BACKGROUND AND HYPOTHESIS:Immune dysregulation contributes to the pathophysiology of schizophrenia (SZ) and bipolar disorder (BD), but specific immune alterations remain unclear. Natural killer (NK) cells, regulated by a balance of activating and inhibitory receptors, are increasingly implicated in neuroimmune interactions. We hypothesized that distinct NK cell receptor expression patterns may identify patient subgroups characterized by preserved brain structure and reduced symptom burden. STUDY DESIGN:We conducted deep immunophenotyping and unsupervised clustering of NK cells from 53 patients (32 BD, 21 SZ) and 25 healthy controls (HC). Frequencies of NK subsets defined by main receptor expression were examined. A subset of 58 participants also underwent MRI to assess cortical thickness and white matter microstructure. Associations between immune profiles, imaging measures and clinical symptoms were tested. STUDY RESULTS:Across clustering models, we consistently identified a patient-specific cluster (PSI) characterized by increased NKG2A+NKp30+ NK cells and reduced double-negative (NKG2A-NKp30-) NK cells. This pattern was independent of classical CD56-based maturation. Patients in the PSI cluster showed preserved cortical thickness and white matter integrity compared with patients outside the cluster, who exhibited widespread reductions relative to HC controls. Furthermore, reduced double-negative NK frequencies correlated with lower burden of negative symptoms. CONCLUSIONS:Co-expression of NKp30 and NKG2A defines a meaningful NK cell phenotype associated with immune regulation, preserved brain structure, and improved clinical outcomes in SZ and BD. This profile may represent a marker of immune resilience and neuroimmune homeostasis, with potential implications for early diagnosis, patient stratification, and novel therapeutic strategies.

Introduction T cell receptor-engineered T cell (TCR-T) therapy is a promising approach for cancer treatment. Expanding the repertoire of functional TCRs across diverse human leucocyte antigen (HLA) alleles and tumour antigens is essential to broaden patient access while minimising off-target toxicities. However, naturally occurring tumour-specific TCRs, which represent an important source of therapeutic candidates, are typically derived from extremely rare peripheral T-cell populations. Therefore, their detection and characterisation remain challenging with conventional methods. Research design and methods Agnostic mass cytometry screening identified a novel HLA-A*11:01-restricted TCR targeting an MSLN epitope. Preclinical efficacy screening of the discovered TCR included cognate peptide and various tumour cell lines expressing MSLN. Safety profiling included alloreactivity testing and an integrated screening approach of predicted off-target epitopes from both (A) conventional alanine-scan (A-scan) and (B) structurally informed TCR-pMHC interaction modelling approaches. Results We used a comprehensive, high-throughput TCR discovery and safety profiling platform to identify an MSLN-targeting TCR and validated a novel HLA-A*11:01-restricted mesothelin epitope as a therapeutic target. The TCR exhibited robust activity against MSLN-expressing tumour cells from ovarian, prostate, lung and pancreatic origins but showed cross-reactivity against an epitope from DHRS11. Conclusions We demonstrate the utility of our described TCR discovery and characterisation platform by applying it to identify a TCR targeting a novel MSLN HLA-A*11:01-restricted epitope. The discovered TCR showed promising anti-tumour activity although DHRS11 cross-reactivity limits its clinical eligibility.

BackgroundIn the heterochronic parabiosis model it has been shown that blood from elderly animals exhibits markedly reduced rejuvenating effects compared to that of young organisms. Furthermore, human plasma from older subjects, when used as a supplement in cell culture media, is significantly less effective than plasma derived from younger individuals. This study analyzed plasma from a cohort of 229 subjects by a proteomic approach to reveal age-related changes.MethodsA mass spectrometry-based proteomic analysis was performed on plasma samples from 3 age-groups: a prepubertal, a healthy young adult group and a cohort of individuals over 75 years old with three different life-experiences. An additional parallel study was conducted by a Milliplex Luminex assay.ResultsThe proteomic analysis revealed a chronic inflammatory state in the elderly population, along with complement activation and impaired regulation of blood coagulation. This inflammatory condition was confirmed by Luminex assay, showing elevated levels of classical pro-inflammatory cytokines in the plasma of elderly individuals. Moreover, the elderly group showed a reduced production of antibody light chains, suggesting concurrent immunosenescence. In the older group we identified 25 upregulated proteins whose elevated abundance, combined with acquired immune aging may constitute a plasma proteomic signature of aging. The degree of upregulation of these signature proteins varied among elderly subgroups with different life-experience. A good physical condition and/or cognitive function correlated with a lower expression of the aging-related proteomic profile. Furthermore, several sex-specific differences were identified in the plasma profiles of young donors. Reversely, among elderly individuals, no major differences were observed, except for an increased level of Pregnancy Zone Protein (PZP) in females.ConclusionsProteomic analysis of plasma revealed protein variations associated with aging, primarily involving inflammation-related pathways, immunosenescence features, and sex-linked differences. This study highlights the pathological characteristics underlying the aging process.

Over the last few decades, scientists' attention has shifted from neuronal to non-neuronal cells to explain the mechanisms at the basis of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). ALS is a multifactorial and multicellular disease in which microglia have a central role, during disease progression. We previously demonstrated that metabotropic glutamate receptor 5 (mGluR5) is dysfunctional in the spinal cord of the SOD1G93A ALS mice, and its in-vivo genetic or pharmacological dampening ameliorates disease outcome and astrocyte and microglia reactivity. Here, we studied the expression of typical phenotype-related markers during the disease progression in spinal cord microglia cells acutely isolated from early asymptomatic and late symptomatic SOD1G93A ALS mice. Moreover, we investigated whether reducing mGluR5 affected the microglia phenotype and function. In contrast to what we previously observed in astrocytes, mGluR5 expression decreased during disease progression in microglia acutely isolated from adult SOD1G93A mice. In-vivo genetic mGluR5 downregulation did not affect microglia phenotype-relevant markers, which evidenced a unique expression distribution. Conversely, mGluR5 reduction ameliorated redox balance and bioenergetics of adult microglia. Microglia cultured from the spinal cord of SOD1G93A pups showed that in-vitro mGluR5 pharmacological manipulation by the negative allosteric modulator CTEP partially modified their bioenergetic and oxidative status. Overall, our results suggest that mGluR5 manipulation ameliorates microglia phenotype and function in ALS by both direct and indirect mechanisms. Consequently, we hypothesised that the improvement of microglia reactive status by in-vivo mGluR5 downregulation or CTEP pharmacological modulation is supported by ameliorated bioenergetic metabolism, and the indirect astrocyte's phenotype change that promotes an improvement of the surrounding environment.

BACKGROUND:Innate immune dysfunction is implicated in schizophrenia (SZ) and bipolar disorder (BD). Alterations in natural killer (NK) cells, monocytes and macrophages occur in both disorders across peripheral and central compartments. This systematic review synthesises current evidence by clinical stage and illness phase. METHODS:Following PRISMA guidelines, PubMed, Scopus and PsycINFO were searched to May 2025. Eligible studies reported peripheral blood, cerebrospinal fluid (CSF) or post-mortem brain findings. FINDINGS:Eighty-one studies met inclusion criteria. In SZ, peripheral data showed altered NK cell subsets and monocyte abnormalities, including elevated counts and inflammatory ratios, particularly in early or acute stages. CSF studies found increased monocyte chemoattractants, and post-mortem analyses revealed macrophage upregulation in frontal and temporal cortices. In BD, NK cell results were limited and inconsistent. Monocyte activation was most evident during symptomatic phases, particularly mania. CSF analyses detected increased monocyte- and macrophage-associated proteins, while post-mortem findings indicated microglial activation in selected cortical and subcortical regions, less consistently than in SZ. INTERPRETATION:Innate immune alterations in SZ and BD partly overlap yet remain disorder- and state-specific. Central compartments and NK cells are underexplored. Stratification by stage and phase may improve interpretability and guide longitudinal, multimodal, cell-specific research for precision immunopsychiatry.

Cell free DNA (cfDNA) is detectable at low concentrations in the plasma of healthy subjects and at high concentrations in disorders characterized by a high rate of necrotic events, such as tumors and vasculitis, leading to the release of necrotic DNA into the surrounding tissue and the bloodstream. Although cfDNA may act as a danger signal by binding to DNA sensors, triggering inflammation and immune responses, elevated cfDNA concentrations instead may exert immunoregulatory activities. Here, we show that exogenously administered cfDNA mediates immunoregulatory functions in vivo, in particular, it protects lupus-prone mice from disease progression and favors tumor growth in tumor-challenged mice. Our data suggest that cfDNA mediates immune regulatory activities by directly interacting with MHC class II molecules on antigen-presenting cells and through recruitment of regulatory T cells. This study unveils unprecedented biologic functions of cfDNA with significant pathogenic relevance and remarkable implications for the treatment of cancer patients.

Introduction:The tumor microenvironment (TME) plays a crucial role in cancer progression, yet the interactions between tumor cells and stromal components, such as fibroblasts, remain poorly understood. Traditional two-dimensional (2D) culture models fail to accurately replicate the complexities of the TME, hindering progress in cancer research and drug development. Methods:This study presents a novel 3D spheroid model, generated using the hanging drop system, that incorporates both tumor cells (B16F10 mouse melanoma) and fibroblasts (NIH/3T3), and aimed at simulating the early-stage TME. Results:We demonstrate that fibroblasts are essential for ECM deposition, which is absent in spheroids composed only of tumor cells. Co-cultured spheroids exhibited a more organized structure, enhanced ECM deposition (type-VI collagen), and more closely resembled the morphology of native tumors compared to monocultures. RNA sequencing analysis revealed that the gene expression profile of B16F10-NIH/3T3 spheroids closely matched that of in vivo tumors, with 693 genes involved in critical pathways such as "pathways in cancer" and those linked to drug resistance. Discussion:These findings highlight the importance of fibroblast inclusion in 3D models to replicate the genetic and structural features of the TME. Our spheroid system provides a more accurate representation of early tumor stages and offers a promising platform for drug screening, reducing the need for in vivo models by allowing the selection of the most effective compounds for further testing. This work underscores the potential of 3D culture systems in advancing our understanding of tumor biology and improving the precision of cancer therapeutics.

Silk fibroin nanoparticles (SFNs) have been widely investigated for drug delivery, but their clinical application still faces technical (large-scale and GMP-compliant manufacturing), economic (cost-effectiveness in comparison to other polymer-based nanoparticles), and biological (biodistribution assessments) challenges. To address biodistribution challenge, we provide a straightforward desolvation method (in acetone) to produce homogeneous SFNs incorporating increasing amounts of Fe2O3 (SFNs-Fe), detectable by Magnetic Resonance Imaging (MRI), and loaded with curcumin as a model lipophilic drug. SFNs-Fe were characterized by a homogeneous distribution of the combined materials and showed an actual Fe2O3 loading close to the theoretical one. The amount of Fe2O3 incorporated affected the physical-chemical properties of SFNs-Fe, such as polymer matrix compactness, mean diameter and drug release mechanism. All formulations were cytocompatible; curcumin encapsulation mitigated its cytotoxicity, and iron oxide incorporation did not impact cell metabolic activity but affected cellular uptake in vitro. SFNs-Fe proved optimal for biodistribution studies, as MRI showed significant nanoparticle retention at the administration site, supporting their potential for locoregional cancer therapy. Finally, technical and economic challenges in SFN production were overcome using a GMP-compliant microfluidic scalable technology, which optimized preparation to produce smaller particle sizes compared to manual methods and reduced acetone usage, thus offering environmental and economic benefits. Moreover, enabling large-scale production of GMP-like SFNs, this represents a considerable step forward for their application in the clinic.

Some studies showed a “rejuvenating” effect of exposing aging tissues to a young environment. In mouse heterochronic parabiosis experiments, in response to young organisms, old animals lived longer than isochrony old age-matched conjoint animals. Comparable “rejuvenating” effects were obtained by injecting young plasma in old mice. This raised great hopes of slowing down the senescence process in humans by the injection of young plasma, as well as to prevent or cure age-related diseases. Some clinical trials are currently being performed or were recently completed. However, these studies are small and of limited duration, and we still lack convincing evidence to support the effectiveness of young plasma injection. It is urgent to perform additional investigations, including the development of an assay to measure the cell proliferation induction capability of different human plasmas, before one can seriously think of a large-scale treatment of humans. We adopted a simple method to measure the potential of different plasmas in supporting cell line proliferation, regardless of the co-presence of a platelet lysate. By comparing plasmas from young and old subjects, we observed a decreased activity in plasmas from old individuals. The young plasma effect may be attributed to specific proteins and growth factors more abundant in younger individuals that could decrease with age. Alternatively, or at the same time, the reduced cell proliferation support could be due to inhibitors present in the old plasma. Studying the different protein content of young and old plasmas was out of the scope of this article. Such differences should be adequately investigated by proteomics using many samples. However, a preliminary study of the different protein content of young and old plasmas was part of the assay validation using a commercially available cytokine array for parallel determination of the relative levels of 105 selected human proteins. We could show the existence of specific differences between young and old plasmas and that plasmas from old individuals presented a higher concentration of “inflammatory” proteins.

Sotos syndrome (SoS) is a neurodevelopmental disorder that results from NSD1 mutations that cause haploinsufficiency of NSD1. Here, we generated an induced pluripotent stem cell (iPSC) line from fibroblasts of a SoS patient carrying the pathogenic variant (c.1633delA). The cell line shows typical iPSC morphology, high expression of pluripotent markers, normal karyotype, and it differentiates into three germ layers in vitro. This line is a valuable resource for studying pathological pathways involved in SoS.

Germline variants in the NSD1 gene are responsible for Sotos syndrome, while somatic variants promote neoplastic cell transformation. Our previous studies revealed three alternative RNA isoforms of NSD1 present in fibroblast cell lines (FBs): the canonical full transcript and 2 alternative transcripts, termed AT2 (NSD1 Δ5Δ7) and AT3 (NSD1 Δ19–23 at the 5′ end). The precise molecular pathways affected by each specific isoform of NSD1 are uncharacterized to date. To elucidate the role of these isoforms, their expression was suppressed by siRNA knockdown in FBs and protein expression and transcriptome data was explored. We demonstrate that one gene target of NSD1 isoform AT2 is ARP3 actin-related protein 3 homolog B (ACTR3B). We show that loss of both canonical NSD1 and AT2 isoforms impaired the ability of fibroblasts to regulate the actin cytoskeleton, and we observed that this caused selective loss of stress fibers. Our findings provide novel insights into NSD1 function by distinguishing isoform function and demonstrating an essential role of NSD1 in regulating the actin cytoskeleton and stress fiber formation in fibroblasts.

Objective: The aim of this study was to characterize T-cell activation, exhaustion, maturation and Treg frequencies in individuals who acquire perinatal HIV (PHIV), in individuals who acquired HIV as adult (AHIV), and in healthy controls. Design: This cross-sectional study included people with HIV at least 14 and younger than 40 years, HIV-RNA less than 50 copies/ml on antiretroviral therapy for at least 6 months, and HC. Methods: We assessed the expression of PD-1, TIM-3, EOMES, CD38+ DR+, maturation status by CD4+ and CD8+ T cells and the frequency of CD4+ and CD8+ Treg cells. Principal component analysis (PCA) and k-means cluster analysis investigated which combination of immunological parameters better associated with each group. Results: Twenty-six PHIV and 18 AHIV with median ages of 26 (8.0) and 28 (6.8) years were consecutively enrolled. PHIV showed significant higher frequency of naive and lower frequency of terminal effector memory CD4+ and CD8+ T cells than AHIV. AHIV exhibited higher expression of exhaustion and activation markers. The statistical analysis returned two clusters with 94% of specificity and 88% of sensitivity identifying PHIV vs. AHIV. The nine healthy controls had a lower expression of exhaustion markers on both CD4+ and CD8+ T lymphocytes than PHIV and AHIV. Conclusion: These data may exclude major alterations of lymphopoiesis in PHIV, with even lower state of immune-activation and exhaustion compared with AHIV. This suggests that recent lack of virological control, may affect immune activation and exhaustion of CD4+ and CD8+ T cells.

Background The combination of Programmed Cell Death 1 (PD-1) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) blockade has dramatically improved the overall survival rate for malignant melanoma. Immune checkpoint blockers (ICBs) limit the tumor’s immune escape yet only for approximately a third of all tumors and, in most cases, for a limited amount of time. Several approaches to overcome resistance to ICBs are being investigated among which the addition of epigenetic drugs that are expected to act on both immune and tumor cells. Guadecitabine, a dinucleotide prodrug of a decitabine linked via phosphodiester bond to a guanosine, showed promising results in the phase-1 clinical trial, NIBIT-M4 (NCT02608437). Methods We used the syngeneic B16F10 murine melanoma model to study the effects of immune checkpoint blocking antibodies against CTLA-4 and PD-1 in combination, with and without the addition of Guadecitabine. We comprehensively characterized the tumor’s and the host’s responses under different treatments by flow cytometry, multiplex immunofluorescence and methylation analysis. Results In combination with ICBs, Guadecitabine significantly reduced subcutaneous tumor growth as well as metastases formation compared to ICBs and Guadecitabine treatment. In particular, Guadecitabine greatly enhanced the efficacy of combined ICBs by increasing effector memory CD8+ T cells, inducing effector NK cells in the spleen and reducing tumor infiltrating regulatory T cells and myeloid derived suppressor cells (MDSC), in the tumor microenvironment (TME). Guadecitabine in association with ICBs increased serum levels of IFN-γ and IFN-γ-induced chemokines with anti-angiogenic activity. Guadecitabine led to a general DNA-demethylation, in particular of sites of intermediate methylation levels. Conclusions These results indicate Guadecitabine as a promising epigenetic drug to be added to ICBs therapy.

Background: Inflammation and immunological alterations, such as T-cell and cytokine changes, are implicated in bipolar disorder (BD), with some evidence linking them to brain structural changes (e.g., cortical thickness (CT), gray matter (GM) volume and white matter (WM) microstructure). However, the connection between specific peripheral cell types, such as T-cells, and neuroimaging in BD remains scarcely investigated. Aims of the study: This study aims to explore the link between T-cell immunophenotype and neuroradiological findings in BD. Methods: Our study investigated 43 type I BD subjects (22 depressive, 21 manic) and 26 healthy controls (HC), analyzing T lymphocyte immunophenotype and employing neuroimaging to assess CT for GM and fractional anisotropy (FA) for WM. Results: In lymphocyte populations, BD patients exhibited elevated CD4+ and CD4+ central memory (TCM) cells frequencies, but lower CD8+ effector memory (TEM) and terminal effector memory (TTEM) cells. Neuroimaging analysis revealed reduced CT in multiple brain regions in BD patients; and significant negative correlations between CD4 + TCM levels and CT of precuneus and fusiform gyrus. Tract-based spatial statistics (TBSS) analysis showed widespread alteration in WM microstructure in BD patients, with negative and positive correlations respectively between FA and radial diffusivity (RD) and CD4 + TCM. Additionally, positive and negative correlations were found respectively between FA and RD and the CD8 + TEM and CD8 + TTEM subsets. Conclusions: Our research revealed distinct T lymphocyte changes and brain structure alterations in BD, underscoring possible immune-brain interactions, warranting further study and therapeutic exploration.

COVID-19 in immunocompromised patients is difficult to treat. SARS-CoV-2 interaction with the host immune system and the role of therapy still remains only partly understood. There are no data regarding the use of monoclonal antibodies and the combination of two antivirals in fighting viral replication and disease progression. We report the cases of two patients, both treated with rituximab for non-Hodgkin lymphoma and granulomatosis with polyangiitis, respectively, and both hospitalized for COVID-19 with positive SARS-CoV-2 RNAemia, who were successfully treated with a salvage combination therapy with sotrovimab, remdesivir and nirmatrelvir/ritonavir.

Background: Metastatic uveal melanoma (MUM) is a highly aggressive, therapy-resistant disease. Driver mutations in Gα-proteins GNAQ and GNA11 activate MAP-kinase and YAP/TAZ pathways of oncogenic signalling. MAP-kinase and MEK-inhibitors do not significantly block MUM progression, likely due to persisting YAP/TAZ signalling. Statins inhibit YAP/TAZ activation by blocking the mevalonate pathway, geranyl-geranylation, and subcellular localisation of the Rho-GTPase. We investigated drugs that affect the YAP/TAZ pathway, valproic acid, verteporfin and statins, in combination with MEK-inhibitor trametinib. Methods: We established IC50 values of the individual drugs and monitored the effects of their combinations in terms of proliferation. We selected trametinib and cerivastatin for evaluation of cell cycle and apoptosis. Synergism was detected using isobologram and Chou–Talalay analyses. The most synergistic combination was tested in vivo. Results: Synergistic concentrations of trametinib and cerivastatin induced a massive arrest of proliferation and cell cycle and enhanced apoptosis, particularly in the monosomic, BAP1-mutated UPMM3 cell line. The combined treatment reduced ERK and AKT phosphorylation, increased the inactive, cytoplasmatic form of YAP and significantly impaired the growth of UM cells with monosomy of chromosome 3 in NSG mice. Conclusion: Statins can potentiate the efficacy of MEK inhibitors in the therapy of UM.