6 secretion and cell proliferation in a concentration-dependent manner (17–200 nM), with a 65–85% efficacy, comparable to that of secukinumab. In axSpA-derived FLS, we observed time-dependent efficacy: At 4 h, all three aptamers suppressed IL-6 to the same extent as secukinumab; at 24 h, RA10-6 maintained high efficacy while Apt21-2 and Apt3-4 showed reduced activity. A combination of receptor-targeting RA10-6 with anti-IL-17A aptamers resulted in synergistic IL-6 suppression. All aptamers reduced MMP-13 to basal levels. RA-derived FLS showed diminished responses to all inhibitors. Conclusions: Aptamers demonstrate high specificity and sustained efficacy in suppressing IL-17A signaling for an in vitro model of spondyloarthritis, with superior performance over antibodies. Disease-dependent differential efficacy in RA FLS reflects heterogeneity consistent with limited clinical anti-IL-17 efficacy in RA. These findings show the strong potential of the studied aptamers as an alternative to monoclonal antibodies for IL-17-associated inflammatory arthropathies, particularly spondyloarthritis. Full article

Background/Objectives: Mycosis fungoides (MF) and Sézary syndrome (SS) are cutaneous T-cell lymphomas (CTCLs) with variable clinical outcomes. Peripheral blood (PB) involvement in MF/SS is an independent predictor of prognosis. Accurate laboratory determination of PB involvement by MF/SS cells, however, is an ongoing challenge. Both flow cytometry (FC) and morphology-based quantification are limited by the overlap of CTCL cells and reactive T-cells. This study looks at the optimization over time of CTCL blood burden evaluation. Methods: This retrospective study reviews CTCL blood assessment at Northwestern Memorial Hospital from 2012 to 2021. Test ordering and reporting practices for morphology-based Sézary cell counts and FC were evaluated. For each assay, quantitative and qualitative results were analyzed and compared including percentages and absolute counts of abnormal T-cell populations and pathologist interpretations. Results: A total of 514 patients were evaluated, with increasing numbers of both tests ordered over time. FC quantitative metrics showed a moderate to high correlation with morphology metrics, especially for absolute CD4+/CD7− counts (correlation coefficient = 0.901, p-value < 0.001). Qualitative pathologist interpretations had moderate agreement between methods (kappa = 0.58). The recent addition of TRBC1 clonality assessment to our FC assay further optimizes the evaluation for CTCL blood burden. Conclusions: Flow cytometry offers a reliable approach for blood staging in MF/SS, and morphologic assessment may be redundant. This study provides a foundation for designing a new FC approach with TRBC1. This comprehensive review of the evolution of our laboratory practices may serve as a guide for other institutions with similar clinical needs. Full article

The blood–brain barrier (BBB) is a structure that regulates the exchange of substances between the peripheral circulation and the central nervous system (CNS), thereby protecting this environment. An increase in BBB permeability may lead to the influx of inflammatory cells, resulting in neuroinflammation and neurodegeneration. The integrity of the BBB is maintained due to the specific properties of brain endothelial cells. Considering the importance of brain endothelial cells in the BBB during inflammatory processes, these cells may be a target for anti-inflammatory agents. Polyphenols are substances exhibiting the ability to decrease inflammation; therefore, in our research, we aimed to examine their effectiveness in a brain endothelial cell culture stimulated with the pro-inflammatory cytokine TNF-α. The tested polyphenols were myricetin, chrysin, resveratrol, and curcumin. ELISA tests revealed that myricetin and chrysin decreased the concentrations of the pro-inflammatory cytokines IL-1ß, IL-6, and IL-8 secreted by brain endothelial cells. The results of flow cytometry indicate that chrysin and resveratrol are the most potent in downregulating the expression of VCAM-1 on the surface of brain endothelial cells. The obtained results confirm the anti-inflammatory potential of polyphenols in brain endothelial cells. The selected polyphenols also contribute to increasing brain endothelial cell viability and act as antioxidants. Full article

Recurrent pregnancy loss (RPL) is defined as the loss of two or more pregnancies before the 22nd gestational week and affects 10–15% of clinical pregnancies. Despite extensive diagnostics, over 50% of RPL cases remain unexplained, suggesting an important role for immunological mechanisms. Sex hormones (SH) are key regulators of immune responses during pregnancy; however, their influence on immune checkpoint proteins (ICPs) is poorly understood. This study evaluated the effects of progesterone, β-estradiol, and dihydrotestosterone (DHT) on ICP expression on immune cells, including Treg, NK, NKT, TC, Th, and T cells, collected from pregnant women and patients with unexplained RPL (uRPL). Peripheral blood mononuclear cells from 20 pregnant women and 20 uRPL patients were cultured for 48 h with SH. The expression of the first generation of ICPs—PD-1 and TIM-3—and the second—LAG-3, TIGIT, and VISTA—on T, NK, and NKT cells was analyzed by the flow cytometry method. In pregnant women, SH exerted modest effects, with DHT increasing VISTA and LAG-3 expression, while progesterone and estradiol mainly upregulated LAG-3 and TIM-3 on cytotoxic cells. In contrast, uRPL immune cells showed pronounced SH sensitivity, characterized by increased TIM-3 and VISTA expression and reduced TIGIT expression, particularly after DHT stimulation. In conclusion, SH modulates ICP expression in a cell-specific manner, with stronger effects observed in uRPL patients’ lymphocytes. These findings highlight a potential role for hormonal and ICP-targeted strategies in RPL management. Full article

Background/Objectives: Fusobacterium nucleatum and Akkermansia muciniphila are key components of the human microbiota, influencing health and disease. F. nucleatum is associated with colorectal cancer (CRC) and poor prognosis through its pro-inflammatory and pro-tumorigenic activity, whereas A. muciniphila is linked to metabolic benefits and anti-inflammatory effects. This study aimed to evaluate the immunomodulatory impact of protein extracts from these bacteria on peripheral T cell responses in healthy individuals and CRC patients. Methods: Peripheral blood mononuclear cells (PBMCs) were exposed to bacterial extracts, individually or in combination, and T cell subsets were analyzed by polychromatic flow cytometry. Results: In healthy donors, F. nucleatum increased Th0, Th2, and Tc9 cell frequencies while reducing Th1, Th1/Th17, and Treg cells. Conversely, A. muciniphila promoted a pro-inflammatory-associated T cell phenotype characterized by higher Th0, Th2, Th17, and Tc17 cells. Combined exposure enhanced Th0, Th17, and Tc17 cells while decreasing Th9 cells. In CRC patients, bacterial extracts induced no significant changes in T cell subsets. Conclusions: These findings indicate that F. nucleatum skews immune responses toward humoral and mucosal defense, whereas A. muciniphila enhances T cell polarization toward subsets usually associated with pro-inflammatory immune responses in healthy subjects. Further studies are needed to clarify their systemic immunological roles and interactions within the tumor microenvironment of CRC. Full article

T cells play an important role in the development and progression of type 1 diabetes (T1D). Checkpoint receptors regulate T cell activity, and their expression may be linked to the cells’ metabolic state. This study aims to investigate the association between T regulatory (Treg) and T conventional (Tconv) cells expressing various checkpoint inhibitors and glucose metabolism in type 1 diabetes patients and healthy controls (HCs). The study included 28 participants, with 16 of them diagnosed with type 1 diabetes, while 12 constituted a healthy control group. Multicolor flow cytometry, spectrophotometric analysis, and bead-based multiplex assays were utilized for the analyses. The study revealed that the most significant difference in T cell subsets in peripheral blood concerned TIGIT. Compared to healthy subjects, the percentages of TIGIT+ Tregs and TIGIT+ Tconvs were lower in T1D patients. Interestingly, hyperglycemia in in vitro cultures reduced percentages of TIGIT+ Tregs and TIGIT+ Tconvs, and to some extent also CTLA-4+ Tregs. A decreased percentage of these subsets was, in turn, associated with reduced glucose uptake and lower activity of the enzymes responsible for various stages of glucose metabolism. The described associations suggest a negative influence of hyperglycemia in T1D on immune regulation via a TIGIT-dependent mechanism. Hyperglycemia seems to reduce the percentage of highly regulatory TIGIT+ Tregs both in vivo and in vitro, and it is associated with reduced glucose consumption by these cells. At the same time, a reduction in the percentage of TIGIT+ Tconvs under such conditions may facilitate higher activity of Tconvs, including aberrant autoimmune reactions. Full article

Regulatory T cells (Tregs), particularly their phenotypically distinct subpopulations, are critical for the establishment of maternal immune tolerance during embryo implantation. Despite advances in assisted reproductive technologies, implantation failure remains a frequent and often unexplained clinical challenge. Variations in Treg frequency and phenotype have been proposed to influence implantation success, particularly under differing hormonal conditions. This study aimed to investigate peripheral blood Treg levels and their subpopulations on the day of blastocyst transfer in both stimulated in vitro fertilization (IVF/ICSI) cycles involving controlled ovarian hyperstimulation (COH) and true natural cycles with frozen embryo transfer (FET), and to examine their associations with systemic hormone levels and anti-Müllerian hormone (AMH). A prospective observational study was conducted including women undergoing IVF/ICSI with fresh embryo transfer (ET) and women undergoing natural cycle FET. Peripheral blood samples were collected on the day of ET and analyzed using 13-colour flow cytometry, enabling detailed subdivision of Tregs into multiple subpopulations based on the expression of differentiation and chemokine markers, including CXCR5. In addition, because common $\gamma$-chain cytokines may influence pregnancy success by modulating the balance between suppressive Treg and non-Treg subsets, intracellular STAT5 signaling was assessed using phospho-specific flow cytometry. Serum estradiol, progesterone, FSH, LH, and AMH levels were measured in parallel. Significant differences were observed in Treg subpopulation distributions between women who conceived and those who did not. Higher frequencies of naïve CXCR5⁻ Tregs were associated with clinical pregnancy, independent of age, and correlated with serum progesterone levels. Moreover, both naïve Treg frequency and enhanced IL-7-dependent STAT5 signaling in naïve Tregs from women undergoing COH were associated with AMH levels, suggesting a link between ovarian reserve and Treg homeostasis mediated by signal transducer and activator of transcription 5 (STAT5) signaling. In conclusion, Treg subpopulations, particularly CXCR5⁻ naïve Tregs, appear to play a central role in implantation success following ET. Their distribution differs between stimulated and natural cycles and is influenced by systemic progesterone levels and STAT5 signaling. These findings suggest that peripheral Treg profiling may represent a potential biomarker of implantation competence and could inform personalized approaches in assisted reproduction. Full article

Background/Objectives: Patients in need of specialized palliative care are clinically highly complex, with pain being the most prevalent problem. Furthermore, in these patients, a self-report for characterization of pain could be difficult to obtain. This cross-sectional, exploratory study investigates the use of clinical parameters and peripheral blood biomarkers for potentially identifying and characterizing pain (assessed using Pain Assessment in Advanced Dementia (PAINAD) and Numeric Scale (NS)) in patients under palliative care, including a population with dementia where pain is often underdiagnosed. Methods: Fifty-three patients with non-oncological diseases were analyzed in a cross-sectional study using medical and nursing records. Among previous biomarkers related to monocytes and platelets assessed by flow cytometry, we selected the most significative ones for pain characterization in a logistic regression analysis (multivariate analysis), alongside patient-specific characteristics such as renal function, nutritional status, and age. Results: Our exploratory findings suggest strong relationships between chronic pain and advanced age, reduced glomerular filtration rate (GFR), and malnutrition within this cohort. Furthermore, the percentage of lymphocytes, total and classical monocytes, the relative expression in monocytes of CD206, CD163, the CD163/CD206 ratio, and the relative expression in platelets of CD59 emerged as potential predictors of pain. Statistical analyses highlighted the challenges of multicollinearity among variables such as age, GFR, and nutritional status. A classification model further suggested that all patients over 65 years in our specific sample reported pain. Conclusions: This pilot study provides preliminary support for prior evidence linking chronic pain to aging, nutritional deficits, and renal impairment, and highlights potential novel peripheral blood biomarkers for pain assessment. This work emphasizes the promise of clinical and molecular biomarkers to improve pain detection and management, contributing to personalized and effective palliative care strategies. Full article

Glaesserella parasuis (G. parasuis) is a key pathogen responsible for swine respiratory disease, and the development of broadly protective vaccines is hampered by its high antigenic diversity. The iron-acquisition protein TbpB is a conserved vaccine candidate, but the cellular immune responses it elicits, particularly T-cell subset dynamics during immunization and challenge, remain insufficiently defined. This study characterized these responses after oral immunization of colostrum-deprived piglets with the TbpB^Y167A mutant. Ten colostrum-deprived piglets were allocated to immunized and non-immunized (PBS) groups, immunized at days 15 and 30 of life and subsequently challenged with G. parasuis (45 days old); peripheral blood mononuclear cells were collected at baseline, after each immunization, and at 1 and 3 days post-infection. Multiparametric flow cytometry was used to quantify major leukocyte subsets and T-cell phenotypes defined by sIgM, CD172a, CD3, TCRγδ, CD8α/β, CD4 and CD27 expression. Booster immunization induced significant expansion of B cells (p < 0.01), TCRγδ T cells (p < 0.01), CD8⁺ αβ T cells (p < 0.001) and CD4⁺ memory T cells (p < 0.01) in immunized piglets compared with controls. After challenge, CD8⁺ cytotoxic T cells in immunized animals rapidly shifted from naïve to memory phenotypes, peaking at 48–72 h (p < 0.01). These biphasic T-cell dynamics are consistent with the protective efficacy previously demonstrated for this vaccine in colostrum-deprived piglets, and support a key contribution of TCRγδ, CD8⁺ cytotoxic and CD4⁺ memory T cells to immunity against G. parasuis and to the design of next-generation vaccines. Full article

Background: Small-cell lung cancer (SCLC) is an aggressive type of lung cancer, and several factors are currently used to predict poor outcomes, including performance status (PS), extensive-stage disease, male sex, advanced age, and elevated lactate dehydrogenase (LDH) levels. In this study, we aimed to explore the role of Tegs and inflammatory indices, such as CRP and NLR, in predicting response to immunotherapy. Methods: Fifty-one therapy-naïve ES-SCLC patients and ten healthy donors were enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated and stained with fluorochrome-conjugated monoclonal antibodies. Multicolor flow cytometry was performed to determine the levels of CD8⁺ T cells and CD4⁺ Tregs, as well as their correlation with inflammatory indices and clinical outcomes. Results: ES-SCLC patients harbored higher percentages of CD8⁺ Teffs (p = 0.005) and FOXP3⁺ Tregs (p < 0.0001) in circulation before therapy compared with healthy donors. In addition, high levels of CD3⁺CD8⁺ T effectors were associated with longer PFS (p = 0.018) and longer OS (p = 0.012) compared with patients bearing low levels, while Tregs were not found to be predictive. More importantly, a survival benefit was observed in ES-SCLC patients with a low Treg/Teff ratio, as longer OS was observed in those with high percentages of CD8⁺ Teffs and low FOXP3⁺CTLA-4⁺ Tregs (p = 0.014) compared with those bearing low CD8⁺ Teffs and high FOXP3⁺CTLA-4⁺ Tregs. A low Treg/Teff ratio was further associated with low eosinophil levels and a low NLR before treatment initiation. Conclusions: These findings suggest a novel, easily obtainable blood-based signature that may help predict response to ICIs in ES-SCLC patients. Full article

Psoriasis is a common inflammatory skin disease with chronic manifestation in which the role of neutrophil extracellular traps (NETs) and alarmins are increasingly recognized as contributors to systemic and cutaneous inflammation. However, the interaction between alarmins and NET-driven immune responses remains poorly defined. The main aim of this study is to define the role of target alarmins (i.e., IL-33 and TSLP) in NETs induction and its subsequent impact on oxidative stress and inflammation in the peripheral blood. In the present study, we recruited active psoriasis patients (n = 56) and control (n = 56) subjects. The frequency of circulating neutrophils, the levels of NET-associated markers (MPO (myeloperoxidase)–DNA complex, CitH3 (citrullinated histone H3), PAD4 (peptidyl arginine deiminase4), NADPH oxidase, and NE (neutrophil elastase)), and alarmin transcripts (IL (interleukin)-33, TSLP (thymic stromal lymphopoietin), S100A7, S100B, HSP (heat shock protein) 60/70 were quantified using flow cytometry, ELISA (Enzyme-linked immunosorbent assay), and qPCR (quantitative polymerase chain reaction), respectively, in each group. The NET formation potential of isolated neutrophils was assessed in the presence or absence of rhIL-33 and rhTSLP by immunocytofluorescence. The effect of rhIL-33- and rhTSLP-primed NETs in augmenting oxidative stress and inflammation was evaluated on peripheral blood mononuclear cells (PBMCs) by ELISA. Significantly higher circulating neutrophils (p < 0.001) and levels of NET-associated markers (i.e., MPO–DNA complex, CitH3, PAD4, NADPH oxidase, and NE) were observed in active psoriasis patients compared to controls. Lesional skin exhibited strong expression of MPO (p < 0.001) compared to normal skin. The alarmins, IL-33 and TSLP, were markedly upregulated in the blood and skin (p < 0.05). The rhIL-33 and rhTSLP treated neutrophils demonstrated enhanced NETosis in patients (p < 0.001). Increased expression of inflammatory cytokines and oxidative stress markers were reported in PBMCs when incubated with rhIL-33- and rhTSLP-primed NETs. Taken together, our investigation demonstrated the novel mechanism wherein the alarmins IL-33 and TSLP exacerbate NET formation that may drive enhanced inflammation and oxidative stress in psoriasis. Full article